Intestinal Damage in COVID-19: SARS-CoV-2 Infection and Intestinal Thrombosis

Authors: Xiaoming Wu1Haijiao Jing1Chengyue Wang1Yufeng Wang1Nan Zuo1Tao Jiang2*Valerie A. Novakovic3 and Jialan Shi1,3,4* Front. Microbiol., 22 March 2022 | https://doi.org/10.3389/fmicb.2022.860931

The intestinal tract, with high expression of angiotensin-converting enzyme 2 (ACE2), is a major site of extrapulmonary infection in COVID-19. During pulmonary infection, the virus enters the bloodstream forming viremia, which infects and damages extrapulmonary organs. Uncontrolled viral infection induces cytokine storm and promotes a hypercoagulable state, leading to systemic microthrombi. Both viral infection and microthrombi can damage the gut–blood barrier, resulting in malabsorption, malnutrition, and intestinal flora entering the blood, ultimately increasing disease severity and mortality. Early prophylactic antithrombotic therapy can prevent these damages, thereby reducing mortality. In this review, we discuss the effects of SARS-CoV-2 infection and intestinal thrombosis on intestinal injury and disease severity, as well as corresponding treatment strategies.

Introduction

COVID-19 has become a worldwide pandemic causing widespread illness and mortality. SARS-CoV-2 mainly infects the respiratory tract through attachment to angiotensin-converting enzyme 2 (ACE2) receptors (Lan et al., 2020). ACE2 is also highly expressed on intestinal epithelial cells, allowing SARS-CoV-2 to infect the intestinal tract (Xiao et al., 2020a). Recent meta-analyses show that 48%–54% of fecal samples from COVID-19 patients have tested positive for viral RNA, and 15%–17% of patients have gastrointestinal (GI) symptoms (Cheung et al., 2020Mao et al., 2020Sultan et al., 2020). Additionally, live virus can be isolated from fecal samples of COVID-19 patients (Wang et al., 2020). Some studies have proposed fecal–oral transmission as the cause of intestinal infection (Guo et al., 2021). However, direct evidence for fecal–oral transmission is still lacking. Meanwhile, the virus has been detected in the blood of both symptomatic and asymptomatic patients (Chang et al., 2020), and disseminated virus could infect extrapulmonary organs (Jacobs and Mellors, 2020). Thus, the potential that intestinal infection occurs via blood transmission should be carefully considered.

Pulmonary infection triggers cytokine storm and induces a prothrombotic state (McFadyen et al., 2020Moore and June, 2020). Venous and arterial thrombosis are common in COVID-19 (Moore and June, 2020). Systematic reviews estimate that 14%–31% of in-hospital patients develop a clinically apparent thrombotic event (Suh et al., 2021Tan et al., 2021), while autopsy reports show a high prevalence of microthrombi in multiple organs, including lung, heart, liver, kidney, and gastrointestinal tract (Bradley et al., 2020Polak et al., 2020). A cohort study showed that COVID-19 patients with intestinal ischemia had markedly elevated D-dimer levels and poor outcomes (Norsa et al., 2020). Additionally, recent studies have shown that mesenteric thrombosis often results in intestinal resection and significantly increases mortality (Bhayana et al., 2020El Moheb et al., 2020). Therefore, it is essential to outline the mechanisms of intestinal thrombosis and its contribution to intestinal damage and disease progression.

In this review, we discuss blood transmission as a potential route for intestinal infection. We then summarize the characteristics and mechanism of intestinal thrombosis formation in COVID-19. Next, we focus on the effects of intestinal infection and thrombosis on intestinal damage and disease severity. Finally, we discuss therapeutic strategies to prevent intestinal damage.

Gastrointestinal Symptoms and SARS-CoV-2 Infection

Multiple studies have reported GI symptoms in COVID-19 patients, including diarrhea, nausea, vomiting, anorexia, and abdominal pain (Cheung et al., 2020Mao et al., 2020Sultan et al., 2020). According to a meta-analysis comprising 10,890 COVID-19 patients, the pooled prevalence estimates of GI symptoms were: diarrhea (7.7%), nausea or vomiting (7.8%), and abdominal pain (2.7%; Sultan et al., 2020) with 10% of these patients reporting GI symptoms as being their initial symptoms (Cheung et al., 2020). These data indicate potential gastrointestinal infection by SARS-CoV-2, which is reported to infect and replicate in epithelial cells of human small intestinal organoids (Zang et al., 2020). Both viral nucleocapsid proteins and viral particles have been detected in infected patient intestinal biopsies (Livanos et al., 2021). Additionally, SARS-CoV-2 RNA and live virus can be found in the stool of patients (Wang et al., 2020). More importantly, SARS-CoV-2 subgenomic mRNA is transcribed in actively replicating cells and has been detected in fecal samples (Wölfel et al., 2020). Further, rectal viral shedding persists for longer than that of the respiratory system (Zhao et al., 2020). All these data demonstrate that SARS-CoV-2 directly infects and replicates in intestinal epithelial cells of patients.

Intestinal Infection and Transmission Routes

With the deepening understanding of COVID-19, GI symptoms have been recognized as early signs of the disease. The high expression of ACE2 in the GI tract, isolation of live virus from fecal samples, and a subset of patients presenting with only GI symptoms seem to suggest fecal–oral transmission. However, problems with the feasibility of this mode of transmission remain. First, studies have shown that SARS-CoV-2 loses infectivity in simulated gastric acid within 10 min (Chan et al., 2020Zang et al., 2020Zhong et al., 2020). Secondly, SARS-CoV-2, as an enveloped virus, is largely unable to withstand the detergent effect of bile salts and the activity of digestive enzymes in the duodenum (Figure 1). Although some studies have suggested that highly viscous mucus in the gastrointestinal tract protects SARS-CoV-2, allowing the virus to retain its infectivity (Guo et al., 2021Zhang H. et al., 2021), there is still a lack of direct evidence. Bushman et al. (2019) had previously investigated the links between the structures of viruses and routes of transmission and found a strong association between fecal–oral transmission and the absence of a lipid envelope. Lastly, although some studies have isolated intact viruses from feces (Wang et al., 2020Zhang Y. et al., 2020Zhou et al., 2020Xiao et al., 2020b), most of them have not further confirmed the infectivity of these viruses (Wang et al., 2020Zhang Y. et al., 2020Xiao et al., 2020b). Zhou et al. (2020) confirmed viral propagation by RT-PCR, but only in a single fecal sample. Previous research has shown that SARS-CoV-2 is completely inactivated in simulated human colonic fluid over the course of 24 h, which may explain the sporadic detection of infection-active SARS-CoV-2 from feces samples.FIGURE 1

Figure 1. Intestinal infection and transmission routes. ① Direct evidence for fecal–oral transmission is still lacking. SARS-CoV-2 may be unable to enter the small intestine from the stomach due to gastric acid, bile and digestive enzymes. ② SARS-CoV-2 released from type II alveolar cells infects alveolar capillary endothelial cells (ECs). The virus replicates in ECs and is released into the blood to form viremia. ③ SARS-CoV-2 is released from infected ciliary cells of the nasal cavity and breaks through the basement membrane, infecting the vascular ECs and eventually entering circulation. ④ Blood transmission after alveolar or nasal infection is a potential route of intestinal infection. Eventually, SARS-CoV-2 is released into the gut and infects surrounding intestinal epithelial cells along the intestinal tract. ⑤ SARS-CoV-2 in the gut can also enter the capillaries and cause viremia, leading to recurrence of disease.

Several lines of evidence suggest that SARS-CoV-2 may infect the intestinal tract via the bloodstream. Deng et al. (2020) detected SARS-CoV-2 RNA in anal swabs from intratracheally but not intragastrically infected rhesus macaques, suggesting blood transmission. Indeed, SARS-CoV-2 RNA has been detected in blood and urine samples of patients (Wang et al., 2020). The virus can also be detected in multiple organs (including heart, brain, and kidney) and is associated with organ injury, indicating that the virus can reach and infect extrapulmonary organs (Puelles et al., 2020). Another study showed that SARS-CoV-2 viremia was associated with intestinal damage, independent of disease severity (Li Y. et al., 2021). Thus, blood transmission could be the cause of intestinal infection. Specifically, SARS-CoV-2 replicating in alveolar epithelial cells and capillary ECs is released into the bloodstream and infects new vascular ECs. The capillary network is then the main route by which the virus enters and infects extrapulmonary organs. The extensive surface area of intestinal capillaries makes intestinal epithelial cells more susceptible to infection than other extrapulmonary organs. Following infection of intestinal capillaries, SARS-CoV-2 is released into the gut and infects surrounding intestinal epithelial cells along the intestinal tract (Figure 1). Once established in the gut, SARS-CoV-2 can also reenter the capillaries, potentially leading to recurrence of disease. Consistent with this, in patients who experienced recurrence, the phylogenetic analysis of infection samples has shown that recurrent virus evolves from the original parent virus (Hu et al., 2020).

Additionally, SARS-CoV-2 RNA can also be detected in the blood and urine of asymptomatic patients, suggesting a second pathway to viremia through the nasal cavity (Chang et al., 2020Hasanoglu et al., 2021). The abundant blood vessels, thin mucous membrane, and higher levels of ACE2 (Huang et al., 2021) make it possible for the virus to initiate viremia from the nasal cavity. Specifically, SARS-CoV-2 is released from infected ciliary cells of the nasal cavity and breaks through the basement membrane, infecting the vascular ECs and eventually entering circulation (Figure 1). Blood transmission after nasal infection is therefore another potential route of intestinal infection.

Intestinal Damage, Malnutrition, and Poor Outcomes

A recent study has shown that a fecal sample positive for SARS-CoV-2 RNA at any time during hospitalization was associated with higher mortality [HR: 3.4 (1.2–9.9); Das Adhikari et al., 2021]. Similarly, another study showed that small-bowel thickening on CT was strongly associated with ICU admission (Wölfel et al., 2020). This relationship did not hold for colon or rectal thickening. These data indicates that small-bowel damage contributes to poor outcomes. As the main organ for nutrient absorption, damage to the small intestine will result in malabsorption and malnutrition, both of which commonly occur in COVID-19 patients (Di Filippo et al., 2021Lv et al., 2021) and are associated with disease severity (Luo et al., 2020Zhang P. et al., 2021). A fecal metabolome study showed that feces of COVID-19 patients were enriched with important nutrients that should be metabolized or absorbed, consistent with malabsorption (Lv et al., 2021). A prospective study showed that 29% of COVID-19 patients (31% of hospitalization patients and 21% of patients quarantined at home) had lost >5% of body weight [median weight loss, 6.5 (5.0–9.0) kg or 8.1 (6.1–10.9) %; Di Filippo et al., 2021]. Those patients with weight loss had greater systemic inflammation, impaired renal function and longer disease duration. A large, multicenter study (including 3,229 patients with GI symptoms) showed that 23% of patients had malnutrition, of whom 56.4% were unable to gain weight after 6 months follow-up (Rizvi et al., 2021). Studies also showed that malnutrition was associated with higher incidences of acute respiratory distress syndrome, acute myocardial injury, secondary infection, shock, and 28-day ICU mortality (Luo et al., 2020Zhang P. et al., 2021). Overall, malabsorption and malnutrition due to damaged small intestine increased disease severity and mortality.

Nutrient absorption in the small intestine is mainly through ATP-dependent active transport. Intestinal infection, hypoxemia, and intestinal ischemia contribute to malabsorption. SARS-CoV-2 adhesion depletes ACE2 levels on intestinal epithelial cells, which alters the expression of the neutral amino acid transporter B0AT1, reducing the intake of tryptophan and the production of nicotinamide (D’Amico et al., 2020). Meanwhile, uncontrolled viral replication consumes large amounts of ATP and nutrients, resulting in decreased nutrients entering the bloodstream. More importantly, anaerobic glycolysis caused by hypoxemia and intestinal ischemia significantly decreases ATP and active transport, leading to malabsorption. Additionally, hypoxemia and intestinal ischemia can also cause anorexia, nausea, vomiting, and enteral nutrition intolerance, reducing food intake. A prospective multicenter study showed that reduced food intake was associated with higher ICU admission and mortality (Caccialanza et al., 2021).

Intestinal Ischemia and Thrombosis

Intestinal ischemia is a common manifestation in COVID-19 patients. Autopsy results have shown that 31.6% of deceased patients had focal ischemic intestinal changes (Chiu et al., 2020). In a separate imaging study, bowel wall thickening and pneumatosis intestinalis, which indicate intestinal ischemia, were found on 38.1% (16 of 42) of abdominal CT images (Bhayana et al., 2020). Of these, 4 (9.5%) patients with pneumatosis intestinalis developed severe intestinal necrosis and needed resection. In another cohort study, 55.8% (58/104) of ICU patients developed an ileus (Kaafarani et al., 2020). Although mechanical factors cannot be ruled out, insufficient intestinal motility due to intestinal ischemia was more likely to be the cause of ileus in COVID-19 patients. In these patients with ileus, 4 (3.8%) developed severe intestinal ischemia and require emergency surgery. Both studies found microthrombi in these resected intestinal samples, which were the main cause of intestinal ischemia and increased mortality.

Additional intestinal ischemia and necrosis follows the formation of mesenteric thrombosis. However, there is currently relatively little data of mesenteric thrombus in COVID-19. Therefore, we have summarized the characteristics of 40 patients in 39 case reports published on PubMed (Supplementary Table 1). The median age of these patients was 50 (20–82) years, 26 (65%) were male, 38 (95%) developed bowel ischemia or necrosis, 30 (75%) needed bowel resection, 7 (17.5%) required no surgery, at least 3 (7.5%) developed sepsis, and 13 (32.5%) died. Other abdominal thrombotic events (such as celiac aortic thrombosis) leading to mesenteric ischemia can also result in severe intestinal necrosis and require intestinal resection (Zamboni et al., 2021).

Mild intestinal ischemia can lead to reduced diet and malabsorption. Severe intestinal ischemia or necrosis leads to the dissemination of gut bacteria, endotoxins, and microbial metabolites into the blood (Figure 2 bottom), aggravating hyperinflammation and the hypercoagulability state. Such patients need emergency excision of the necrotic bowel, which significantly increases mortality.FIGURE 2

Figure 2. Intestinal thrombosis leads to intestinal mucosal necrosis and dissemination of gut bacteria, endotoxins, and microbial metabolites in blood. (Top) Mesenteric vascular endotheliitis (initiated by viremia and accelerated by cytokines), hyperactivated platelets and high levels of phosphatidylserine (PS) promote a high rate of mesenteric thrombus in COVID-19 patients (mesenteric vein is shown in Supplementary Figure 1). (Bottom) Intestinal microthrombi and hypoxemia rapidly lead to intestinal mucosal ischemia and necrosis. The damaged gut–blood barrier leads to dissemination of gut bacteria, endotoxins, and microbial metabolites in blood.

Long-Term Gastrointestinal Sequelae

Long-term GI complications are common in recovering COVID-19 patients. In one systematic review of post-acute COVID-19 manifestations, diarrhea was among the top 10 most common complaints, with a prevalence of 6%. Other long-term GI symptoms include nausea, vomiting, abdominal pain, loss of appetite, and weight loss (Aiyegbusi et al., 2021Huang et al., 2021). The exact mechanisms of the GI sequelae remain unclear. Recently, persistent endotheliopathy, higher levels of thrombin (Fogarty et al., 2021), and residual SARS-CoV-2 viral antigens in the GI tract (Cheung et al., 2022) were described in convalescent COVID-19 patients. These data suggest that prolonged intestinal infection, persistent endothelial injury (abnormal intestinal–blood barrier), and microthrombi could be causes of the persistent GI symptoms.

The Mechanisms of Intestinal Thrombosis

Damaged Endothelial Cells

Resected bowel samples from COVID-19 patients routinely exhibit thrombi and endotheliitis, indicating the important role of EC injury in mesenteric thrombosis (Bhayana et al., 2020Chiu et al., 2020Kaafarani et al., 2020). SARS-CoV-2 infection (Varga et al., 2020) and elevated inflammatory cytokines (He et al., 2016) damage mesenteric vascular ECs. In response, EC cell margins retract, extending phosphatidylserine (PS) positive filopods and releasing endothelial microparticles (MPs; Figure 3BHe et al., 2016). The PS+ filopods and MPs can be co-stained by Xa and Va and support fibrin formation (Figures 3BD). The exposed PS then activates tissue factor on ECs, triggering the extrinsic coagulation pathway (Versteeg et al., 2013). Next, higher levels of FVIII and vWF released from damaged EC contribute to the hypercoagulable state and platelet aggregation, respectively (Goshua et al., 2020). Thrombomodulin is then released from ECs in its soluble form, which has an attenuated capacity to activate Protein C due to a lack of other cofactors on ECs, such as endothelial protein C receptor (Versteeg et al., 2013). Finally, upregulation of endothelial cell adhesion molecules recruits neutrophils and platelets and further contributes to thrombosis (Tong et al., 2020Li L. et al., 2021).FIGURE 3

Figure 3. Phosphatidylserine exposure on activated/apoptotic cells and microparticles (MPs) promotes fibrin formation. (A) Phosphatidylserine is usually confined to the inner leaflet of the cell membrane. This asymmetry is maintained through ATP-dependent inward transport of PS by flippases and outward transport of non-PS by floppases (left). Upon stimulation, calcium transients will inhibit ATP-dependent transport and stimulate the nonselective lipid transporter scramblase (ATP-independent), resulting in PS exposure (right). (B–D) Human umbilical vein ECs were treated with healthy human plasma and TNF-ɑ (our previous study; He et al., 2016). (B) ECs retracts the cell margins, extends PS positive filopods and releases endothelial-MPs. (C) The PS+ filopods and MPs can be co-stained by Xa and Va. (D) ECs (green) were incubated with MPs-depleted plasma (MDP) in the presence of calcium for 30 min and stained with Alexa Fluro 647-anti-fibrin for 30 min. Considerable fibrin stands among cultured ECs along with filopodia. (E) Confocal images showed PS expression on platelets of patients stained with Alexa 488 lactadherin (our previous study; Ma et al., 2017). MPs from the activated platelet (*) had formed at the margin area located between the distinct outlines. (F) MPs from plasma were co-stained by Xa and Va (or lactadherin and annexin V; our previous study; Gao et al., 2015). (G) MPs that were incubated with recalcified MDP for 30 min and stained with Alexa Fluro 647-anti-fibrin for 30 min. Converted fibrin networks were detected around MPs. The inset bars represent 5 μm in (B–D,G) and 2 μm in (E,F).

Hyperactivated Platelets and Phosphatidylserine Storm

Although COVID-19 patients exhibit mild thrombocytopenia, the remaining platelets are hyperactivated (Manne et al., 2020Taus et al., 2020Zaid et al., 2020). Studies have shown that platelets from COVID-19 patients have increased P-selectin and αIIbβ3 expression. P-selectin on activated platelets interacts with integrin αIIb3 on monocytes to form platelet-monocyte complexes, which induce monocyte tissue factor expression (Hottz et al., 2020). The activated platelets can also induce neutrophils to release neutrophil extracellular traps (NETs; Middleton et al., 2020). Furthermore, platelets from COVID-19 patients aggregate and adhere more efficiently to collagen-coated surfaces under flow conditions (Manne et al., 2020Zaid et al., 2020). Meanwhile, activated platelets release α- and dense-granule contents including FV, FXI, fibrinogen and vWF (Zaid et al., 2020). In addition, activated platelets also produce inflammatory cytokines, fueling cytokine storm (Taus et al., 2020Zaid et al., 2020). Most importantly, activated platelets expose higher levels of PS and release higher numbers of PS+ MPs (Figures 3EGZaid et al., 2020Althaus et al., 2021).

Phosphatidylserine is the most abundant negatively charged phospholipid in mammalian cells and is usually confined to the inner leaflet of the cell membrane (Versteeg et al., 2013). This asymmetry is maintained through ATP-dependent inward transport of PS by flippases and outward transport of other phospholipids by floppases (Figure 3A left). Upon stimulation, transiently increased calcium inhibits ATP-dependent transport and stimulates the nonselective lipid transporter scramblase (ATP-independent), resulting in PS exposure on the outer membrane (Figure 3A right). During this process, microvesicles derived from the budding of cellular membranes will be released. These MPs are typically <1 μm and express PS (Burnier et al., 2009). The exposure of PS on the surface of cells and MPs provides a catalytic surface for factor Xa and thrombin formation in vivo (Versteeg et al., 2013). We have previously demonstrated that PS mediates 90% of Xa and thrombin formation and significantly increases thrombosis in vivo (Shi and Gilbert, 2003).

Cytokines and virus infection can activate blood cells and ECs, resulting in higher levels of PS+ cells and MPs. As COVID-19 progresses, the developing cytokine storm activates more blood cells, leading to PS storm. Platelets are highly sensitive to circulating cytokines, releasing large amounts of cytokines and PS exposed MPs into the plasma (Taus et al., 2020Althaus et al., 2021) and thus are a major contributor to PS storm. Previous studies found an unusual elevation of FVa in severe COVID-19 patients (248 IU/dl, higher than any previous disease; Stefely et al., 2020von Meijenfeldt et al., 2021). The degree of FVa elevation in these patients may be the result of PS storm.

Collectively, SARS-CoV-2 infection is the initiating factor for injury of the intestinal vascular ECs, which is then aggravated by systemic cytokines, leading to endotheliitis. Subsequently, the hyperactivated platelets in circulation rapidly accumulate around the damaged ECs, inducing tissue factor expression, NET release, and activating the intrinsic/extrinsic coagulation pathways. Simultaneously, the high levels of PS expression in circulating cells and MPs further promote thrombin and fibrin formation (Figure 2 top).

Early Antithrombotic Treatment

Vaccines and antithrombotic therapy are effective measures to reduce intestinal damage and fight against the COVID-19 pandemic (Baden et al., 2021Chalmers et al., 2021). Vaccines induce adaptive immunity to clear the virus, reducing intestinal infection and intestinal damage. However, the usefulness of vaccines is limited by incomplete vaccine acceptance and viral mutations (Hacisuleyman et al., 2021Wang et al., 2021). Vaccines are also ineffective for already infected patients. Therefore, more attention should be paid to antithrombotic therapy. Studies had shown that thrombotic events mainly occurred within 7 days of COVID-19 diagnosis (both inpatients and outpatients; Mouhat et al., 2020Ho et al., 2021). Meanwhile, two large randomized controlled trials (RCTs) from the same platform showed that therapeutic anticoagulation reduced mortality in moderate cases but not in severe ones, suggesting that delayed anticoagulant therapy may lead to treatment failure (REMAP-CAP Investigators et al., 2021a,b). More importantly, a recent study reported three asymptomatic COVID-19 patients who developed abdominal (or intestinal) thrombosis leading to intestinal necrosis (Zamboni et al., 2021). All these data suggest that antithrombotic therapy should be initiated once COVID-19 is diagnosed (excluding patients with contraindications). Early prophylactic antithrombotic therapy can reduce the activation of vascular ECs and blood cells, preventing intestinal thrombosis, ensuring sufficient intestinal perfusion, maintaining the normal gut–blood barrier, avoiding malabsorption, malnutrition, and intestinal flora entering the bloodstream. Further, attenuated injury and decreased microthrombi in convalescent patients may lower the risk of long-term GI sequelae. Meanwhile, unobstructed systemic circulation can also accelerate the removal of SARS-CoV-2, inflammatory cytokines and damaged blood cells by the mononuclear phagocyte system.

Anticoagulation

Table 1 summarizes the RCTs of anticoagulant therapy in COVID-19 patients. For outpatients, early anticoagulant therapy reduced hospitalization and supplemental oxygen (Gonzalez-Ochoa). While, delayed treatment had no similar effect (ACTIV-4B and Ananworanich). Thus, oral anticoagulant therapy should be initiated in outpatients once COVID-19 is diagnosed. For non-critically ill patients, therapeutic doses of low molecular weight heparin (LMWH) reduced thrombotic events and mortality, and increased organ support-free days (REMAP-CAP, ACTIV-4a, ATTACC; RAPID; HEP-COVID). However, therapeutic doses of rivaroxaban did not improve clinical outcomes and increased bleeding (ACTION). This is potentially because novel oral anticoagulants do not share the anti-inflammatory and antiviral functions of heparin. Intestinal damage might also result in abnormal absorption of oral anticoagulants. Therefore, therapeutic LMWH should be the first choice for non-critically ill patients. For critically ill patients, RCTs showed that moderate and therapeutic doses were not superior to prophylactic ones. Results from several other studies suggest that the overwhelming thrombosis leads to failure of anticoagulant therapy at therapeutic doses (Leentjens et al., 2021Poor, 2021). Faced with this dilemma, an editorial in N Engl J Med argued that profibrinolytic strategies should be considered (Ten Cate, 2021). More studies are needed to explore optimal antithrombotic therapy in critically ill patients.TABLE 1

Table 1. Randomized clinical trials of anticoagulant therapy in COVID-19 patients.

Inhibition of Platelet Activation

As COVID-19 progresses, cytokine storm activates platelets, which not only participate in primary hemostasis, but also are the major components of PS storm. Autopsy results show a high prevalence of platelet-fibrin-rich microthrombi in lung and extrapulmonary organs, including the gastrointestinal tract (Bradley et al., 2020Polak et al., 2020). Early inhibition of platelet activation can reduce platelet activity and prevent PS storm, thus decreasing thrombosis and mortality. Several observational studies have shown that aspirin decreases mechanical ventilation, ICU admission, and mortality (Chow et al., 2020Santoro et al., 2022). The RCTs testing antiplatelet agents were still preliminary. A recent RCT suggested that aspirin was associated with an increase in survival and reduction in thrombotic events (RECOVERY Collaborative Group, 2022). In addition, anti-inflammatory therapy (e.g., dexamethasone, 6 mg once daily; RECOVERY Collaborative Group et al., 2020) inhibits cytokine storm, as well as platelet activation, reducing mortality. Overall, inhibition of platelet activation is also important to reduce mortality through the prevention of thrombosis and organs damage.

Factors Influencing Antithrombotic Treatment

Thrombotic Risk Factors or Co-morbidities

Studies have shown that obesity, hyperglycemia and diabetes are associated with increased thrombotic events (including intestinal thrombosis), COVID-19 severity, and mortality (Drucker, 2021Stefan et al., 2021). Other thrombotic risk factors include previous venous thromboembolism, active cancer, known thrombophilic condition, recent trauma or surgery, age ≥70 years, respiratory/cardiac/renal failure, and inflammatory bowel disease (Susen et al., 2020). These factors or co-morbidities heighten basal inflammatory levels and endothelial damage, leading to premature cytokine and PS storms, ultimately increasing thrombosis and mortality. Thus, more active antithrombotic therapy strategies should be adopted in these patients. For patients with mild COVID-19 with these factors, the French Working Group on Perioperative Hemostasis and the French Study Group on Thrombosis and Hemostasis recommend higher (intermediate) doses of anticoagulant therapy (Susen et al., 2020). For moderately ill patients, therapeutic doses of anticoagulant therapy should be initiated as soon as possible to prevent excessive microthrombus formation. The need for extended thromboprophylaxis in discharged patients remains controversial. However, a recent RCT showed that rivaroxaban (10 mg/day, 35 days) improved clinical outcomes in discharged COVID-19 patients with higher thrombotic risk factors (Ramacciotti et al., 2022), supporting extended thromboprophylaxis in patients with these risk factors or co-morbidities.

Vaccination

Although more than half the world population has received at least one dose of the vaccines, there are relatively little data of antithrombotic therapy in vaccinated patients. Studies of viral dynamics show that the viral loads of vaccinated patients are as high as that of unvaccinated patients, but drop significantly faster (Brown et al., 2021Klompas, 2021). Thus, vaccinated patients have shorter hospital stays, and are less likely to progress to critical illness and death (Tenforde et al., 2021Thompson et al., 2021). Nevertheless, antithrombotic therapy is still beneficial for the vaccinated patients. Firstly, heparin has anti-inflammatory and antiviral functions and can interfere with the binding of SARS-CoV-2 to ACE2 and shorten the duration of virus infection (Kwon et al., 2020Pereyra et al., 2021). Secondly, antithrombotic therapy protects cells from damage, PS exposure, and microthrombi formation, maintains unobstructed blood circulation, and facilitates virus clearance (by vaccine-induced adaptive immunity). Thirdly, thrombosis remains an important factor in disease progression. Antithrombotic therapy further reduces thrombosis and mortality, especially in vaccinated patients with high risk factors or co-morbidities. Lastly, although vaccines reduce the incidence, a subset of vaccinated patients will still develop long-term sequelae or Long Covid (Ledford, 2021Antonelli et al., 2022). Persistent viral infection and microthrombi are the primary causes (Ledford, 2021Xie et al., 2022), and early antithrombotic therapy is still needed to prevent them.

Conclusion and Future Research

During COVID-19 disease progression, SARS-CoV-2 infiltrates the blood stream from the initial respiratory tract infection, causing viremia, hyperactivated platelets and PS storm. The virus settles into the vascular beds of extrapulmonary organs, ultimately causing infection of intestinal epithelial cell. Damaged ECs, combined with hyperactivated platelets and PS storm, promote intestinal thrombosis, resulting in intestinal ischemia or necrosis. The damaged gut–blood barrier leads to malabsorption, malnutrition and intestinal flora entering the bloodstream, which significantly increase disease severity and mortality. Prolonged intestinal infection, persistent endothelial injury and microthrombi contribute to the long-term GI sequelae after discharge. Early prophylactic antithrombotic therapy can prevent microthrombi, ensuring sufficient intestinal perfusion, maintaining the normal intestinal function, and reducing the risk of long-term GI sequelae. More active antithrombotic therapy should be adopted in patients with other thrombotic risk factors or co-morbidities. Even in vaccinated COVID-19 patients, antithrombotic therapy is also important to decrease (intestinal) thrombosis, mortality and the risk of long-term GI sequelae.

With the Omicron pandemic, patients requiring hospitalization and ICU treatment decline rapidly. However, people are increasingly concerned about Long Covid. In terms of long-term GI sequelae, the detailed mechanisms of prolonged intestinal infection and persistent microthrombi remain unclear. And whether anticoagulant therapy can decrease GI symptoms in patients with long-term GI sequelae deserves further study. Finally, the impact of vaccines on long-term GI sequelae remains unclear in previously infected and breakthrough infected patients.

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Review of Mesenteric Ischemia in COVID-19 Patients

Authors: Amit GuptaOshin SharmaKandhala SrikanthRahul MishraAmoli Tandon & Deepak Rajput  Indian Journal of Surgery (2022) Published: 

Abstract

The new coronavirus (COVID-19) infection, first detected in Wuhan, China in 2019 has become a pandemic that has spread to nearly every country in the world. Through October 11, 2021, more than 23 billion confirmed cases and 4.8 million fatalities were reported globally. The bulk of individuals afflicted in India during the first wave were elderly persons. The second wave, however, resulted in more severe diseases and mortality in even younger age groups due to mutations in the wild virus. Symptoms may range from being asymptomatic to fatal acute respiratory distress syndrome (ARDS). In addition to respiratory symptoms, patients may present with gastrointestinal symptoms such as stomach pain, vomiting, loose stools, or mesenteric vein thrombosis. The frequency of patients presenting with thromboembolic symptoms has recently increased. According to certain studies, the prevalence of venous thromboembolism among hospitalized patients ranges from 9 to 25%. It was also shown that the incidence is significantly greater among critically sick patients, with a prevalence of 21–31%. Although the exact origin of thromboembolism is unknown, it is considered to be produced by several altered pathways that manifest as pulmonary embolism, myocardial infarction, stroke, limb gangrene, and acute mesenteric ischemia. Acute mesenteric ischemia (AMI) is becoming an increasingly prevalent cause of acute surgical abdomen in both intensive care unit (ICU) and emergency room (ER) patients. Mesenteric ischemia should be evaluated in situations with unexplained stomach discomfort. In suspected situations, appropriate imaging techniques and early intervention, either non-surgical or surgical, are necessary to avert mortality. The purpose of this article is to look at the data on acute mesenteric ischemia in people infected with COVID-19.

Introduction

Aside from the respiratory system, the gastrointestinal system is the most common site of SARS-COV-2 infection. This might be because enterocyte and vascular endothelial membranes have large amounts of angiotensin-converting enzyme receptor 2, a membrane integral protein. As a result, the COVID virus induces direct enterocyte invasion as well as indirect endothelial injury-induced thrombosis/intestinal ischemia in the bowel [1]. ICU patients are more prone than non-ICU patients to suffer acute mesenteric ischemia. This might be because, in addition to the direct viral activity on vascular endothelium, ICU patients have extra persistent pro-inflammatory effects. Cases have been observed even among individuals who have recovered from infection [2]. A rising number of cases of acute mesenteric ischemia in COVID-19 patients have been reported in the literature since the outbreak of this pandemic (list of reported cases are summarized in the Table 1). AMI risk was shown to be increased with age, male sex, and comorbidities such as hypertension, obesity, and diabetes mellitus. Because of delayed clinical manifestation, AMI-related mortality is quite significant, with 60–80% [3].Table 1 Summary of the cases reported on mesenteric ischemia in COVID-19 patientsFull size table

Case summary

A 55 years old man with no known comorbidity presented to the emergency department of our institute with severe pain abdomen and multiple episodes of vomiting. He reported the recent recovery from the non-complicated COVID-related illness. He did not report any intake of anticoagulants. On clinical examination, abdomen was unremarkable. X-ray chest, x-ray erect abdomen, and ultrasound abdomen were unremarkable. Mesenteric ischemia was suspected and the patient was subjected to CT angiography abdomen, which revealed thrombus at the origin of the superior mesenteric artery and impending gangrene of the small bowel (Fig. 1). Emergency laparotomy was done and intraoperatively found the gangrenous bowel involving the distal jejunum and almost the entire ileum sparing the terminal ileum (Fig. 2). Resection of the gangrenous small bowel and end jejunostomy was done. Later, he was given ICU care, but unfortunately, the patient succumbed to multi-organ dysfunction syndrome.

figure 1
Fig. 1
figure 2
Fig. 2

Pathophysiology

Although the specific etiology of hypercoagulable state and subsequent mesenteric ischemia in COVID-19 patients is unknown, these thromboembolic events can be related to alterations in all three Virchow triad characteristics (vascular endothelial injury, hypercoagulability, and stasis). A variety of variables complicate the etiology of thrombus development, one of which is vascular endothelial injury. Capillary permeability, hemostasis, and fibrinolysis are all maintained by the vascular endothelium (Fig. 3). Direct invasion causes endothelial cells to be damaged and lysed, resulting in an imbalance between pro and anticoagulant states [4]. Furthermore, vascular endothelial cells displayed morphological changes such as cellular expansion, retraction, and intercellular connection breakage [5]. The elevated levels of pro-inflammatory markers, von Willebrand factor, tissue factor, fibrinogen, and circulating microvesicles in the COVID-19 patients explain their hypercoagulability [6]. Antiphospholipid antibodies are elevated in some situations [7]. Patients who are critically ill, on limited oxygen support, and mechanical breathing are less mobilized, which increases the risk of deep venous thrombosis [3].

figure 3
Fig. 3

These mesenteric vascular thromboses cause acute hypoxia in the intestinal wall, which stimulates the renin-angiotensin system, causing mesenteric vasospasm and an elevated risk of hypoxic injury. SARS-COV binds to ACE 2 receptors in intestinal cells, causing cell lysis [8]. As a result, both hypoxia and direct invasion can trigger intestinal cell death. The loss of this epithelial barrier function in the gut promotes increased contact with enteric bacteria/endotoxins and viral particle penetration into the circulation [5]. The hypoxia continues, resulting in transmural infarction, perforation, and peritonitis. In one example of mesenteric ischemia induced by invasive mucormycosis, the presence of fungal components in the mesenteric microcirculation was documented [2]. See the flow chart summarizing the pathophysiology of mesenteric ischemia in covid-19 infection.

Clinical Presentation

Patients with mesenteric ischemia may exhibit a range of symptoms, from nonspecific complaints to peritonitis-like symptoms. Most of the patients developed symptoms a few days after being discharged successfully with proper symptomatic inpatient care. Although the respiratory symptoms predominate mesenteric ischemia presents with nonspecific abdominal symptoms such as loose stools, abdominal pain, nausea, vomiting, abdominal distension, and bleeding per rectum may occur in addition to the usual clinical presentation with respiratory features [6]. When opposed to arterial thrombosis, venous thrombosis has a delayed onset of symptoms. At first, sudden onset pain in the abdomen may be the sole symptom, and it may develop after 5–14 days. Abdominal clinical examination is nonyielding in the majority of cases. Abdominal signs would not develop unless the bowel gangrene or bowel perforation with peritonitis occurs [9].

Investigations

Blood investigations

Despite extensive study on the subject of acute mesenteric ischemia, the associated biomarkers were shown to be neither sensitive nor selective [10]. Elevated lactic acid levels and fibrin degradation products like D-dimer have low specificity and remain elevated in severe COVID-19 without AMI. However, biomarkers associated with hypercoagulable conditions aid in the initiation of preventive treatment and, to a lesser extent, in the management of COVID-related thrombotic events. Increased biomarkers of inflammation and infection include leukopenia (due to corticosteroid usage) and other signs such as C-reactive protein, procalcitonin, and IL-6. D-dimer, ferritin, prothrombin time, and lactate dehydrogenase are additional significant markers. The severity of increased lactate dehydrogenase and ferritin levels is associated with high mortality[8].

Radiological imaging

In the emergency room, an X-ray of the abdomen and an ultrasound are helpful for early examinations. X-ray of the erect abdomen helps in initial assessment in cases presented with features of obstruction or perforation. Ultrasound in the early phase may show SMA occlusion and bowel spasm or ultrasound findings in the early stages of acute mesenteric ischemia may appear normal [11]. In the intermediate phase, USG is not useful because of the presence of a large amount of gas-filled intestinal loops. In the late phase, USG may reveal fluid-filled lumen, bowel wall thinning, evidence of extra-luminal fluid, decreased or absent peristalsis. Therefore, USG may be helpful in the diagnosis of advanced bowel obstruction, gangrene, and perforation with peritoneal collection [12]. Ultrasonography revealed some other important features with distended and sludge-filled gall bladder with bile stasis. Portal venous gas also can be detected on ultrasonography which can be better characterized with the help of computed tomography [13].

Computed tomography

The gold standard investigation is CT angiography. CT observations commonly encountered in acute mesenteric ischemia secondary to COVID-19 includes thrombus in the aorta/SMA/portal circulation, augmentation of the bowel wall, thickness of the bowel wall with distention(> 3 cm), edema, and stranding of the mesentery, pneumatosis intestinalis or portal venous gas suggesting bowel wall ischemia, and non-enhancing thick bowel wall seen in bowel infarction, bowel perforation secondary to bowel infarction may present discontinuity of bowel wall with localized air collection. One should remember that pneumatosis intestinalis may also occur due to mechanical ventilation. Pneumoperitoneum occurs when there is severe intestinal necrosis and perforation. There were additional reports of nonspecific features such as a dilated gut with a fluid-filled lumen, distended gallbladder with bile stasis, features of solid organ ischemia, and pancreatitis [14]. MRI, despite its accessibility, has drawbacks such as a longer acquisition time and lower resolution than CT angiography [12].

Management

A summary of cases of acute mesenteric ischemia has been tabulated (Table 1). Management of acute mesenteric ischemia in COVID-19 includes the following:

  • Supportive measures: Crystalloid rehydration and empirical antibacterial treatment should begin before angiography or any surgical resection. Comorbidity management, hemodynamic support in unstable patients, and electrolyte balance correction are all critical components of patient care [10].
  • Anticoagulation: There is insufficient data in 19 patients to warrant thromboprophylaxis. According to the Tang et al. study, low-dose heparin prophylaxis decreased thrombotic events and mortality in those with D-dimer levels over 3 mg/ml. Despite the increased risk of bleeding, mesenteric ischemia should be treated with intraoperative and postoperative anticoagulation [15].
  • Revascularisation: Revascularization with catheter-directed thrombolysis and thrombectomy by percutaneous/surgical intervention can be explored in instances where there is no indication of significant intestinal ischemia. Catheter-directed thrombolysis with unfractionated heparin and recombinant tissue plasminogen activators can accomplish this. Because of the increased risk of re-thrombosis, vascular clearance is not indicated in instances of superior mesenteric vein thrombus [15].
  • Resection of the gangrenous bowel: Depending on clinical suspicion, a CT angiography examination of mesenteric vasculature and bowel health can be performed, and an emergency exploration call should be placed. Intraoperatively, if the patient is normotensive, has no sepsis or peritonitis, and the remaining bowel viability is unquestionable, the gangrenous bowel is to be removed, and the remaining bowel can be considered for re-anastomosis. In unfavorable circumstances, a stoma should be created following gangrenous bowel resection [11]. The margin dissection in venous thrombosis should be broader than in arterial thrombosis. To assure the bowel’s survivability, abdominal closure should be temporary, and a relook laparotomy should be done 48 h later. Histopathological examination of the resected intestine may indicate patchy or widespread necrotic changes from mucosa to transmural thickness. In the submucosal vasculature, fibrin-containing microthrombi with perivascular neutrophilic infiltration is observed.
  • Management of short bowel syndrome: The therapy varies depending on the length of colon left after excision of infarcted bowel caused by mesenteric ischemia.
  • Medical- In severe diarrhea, fluid and electrolyte loss must be replaced. TPN for feeding and histamine-2 receptor antagonists or PPIs for stomach acid secretion reduction. Loperamide and diphenoxylate are anti-motility medicines that delay small intestine transit whereas Octreotide reduces the volume of gastrointestinal secretions.
  • Non-transplant surgical therapy- Done to improve the absorption capacity of the remaining intestine by restoring intestinal continuity. Increased nutrient and fluid absorption is the goal. Segmental reversal of the small bowel, fabrication of small intestinal valves, and electrical pacing of the small bowel are all procedures used to delay intestinal transit. Longitudinal intestinal lengthening and tailoring technique (LILT) and serial transverse arthroplasty process are two intestinal lengthening procedures (STEP).
  • Intestinal transplantation- Life-threatening problems such as liver failure, thrombosis of major central veins, frequent episodes of severe dehydration, and catheter-related sepsis are reasons for intestinal transplantation [16].

Prognosis

Acute mesenteric ischemia has a poor prognosis, and life is reliant on prompt diagnosis and treatment. If detected within 24 h, the likelihood of survival is 50%, but it declines to 30% beyond that [17].In operated cases, COVID infection acts as an independent risk factor and is responsible for higher mortality [18].

Conclusion

SARS-COV-2 infection even though initially thought to be respiratory infection; later cases detected presenting with multisystem involvement. The presentation may vary from asymptomatic or mildly symptomatic to severe respiratory distress syndrome or thromboembolic phenomenon requiring ICU care. The exact mechanism of thromboembolism is not established. However, the increasing number of acute mesenteric ischemia is quite alarming. The treating physician should be overcautious in patients presenting with abdominal symptoms either currently affected or recovered from COVID-related illness. In high-risk patients, early start of prophylactic anticoagulation may be beneficial. Earlier intervention is known acute mesenteric ischemia cases with operative or minimally invasive procedures may give higher survival benefits. It mandates more research to determine the causes of thromboembolism, as well as preventive and therapeutic anticoagulation in these individuals.

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Elevated clotting factor V levels linked to worse outcomes in severe COVID-19 infections

Authors: Jonathan A. Stefely,Bianca B. Christensen,Tasos Gogakos,Jensyn K. Cone Sullivan … See all authors First published: 24 August 2020 https://doi.org/10.1002/ajh.25979

Abstract

Coagulopathy causes morbidity and mortality in patients with coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Yet, the mechanisms are unclear and biomarkers are limited. Early in the pandemic, we observed markedly elevated factor V activity in a patient with COVID-19, which led us to measure factor V, VIII, and X activity in a cohort of 102 consecutive inpatients with COVID-19. Contemporaneous SARS-CoV-2-negative controls (n = 17) and historical pre-pandemic controls (n = 260-478) were also analyzed. This cohort represents severe COVID-19 with high rates of ventilator use (92%), line clots (47%), deep vein thrombosis or pulmonary embolism (DVT/PE) (23%), and mortality (22%). Factor V activity was significantly elevated in COVID-19 (median 150 IU/dL, range 34-248 IU/dL) compared to contemporaneous controls (median 105 IU/dL, range 22-161 IU/dL) (P < .001)—the strongest association with COVID-19 of any parameter studied, including factor VIII, fibrinogen, and D-dimer. Patients with COVID-19 and factor V activity >150 IU/dL exhibited significantly higher rates of DVT/PE (16/49, 33%) compared to those with factor V activity ≤150 IU/dL (7/53, 13%) (P = .03). Within this severe COVID-19 cohort, factor V activity associated with SARS-CoV-2 load in a sex-dependent manner. Subsequent decreases in factor V were linked to progression toward DIC and mortality. Together, these data reveal marked perturbations of factor V activity in severe COVID-19, provide links to SARS-CoV-2 disease biology and clinical outcomes, and nominate a candidate biomarker to investigate for guiding anticoagulation therapy in COVID-19.

1 INTRODUCTION

Typically, COVID-19, caused by SARS-CoV-2, presents as a respiratory illness, but coagulopathy can cause morbidity and mortality.17 Line clots, arterial clots, pulmonary thrombosis with microangiopathy, pedal acro-ischemia (“COVID-toes”), bleeding, and venous thromboembolism (VTE)—including deep venous thrombosis (DVT) and pulmonary embolism (PE)—have been associated with COVID-19, especially in severe cases.813 However, the underlying mechanisms remain unclear. Hypothesized mechanisms for thrombosis invoke inflammation, endothelial dysregulation, patient immobilization, antiphospholipid antibodies, and coagulation factor VIII dysregulation.1420 However, direct links between the SARS-CoV-2 virus and coagulopathy remain unmapped. Common laboratory findings include elevations of D-dimer and the acute phase reactants fibrinogen and factor VIII,2128 but additional and more specific biomarkers for guiding prognosis and anticoagulation therapy would be valuable.

Near the beginning of the COVID-19 pandemic in Massachusetts, USA in March of 2020, we obtained an early specimen from a patient with severe COVID-19 on a ventilator. Coagulation laboratory testing revealed an unexpected and unusual elevation of factor V activity at 248 IU/dL (reference range 60-150 IU/dL), and 4 days later this patient developed a saddle PE. This was the highest factor V activity level ever observed in our high-volume coagulation laboratory. Since initiating daily interpretation for every patient tested by our high-volume coagulation laboratory starting in 1994, we had never seen factor V activity >200 IU/dL before, and factor V elevations above 150 IU/dL (above the reference range) were uncommon prior to the pandemic. In the coagulation cascade, activated factor V interacts with activated factor X to form the prothrombinase complex, which catalyzes formation of thrombin and leads to fibrin clot formation. Dysregulation of factor V due to factor V Leiden is a well-known cause of a prothrombotic state.29 Concurrent elevations of factor V activity and factor VIII activity have also been linked to increased VTE risk in one study.30 Thus, we hypothesized that venous thromboembolism and possibly other complications of severe COVID-19 are associated with perturbations of factor V activity.

2 METHODS

2.1 Study population and design

2.1.1 COVID-19 cases

The primary patient specimens in this prospective cohort study were collected over approximately 1 month at the beginning of the COVID-19 pandemic in Massachusetts, USA (March 23, 2020 to April 27, 2020) under an institutional review board-approved study protocol. All authors had access to and analyzed the primary data set, which is also included here as a resource (Table S1). The study site was the Massachusetts General Hospital (MGH), an approximately 1000-bed academic medical center and one of the primary regional referral centers for patients with severe COVID-19. Both SARS-CoV-2 polymerase chain reaction (PCR) positive (“COVID-19”) cases and SARS-CoV-2 PCR negative (“contemporaneous control”) cases were collected from the population of patients with specimens submitted to the MGH Special Coagulation Laboratory. During most of the study period, the inpatient hematology team sent special coagulation testing specimens to our laboratory from all patients in the intensive care units with COVID-19 because of reports of coagulopathy associated with COVID-19. The resultant cohort of 102 inpatients with COVID-19 is comprised of all 102 SARS-CoV-2 positive patient specimens submitted to our coagulation laboratory during the study period without any exclusion criteria. We did not specify additional inclusion criteria other than a positive SARS-CoV-2 test. We measured a panel of coagulation parameters in the earliest available specimen from each of these 102 inpatients with COVID-19.

2.1.2 Contemporaneous control cases

Our study period during the initial peak of the COVID-19 pandemic limited access to contemporaneous specimens from confirmed SARS-CoV-2 negative (“contemporaneous control”) patients submitted to our coagulation laboratory because hospital policies temporarily discontinued elective procedures and outpatient visits for patients without COVID-19. Nevertheless, we were able to obtain a group of specimens from SARS-CoV-2 negative controls (n = 17). We included all submitted specimens from SARS-CoV-2 negative patients on ventilators during the study period (n = 7), which was done by design to include patients with similar illness severity compared to our COVID-19 patients.

2.1.3 Historical control cases

For factors V, X, and VIII, D-dimer, and fibrinogen we also retrospectively obtained historical values from patients with specimens submitted to our laboratory prior to the COVID-19 pandemic. Factor V activity values were obtained from all patient specimens during the 4 years prior to the COVID-19 pandemic (April 2016 – February 2020) (n = 446), as well as all factor VIII activities from March 2019 – February 2020 (n = 478), all factor X activities from May 2016 – February 2020 (n = 346), and all fibrinogen (n = 260) and D-dimer (n = 373) measurements from days 1-14 of January 2020.

2.2 Determination of clinical variables

Patients with COVID-19 and contemporaneous controls were followed forward from the time of their first coagulation laboratory specimen to a median of 78 days (range 64-99 days) to determine clinical outcomes such as the development of DVT/PE. Clinical variables were determined by review of electronic medical records and reviewers were blinded to the results of the research coagulation factor assays. For COVID-19 cases, the date of symptom onset was determined by manual chart review, as documented in the admission note or the first note of the infectious disease consult. When discrepant dates were reported, the date reported in the note closest to admission was chosen. Ventilator use, extracorporeal membrane oxygenation (ECMO) use, and anticoagulation use at the time of the coagulation specimen collection were recorded. Line clots any time during the admission were recorded. DVT/PE and arterial clots were recorded if they occurred any time during the admission or if they were part of the reason for admission (the latter only occurred in SARS-CoV-2 negative patients, some of which were admitted for DVT/PE or stroke). Death was recorded. Discharge was noted if the patient was discharged to home or to a rehabilitation facility.

2.3 Determination of laboratory variables

Factor V, VIII, and X activities and activated partial thromboplastin time (aPTT) waveforms were measured in the same leftover clinical specimens using validated clinical laboratory assays (details below). The remaining parameters in the study were determined by review of existing clinical data. Note, SARS-CoV-2 real-time PCR (RT-PCR) cycle threshold (Ct) values for the diagnostic specimen were obtained from the instrument runs on either a Roche Cobas 6800 or a Cepheid GeneXpert Infinity System. If the Ct values for the diagnostic specimen were not available, the Ct values for the specimen closest to onset of symptoms were recorded. Prothrombin time (PT), aPTT, heparinase aPTT (all by Stago, Asnieres, France), and the activities of factors II, VII, IX, XI, and XII were recorded only if determined on a specimen collected within 6 hours of the study specimen. Both D-dimer (bioMerieux, Marcy-l’Étoile France) and fibrinogen (Stago) values were recorded at the closest time point to the study specimen and were only included if they were measured within 2 days of the study specimen. The following results were recorded at the closest time to the study specimen during the admission: PTT-LA, STACLOT-LA, protein S and antithrombin activity (all by Stago), platelet count, anticardiolipin and beta-2 glycoprotein I (INOVA, San Diego CA), chromogenic protein C activity and activated protein C resistance/factor V Leiden (APC V, Chromogenix, West Chester, OH). The International Society on Thrombosis and Haemostasis (ISTH) DIC scores were determined according to published guidelines.31

2.4 Coagulation factor assay methods

Factor assays were one-stage, PT-based for factors II, V, VII and X, and aPTT-based for factors VIII, IX, XI, and XII, using an ACL TOP 750 analyzer, Hemosil calibrator, Synthasil or Recombiplastin, all from Instrumentation Laboratory (Bedford MA, USA), and factor-deficient plasma from Precision Biologic (Dartmouth, NS, Canada). Three dilutions (1:10, 1:20, and 1:40) were automatically performed for each factor assay.

2.5 APTT waveform analyses

The ACL TOP analyzer automatically generates an aPTT waveform every time an aPTT is performed. Since the ACL TOP does not provide a quantitative measurement of the initial slope, waveforms were manually reviewed to determine if the initial slope was flat (normal) or sloped (abnormal and suggestive of DIC).3233 These determinations were made while blinded to all aspects of the study. The ACL TOP also provides a quantitative measurement of the aPTT waveform’s first derivative peak and second derivative peak and trough.3435

2.6 Statistical methods

For quantitative variables, P values were determined with a two-sided, heteroscedastic Student t test for normally-distributed data, and Mann-Whitney U-test for non-parametric data. Fisher’s exact test was used for categorical variables.

3 RESULTS

3.1 A cohort of patients with severe COVID-19

To begin testing the hypothesis that factor V activity elevation is associated with COVID-19, we measured a panel of coagulation parameters in the earliest available specimen from the first 102 SARS-CoV-2 positive patient specimens submitted to our coagulation laboratory without any exclusion criteria, 17 contemporaneous controls, and 260 to 478 historical controls per test prior to the COVID-19 pandemic.

This cohort of patients with COVID-19 was almost entirely comprised of severe cases based on the observed rate of ventilator use (92%) and ECMO use (7%) at the time of the analyzed coagulation specimen (Table 1). Our prospective follow-up revealed development of line clots (arterial or venous) in 47% (48/102) of the COVID-19 cases, suggesting widespread coagulopathy (Table 2). Furthermore, DVT and/or PE occurred in a striking 23/102 (23%) of these patients with COVID-19. Additionally, 22/102 (22%) of these patients with COVID-19 died before the end of the study period. The primary data set for this cohort, including clinical features and laboratory data, are provided as a resource (Table S1).TABLE 1. COVID-19 cohort characteristics

Patients with COVID-19 (n = 102)Contemporaneous controls (n = 17)
Age (years) median (range)61 (27-87)57 (15-85)P > .05
Male sex − no. (%)68 (67)9 (53)P > .05
Ventilator use − no. (%)94 (92)7 (41)P < .001
ECMO use − no. (%)7 (7)4 (24)P > .05
Anticoagulation at the time of the coagulation lab specimen
Prophylactic SQ heparin or enoxaparin − no. (%)59 (58)2 (12)P > .05
Therapeutic heparin or enoxaparin − no. (%)26 (25)4 (24)P > .05
Other dose of heparin or enoxaparin − no. (%)6 (6)0 (0)P < .001

TABLE 2. Clinical outcomes and features

Patients with COVID-19 (n = 102)Contemporaneous controls (n = 17)
Line clot − no. (%)48 (47)3 (18)P < .05
VTE (DVT or PE) − no. (%)23 (23)7 (41)P > .05
Arterial clot − no. (%)9 (9)3 (18)P > .05
Discharge − no. (%)75 (74)12 (71)P > .05
Death − no. (%)22 (22)5 (29)P > .05
  • a Arterial clots included ischemic strokes and mesenteric ischemia.

3.2 Factor V is elevated in patients with severe COVID-19

Using a validated clinical laboratory assay, we found factor V activity to be markedly elevated in many patients in this severe COVID-19 cohort (median 150 IU/dL, n = 102) compared to the expected reference median value of 100 IU/dL activity (Figures 1A,B). Forty-nine of these cases (48%) fell above the reference range of 60-150 IU/dL. The degree of factor V elevation seen in these COVID-19 cases was notably higher than those seen previously at our hospital before COVID-19 (Figure 1A). Compared to all patient specimens tested in our laboratory during the 4 years prior to the COVID-19 pandemic (April 2016 – February 2020) (n = 446), factor V activity was significantly higher in our cohort of patients with severe COVID-19 (COVID-19 median 150 IU/dL, historical control median 81 IU/dL, P < .001) (Figure 1A). Among COVID-19 patients, 16/102 (16%) had factor V > 200 IU/dL, which was not seen in any of the contemporaneous or historical controls, and which has never been observed at MGH before (extending back to 1994 when daily review of all coagulation results began).

Details are in the caption following the image
FIGURE 1Open in figure viewerPowerPointFactor V activity is markedly elevated in patients with severe COVID-19. A, Box plot indicating factor V activity in a cohort of severe COVID-19 cases compared to contemporaneous SARS-CoV-2 negative controls and historical controls prior to the COVID-19 pandemic. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; notches represent the 95% confidence interval for each median; data points are plotted as open circles. n = 446, 102, 17 sample points (left to right in figure). P values, two-sided, heteroscedastic Student t test. B, Histogram of factor V activity values in the COVID-19 cohort (n = 102), contemporaneous controls (n = 17), and historical controls (n = 446). C, Scatter plot of the activities of factor V and factor VIII in a cohort of patients with severe COVID-19. The reference ranges are indicated by gray-blue (lower limit) and red (upper limit) lines. D, Table of cases with elevations of factor V or factor VIII activity and the rate of DVT/PE in these groups. E, Matrix of correlations (Spearman’s rho) for the indicated coagulation parameters. Asterisks indicate significant correlations with a Bonferroni-corrected P value < .05. F, Example of a normal aPTT waveform and the first and second derivatives of this waveform. The solid black line tracks light absorbance over time during the aPTT. Initially, the line is flat. The abrupt rise in the black line is when clot formation occurs, and the time at which it occurs is the aPTT result in seconds. When the clot occurs, the sample changes from a liquid (plasma) to a solid (clot), which absorbs more light. After clot formation, the sample undergoes no further changes, therefore the light absorbance remains unchanged and the line is flat again. The waveform and its first and second derivatives are automatically calculated by the analyzer. G, Comparison of a normal aPTT waveform and an abnormal aPTT waveform in COVID-19 patients from the current study. The portion within the rectangle is expanded in panel H. H, Expanded view of the initial portion of the aPTT waveforms in panel G, showing the abnormal slope. When the initial slope of the line rises upward instead of remaining flat before clot formation, this indicates an abnormal waveform that is suggestive of DIC [Color figure can be viewed at wileyonlinelibrary.com]

Our factor V assay is regularly validated for consistency across time. However, to alleviate concerns for a temporal drift in assay performance, we also measured factor V activity in contemporaneous SARS-CoV-2 negative control cases (median 105 IU/dL, n = 17), which were found to be overall similar to the historical controls (P > .1). Furthermore, factor V activity was significantly elevated in our cohort of patients with severe COVID-19 (median 150 IU/dL) compared to these contemporaneous controls (P <,001) (Figure 1A).

A sub-group analysis also demonstrated that factor V was significantly elevated in the COVID-19 cases (median 150 IU/dL) compared to both the contemporaneous control cases on ventilators (n = 7, median 54 IU/dL, P < .05) and the non-ventilated contemporaneous control cases (n = 10, median 107 IU/dL, P < .001). These findings suggest that the elevation of factor V in severe COVID-19 cannot be simply explained by a general state of severe illness or by ventilator use. Together with the rarity of factor V elevations before the COVID-19 pandemic, these findings suggest a more specific relationship between COVID-19 and factor V elevation.

3.3 Factor V elevation in severe COVID-19 is associated with DVT/PE

We examined DVT/PE events in this cohort to begin testing the hypothesis that elevated factor V activity is a risk factor for DVT/PE in severe COVID-19. Patients with COVID-19 and factor V activity above the upper limit of the reference range (>150 IU/dL) exhibited significantly higher rates of DVT/PE (16/49, 33%) compared to those with factor V activity less than or equal to 150 IU/dL (7/53, 13%) (P = .03). Moreover, among patients with COVID-19, factor V trends toward higher activities in patients who went on to develop DVT/PE (median 165 IU/dL, n = 23) compared to those that did not develop DVT/PE (median 145 IU/dL, n = 79) (P = .05). Together, these findings nominate factor V as a candidate biomarker for future clinical trials investigating VTE and anticoagulation therapies in patients with COVID-19.

The VTE rates were lower in patients with COVID-19 treated with anticoagulation (19/91, 21%) compared to those not treated with anticoagulation (4/11, 36%) at the time of the factor V activity specimen, but this difference was not statistically significant in this cohort (P = .3). Similarly, when restricting the analysis to COVID-19 cases with elevated factor V activity (>150 IU/dL), VTE rates were lower in patients treated with anticoagulation (13/44, 30%) compared to those not treated with anticoagulation (3/5, 60%), but this difference was not significant in this cohort (P = .3). Nonetheless, these findings provide a foundation for larger prospective studies of anticoagulation in cases of COVID-19, especially in cases with elevated factor V activity.

3.4 Factor V activity relationships in COVID-19

A study prior to the COVID-19 pandemic suggested that concurrent elevations of both factor V and its homolog factor VIII can increase risk for VTE in general,30 and factor VIII has been shown to be elevated in COVID-19. Thus, we also measured factor VIII activity in our cohort of patients with COVID-19 and the contemporaneous controls. Factor VIII activity was elevated in the COVID-19 cases (median 298 IU/dL, n = 100) compared to the reference range (50-200 IU/dL), the contemporaneous controls (median 222 IU/dL, n = 17, P < .01), and the historical controls (median 125 IU/dL, n = 478, P < .001) (Figure S1). The activities of factors V and VIII were not significantly correlated in our cohort (Spearman’s rho = 0.16; P > .05), suggesting distinct regulation (Figure 1C). Yet, 43/100 (43%) of the COVID-19 cases showed elevations of both factor V (>150 IU/dL) and factor VIII (>200 IU/dL) above their reference ranges. Thus, some patients with severe COVID-19 could be at risk for DVT/PE because of elevations of both factor V and factor VIII. In this cohort of COVID-19 cases, DVT/PE occurred in 13/43 (30%) of cases with elevations of both factor V and factor VIII but did not occur in the 11 cases with factor V < 150 IU/dL and factor VIII <200 IU/dL (P = .048) (Figure 1D).

We also measured the activity of factor X because its active form physically interacts with activated factor V and we questioned whether all coagulation factors were elevated. However, factor X activity was not altered in COVID-19 cases (median 106 IU/dL) compared to the reference range (60-150 IU/dL).

Additional coagulation parameters were extracted from existing clinical laboratory data (Table 3). Elevations of fibrinogen and D-dimer have been a point of emphasis in studies of COVID-19 coagulopathy. We also observed an elevation of D-dimer in COVID-19 cases (median 2849 ng/mL, n = 101) compared to the reference range (< 500 ng/mL) and historical controls (median 546, n = 373, P < .001). Likewise, we observed an elevation of fibrinogen in COVID-19 cases (median 763 mg/dL, n = 91) compared to the reference range (150-400 mg/dL), historical controls (median 349, n = 260, P < .001), and contemporaneous controls (median 212 mg/dL, n = 9, P < .001). In patients with COVID-19, we observed a correlation between the acute phase reactants fibrinogen and factor VIII (Figure 1E). Factor V showed a moderate correlation with its functional partner factor X, but factor V was not significantly correlated with the acute phase reactants fibrinogen and factor VIII (P > .05) (Figure 1E). Notably, among the coagulation parameters analyzed (Table 3, Figures 1 and S1), the elevation of factor V in these COVID-19 cases was the most significant difference compared to the contemporaneous controls and distinguished itself as the most striking difference compared to our laboratory’s historical results prior to the COVID-19 pandemic.TABLE 3. Coagulation parameters

Reference rangePatients with COVID-19(n)Contemporaneous controls(n)P value
Primary prospective study test results
Factor V activity (IU/dL) median60–15015010210517P < .001
Factor VIII activity (IU/dL) median50-20029810022217P < .01
Factor X activity (IU/dL) median60–1501061027817P < .01
Secondary retrospective study test results (obtained from existing clinical data when available)
D-dimer (ng/mL) median< 5002849101242010P > .05
Fibrinogen (mg/dL) median150–400763912129P < .001
PT (seconds) median11.5-14.515.19714.117P > .05
aPTT (seconds) median22-3638.110131.917P > .05
Abnormal aPTT waveform slope − no. (%)Normal14 (15)945 (33)15P > .05
aPTT waveform first derivative (TU/sec) median150-2914619425715P < .001
aPTT waveform second derivative peak (TU/seĉ2) median488-102614859499315P < .001
aPTT waveform second derivative trough medianNA5859443015P < .05
Platelet count (K/μL) median150–40027510116916P < .01
ISTH DIC score median< 528646P < .05
Antithrombin activity (IU/dL) median80-13079797810P > .05
Protein S activity (IU/dL) median70-15050.51891.56P < .05
Protein C activity (IU/dL) median70–1508019118.56P > .05
Lupus anticoagulant − no. (%)Negative25 (57)442 (15)13P < .05
Anticardiolipin antibody − no. (%)Negative21 (54)391 (9)11P < .05
Beta-2-glycoprotein antibody − no. (%)Negative3 (10)290 (0)5P > .05
Activated protein C resistance (factor V Leiden screen) − no. (%)Negative0 (0)90 (0)6NA
Factor II activity (IU/dL) median60–150955NA0NA
Factor VII activity (IU/dL) median60-150525NA0NA
Factor IX activity (IU/dL) median60-160135161261NA
Factor XI activity (IU/dL) median60–1609816571NA
Factor XII activity (IU/dL) median60–160518NA0NA

Nine COVID-19 patients were tested for activated protein C resistance (factor V Leiden), and all were normal (Table 3). As some of these patients had factor V activity above 200 IU/dL, it appears that factor V Leiden is not involved in the unusual factor V elevation.

3.5 COVID-19 progression toward DIC and death is associated with lower FV

Two patients with severe COVID-19 in our cohort had a second factor V activity measured later during their hospital course, in each case after worsening of clinical status as measured by increased ventilation requirements or increased vasopressor requirements. In one case, the initial factor V activity was 248 IU/dL, and 5 days later after severe clinical decompensation it dropped to 28 IU/dL. In a second case, the initial factor V activity was 206 IU/dL and after slight clinical worsening it decreased to 171 IU/dL. Based on these cases, we hypothesized that while patients with severe COVID-19 might initially present with markedly elevated factor V activity, a subsequent decline in factor V activity could be associated with clinical decompensation.

In our severe COVID-19 cohort, cases with factor V activity ≤150 IU/dL had a higher mortality (16/53, 30%) than those with factor V activity >150 IU/dL (6/49, 12%, P < .05). To investigate if this relationship with mortality could be due to consumption of factor V at the beginning stages of DIC, the aPTT waveform slope was assessed, which if abnormal, is associated with DIC or the prediction of DIC.3233 We examined the aPTT waveform shape and the peaks of the first and second derivatives of the aPTT waveform (Figure 1F). A sub-set of patients with severe COVID-19 showed an abnormal slope at the beginning of the aPTT waveform (Figures 1G,H), suggesting progression toward DIC. Factor V was lower in COVID-19 patients with an abnormal waveform slope, compared to COVID-19 patients with a normal slope (median 116 IU/dL vs 158 IU/dL, P = .005). Since these tests were performed on the earliest available specimen, ISTH DIC scores were calculated for all COVID-19 patients and contemporaneous controls, and none of them had scores indicating acute overt DIC at the time that the earliest specimen was collected. Thus, an abnormal slope in the aPTT waveform and/or factor V below 150 IU/dL may be early markers of a DIC-like process that appear before routine laboratory tests can diagnose DIC (D-dimer, fibrinogen, platelet count, and PT).

3.6 Factor V levels in severe COVID-19 are linked to SARS-CoV-2 load in a sex-dependent manner

Note, SARS-CoV-2 differentially affects patients based on their sex, with men often presenting with more severe COVID-19.636 Coagulation parameters also vary based on sex.37 A review of our historical cases prior to the COVID-19 pandemic showed a small, but significant, difference in factor V activity in males compared to females (median 78 IU/dL and 84 IU/dL, respectively; P < .05). (Figure S2A). Thus, we investigated the possibility of a sex-dependent interaction between SARS-CoV-2 and factor V activity. Interestingly, males show a weak anticorrelation (Spearman’s R ~ −0.3) between SARS-CoV-2 RT-PCR Ct values and factor V activity (Figures S2B,C), suggesting that male COVID-19 patients with higher viral loads (lower Ct values) have higher factor V activity. The opposite trend is seen in women, where there is a weak correlation (Spearman’s R ~ 0.4) between Ct values and factor V activity (Figures S2D,E). These findings suggest a complex sex-dependent biological interaction between SARS-CoV-2 and the coagulation system of the infected patient. While many questions about possible biological mechanisms remain to be answered, these findings, together with the unique nature of the marked factor V activity elevations in severe COVID-19, raise the possibility of a specific link between SARS-CoV-2 disease biology and dysregulation of human coagulation.

4 DISCUSSION

In this COVID-19 cohort, representing severe cases with a high rate of line clots, VTE, and mortality, we observed marked elevation of factor V activity. To our knowledge, this is a novel characteristic of COVID-19. Previous studies linked elevations of D-dimer and the acute phase reactants fibrinogen and factor VIII to severe COVID-19,2124 but these are non-specific findings that appear in many disease states and thus might not on their own explain the coagulopathy of COVID-19.26810 In contrast, since initiating daily interpretation for every patient tested by our high-volume coagulation laboratory starting in 1994, we had not seen factor V activity >200 IU/dL prior to the COVID-19 pandemic, suggesting that factor V elevation could be a relatively specific finding in severe COVID-19. The observed relationships between factor V activity and SARS-CoV-2 viral load also raises the possibility of a specific relationship between factor V and COVID-19.

Recently it was discovered that megakaryocytes are abundant in the lungs, heart, and other organs of patients with COVID-19.38 Since megakaryocytes produce platelets, which normally contain about 20%-25% of the factor V in blood, this might be related to the mechanism for the high factor V in our COVID-19 cohort. Normally, factor V in blood is produced by the liver and then some of the factor V is endocytosed by megakaryocytes.

Dysregulation of factor V due to factor V Leiden is a well-known cause of a prothrombotic state.29 Concurrent elevations of factor V and factor VIII activity have also been linked to increased VTE risk in general in a pre-COVID-19 cohort.30 In the present cohort of severe COVID-19 cases, we observed a statistically significant association between DVT/PE event rates and factor V activity elevations above the reference range. Moreover, we observed a trend toward higher factor V activities in COVID-19 cases complicated by DVT/PE. These findings nominate factor V as a candidate for mechanistic studies of COVID-19 coagulopathy and as a candidate biomarker for VTE risk in COVID-19. Further study is needed to determine if factor V activity can help guide initiation and dosing of anticoagulants in COVID-19.223940 For example, in light of the findings presented here, one could hypothesize that patients with severe COVID-19 who have elevated factor V activity (>150 IU/dL) would benefit more from anticoagulation, such as low-molecular weight heparin doses above typical prophylactic doses, yet this hypothesis remains to be tested and must be balanced with the risk of bleeding in such cases.

In our severe COVID-19 cases, further progression toward a DIC-like state as assessed by aPTT waveform analysis was associated with a decrease in factor V activity, and relatively lower factor V activity was also associated with death. An abnormally sloped waveform is an early predictor of DIC.3233 In patients with COVID-19 and contemporaneous controls, first derivative peak, second derivative peak, and second derivative trough values for each aPTT waveform were also lower in patients with an abnormally sloped waveform (predicting DIC) compared to those with a normal waveform (data not shown). This is consistent with a prior report before the pandemic (not in COVID-19 patients) showing that the first and second derivative peaks are decreased in infectious DIC, but higher in patients with infections without DIC.34 Taken together, the results support that the abnormal slope identified in our study predicts DIC, which consequently may explain the significantly lower factor V and higher mortality seen in our patients with an abnormal waveform slope. These findings suggest that in severe COVID-19 cases, while elevations in factor V are common and are associated with hypercoagulability, normal or low factor V activity may be associated with progression toward DIC and risk of death. As such, measuring factor V activity could potentially be useful in two ways: first for identifying COVID-19 coagulopathy and the risk for DVT/PE, and second, for monitoring progression toward DIC in the most severe cases. Thus, factor V activity assays could have diagnostic and prognostic potential in COVID-19.

We re-measured DIC scores on the day of death for the 22 patients with COVID-19 who died, and their DIC scores had increased on average by one point and all had positive D-dimers, but the scores remained below the ISTH cut-off for acute DIC (data not shown). This could be because fibrinogen and factor V are higher with COVID-19 than with other patients at risk for DIC, therefore making it more difficult for two of the four DIC score components to cross the DIC cut-off (fibrinogen and PT, since the PT is shortened by higher fibrinogen and factor V levels). However, platelet counts also did not reach the DIC cut-off in most cases. As noted, the DIC scores could suggest that the aPTT waveform is detecting a DIC-like state that routine laboratory tests do not detect as easily.

Another reason that it is important for hematologists to know that factor V can be elevated with COVID-19 is that it can cause misdiagnosis when interpreting coagulation factor panels. In our experience, factor V elevation in COVID-19 can cause an erroneous diagnosis of vitamin K deficiency in patients with liver dysfunction or DIC (factors II, VII, and X low with normal or elevated factor V). Usually factor V would be low in liver dysfunction or DIC, and the fact that it is normal or elevated gives the false appearance of a deficiency of only the vitamin K dependent PT factors. Thus our findings are important for clinical interpretation of coagulation panels for patients with COVID-19, and could alter management decisions for some patients with suspected liver dysfunction, DIC, or vitamin K deficiency.

Antiphospholipid antibodies (lupus anticoagulant, anticardiolipin, and beta-2 glycoprotein I antibodies) were detected in a high percentage of COVID-19 patients (Table 3). Repeat testing after 12 weeks would be needed to determine if these are transient due to infection or if they persist and could increase the risk for thrombosis.

A limitation of this study is the lack of mildly symptomatic or asymptomatic COVID-19 cases in our cohort, and the relatively small number of contemporaneous controls. Our ability to collect an equivalent contemporaneous control census was limited due to a markedly decreased non-COVID-19 inpatient census at the height of the pandemic at our hospital. Nevertheless, our contemporaneous control group was as severely ill as the COVID-19 group, as indicated by the similar rates of death, discharge, venous or arterial thrombosis, ECMO, and similar ages and sex ratio. The rate of line clots with COVID-19 was markedly high, and significantly higher than in the contemporaneous controls, which might help answer the question as to whether the risk for thrombosis is higher in COVID-19 than in other similarly ill ICU patients without COVID-19. Strengths of this study include the number of severe COVID-19 cases in our cohort, the depth of our coagulation testing for this cohort, and the large number of historical controls, which provide a comprehensive view of pre-COVID-19 pandemic factor V activities and other coagulation parameters. Our de-identified primary data set is included here as a resource (Table S1).

In summary, factor V activity was significantly higher in severe COVID-19 patients than in contemporaneous controls as well as historical controls, and high factor V activity was associated with thromboembolic complications of COVID-19. In contrast, patients with COVID-19 and a relatively lower factor V activity had a higher mortality and a higher incidence of an abnormally sloped waveform, which is an early predictor of DIC. Thus, our study reveals factor V perturbations as a previously unrecognized feature of severe COVID-19, adds a mechanistic candidate to ongoing investigations of COVID-19 coagulopathy with potential links to SARS-CoV-2 disease biology, and provides a foundation for future studies of COVID-19 coagulopathy diagnosis and biomarkers for guiding anticoagulation therapy in severe COVID-19.

ACKNOWLEDGEMENTS

We thank all members of the MGH Special Coagulation Laboratory for their selfless dedication to patient care during the COVID-19 pandemic and for their support of this research study, in particular: Briana Malley, Barbara Pereira, Stoja Islamovic, Ryan Mize, and Fils-Amie Lucien. We thank Sarah E. Turbett and Melis N. Anahtar for help with viral load data collection and input in analysis of the viral load data.

Nuts and bolts of COVID-19 associated coagulopathy: the essentials for management and treatment

Authors: Patrick J Lindsay, a Rachel Rosovsky, b Edward A Bittner, c and Marvin G. Chang c

ABSTRACT

Introduction

COVID-19-associated coagulopathy (CAC) is a well-recognized hematologic complication among patients with severe COVID-19 disease, where macro- and micro-thrombosis can lead to multiorgan injury and failure. Major societal guidelines that have published on the management of CAC are based on consensus of expert opinion, with the current evidence available. As a result of limited studies, there are many clinical scenarios that are yet to be addressed, with expert opinion varying on a number of important clinical issues regarding CAC management.

Methods

In this review, we utilize current societal guidelines to provide a framework for practitioners in managing their patients with CAC. We have also provided three clinical scenarios that implement important principles of anticoagulation in patients with COVID-19.

Conclusion

Overall, decisions should be made on a case by cases basis and based on the providers understanding of each patient’s medical history, clinical course and perceived risk.

Introduction

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in December 2019 in Wuhan, China. The disease was initially identified as a cluster of pneumonia cases; however, the disease dispersed rapidly and was formally declared a pandemic by the World Health Organization (WHO) in March 2020. As of June 2021, there have been greater than 172 million cases reported worldwide, including more than 3.7 million deaths [1]. The initial manifestations of SARS-CoV-2 vary, and usually occur between one and 14 days from exposure to the virus. Risk factors identified for COVID-19 include male gender, obesity, cardiovascular disease, diabetes mellitus, and older age [2–5]. The most common initial symptoms include fever, cough, fatigue, and loss of smell or taste [6]. During this period, the virus infects the epithelial cells through the angiotensin-converting enzyme 2 receptors and may eventually present as a viral pneumonia.

Although the disease primarily affects the respiratory system, multi-system organ involvement can occur with increasing severity of disease [6–8]. COVID-19-associated coagulopathy (CAC) is a well-recognized hematologic complication among patients with severe COVID-19 disease, where macro- and micro-thrombosis can lead to multiorgan injury and failure [7,9]. This identification has led to significant clinical questions regarding the optimal prevention and management of CAC, which in turn, has led to many ongoing clinical trials. To help guide the management of these patients in the interim, numerous major societies have put forth recommendations regarding diagnosis, monitoring, and treatment of CAC. In this report, we discuss the pathogenesis, prevalence, diagnosis, and treatment of CAC to help providers understand this complicated condition and to apply best practices to the care of their patients.

From a practical perspective, societal guidelines are based on consensus of expert opinion, with the current evidence available. Major societal guidelines that have published on the management of CAC include but are not limited to Centers for Disease Control and Prevention (CDC), International Society on Thrombosis and Hemostasis interim guidance (ISTH-IG), American Society of Hematology (ASH), American College of Chest Physicians (ACCP), Scientific and Standardization Committee of ISTH (SCC-ISTH), Anticoagulation Forum (ACF), and American College of Cardiology (ACC) [9–13]. As a result of limited studies, there are many clinical scenarios that are yet to be addressed, with expert opinion varying on a number of important clinical issues regarding CAC management. In this review, we utilize current societal guidelines to provide a framework for practitioners in managing their patients with CAC. To supplement the manuscript, we have provided three clinical scenarios (supplemental material) which implement important principles of anticoagulation in patients with COVID-19. Overall, decisions should be made on a case by cases basis and based on the providers understanding of each patient’s medical history, clinical course and perceived risk.

Prevalence

There is an increasing body of evidence suggesting patients with COVID-19 demonstrate a higher incidence of thromboembolic disease compared to historical data [14,15], with patients admitted to the ICU being at highest risk [16,17]. The true incidence of thromboembolism in patients admitted to hospital remains controversial with rates as low as 1% in those admitted to the medical ward and up to 69% of patients in the ICU [5,18]. In one study of 107 ICU patients, 91% of whom received VTE prophylaxis and 9% who received therapeutic anticoagulation, the prevalence of PE was 20.4%. In comparison to patients admitted to ICU for other reasons, patients with COVID-19 were 3–4 fold more likely to develop a pulmonary embolism (PE) [19]. However, there is also data to suggest that the prevalence of VTE is similar to that of patients admitted to hospital with similar non COVID illnesses, of similar severity [20,21]. The reported increase in prevalence of VTE remains despite thromboprophylaxis in some studies and not in others [15,22]. In addition to macro-vascular thrombosis, autopsy studies have demonstrated significant microvascular thrombosis in the lungs of patients who have died from COVID-19’s [7,23]. It is hypothesized that these microvascular thrombi cause end organ dysfunction such as renal failure [7,23,24]. Although the prevalence of microvascular thrombosis is yet to be determined, it may be greater than in patients with non COVID respiratory viral illnesses [24].

The majority of data on the prevalence of thromboembolic disease in patients with COVID-19 have been observational studies of VTE. However, there is emerging evidence suggesting an increase in arterial thromboses in COVID-19 patients as well. One study identified five cases of acute ischemic stroke in a two-week period in COVID-19 patients under the age of 50, in comparison to 0.7 large vessel strokes per two-weeks prior to the pandemic [25]. There are also studies reporting an increase in the prevalence of acute limb ischemia in patients hospitalized with COVID-19 compared to the general population [26,27].

The micro- and macro-thrombosis associated with CAC often leads to multisystem complications resulting in increased morbidity and mortality [8]. Despite many policies aimed at curbing viral spread, many countries/regions still see their weekly ICU admissions related to COVID-19 increasing [1]. With the introduction of vaccinations and targeted novel treatments for COVID-19, the prevalence and incidence of CAC and its related complications will hopefully decrease.

Pathogenesis

Although the pathogenesis of CAC has not been fully elucidated, there are multiple contributing factors that include hypercoagulability, endothelial dysfunction and abnormal blood flow (especially in the pulmonary vasculature) [28]. In severe COVID-19 disease, excess proinflammatory cytokines trigger the coagulation system resulting in a hypercoagulable state [29]. In addition to direct damage by viral invasion, this cytokine storm also results in endothelial injury and dysfunction, leading to endothelial cell activation [30]. The dysfunctional endothelial cells produce excess thrombin as well as shutdown fibrinolysis, leading to a prothrombotic state [31]. Furthermore, infection induced inflammatory changes in endothelial cells have been shown to increase coagulation biomarkers, including factor VIII, von Willebrand Factor (vWF), fibrinogen and P-selectin [24,32]. All of these mechanisms create an imbalance in the normal hemostatic system, with a resulting prothrombotic state, manifesting as both macro and micro-vascular thrombosis [30].

In patients with CAC, biomarkers supporting the hypercoagulability pathogenesis have been demonstrated to be elevated in vivo. Specifically, the procoagulant markers Factor VIII, Von Willebrand antigen and Von Willebrand activity have been found to be markedly elevated in patients with COVID-19 who develop thromboembolism [33]. Biochemically, this hypercoagulable state appears to be most significant in those patients with the most severe form of the disease. One study demonstrated that many prothrombotic markers are increased above their upper limit of normal in patients with COVID-19, with the greatest increase in patients admitted to the intensive care unit (ICU) as compared to non-ICU patients [34]. In support of this recognition, descriptive studies using viscoelastic hemostasis assays including thromboelastography (TEG) and rotational thromboelastometry (ROTEM) in patients with COVID-19 are more consistent with a hypercoagulable state rather than acute disseminated intravascular coagulation (DIC). For example, in a study of 24 ICU patients with COVID-19, TEG parameters demonstrated a hypercoagulable state with decreased K (kinetic) time (increased fibrinogen activity), increased alpha angle (increased fibrinogen activity), increased maximum amplitude (Ma) (increased platelet activity), and decreased LY30 (decreased fibrinolysis) [33]. These findings suggest decreased time to clot accumulation, increased strength and stability of the clot, and decreased breakdown of the clot. This study also found that fibrinogen, D-dimer, C-reactive protein, Factor VIII, vWF and protein C were all increased, while platelet count was normal or increased, prothrombin time (PT) and partial thromboplastin time (PTT) were near normal and antithrombin (AT) was marginally decreased. These discoveries are all consistent with a hypercoagulable state rather than DIC where one would expect decreased platelet count and increased PT and PTT. In another study of 21 ICU patients with COVID-19, the TEG MA was significantly higher in those with high thrombotic events (≥2 thrombotic events, defined as an arterial, central venous, or dialysis catheter or filter thromboses) compared to those with low thrombotic events (0–1 thrombotic events) [35]. Moreover, the mean fibrinogen and D-dimer levels were elevated in those with high thrombotic events although there was no significant differences in PT, international normalized ratio (INR), PTT or platelets between the two groups.

D-dimer elevation has also been associated with severity of COVID-19 and may be useful as a prognostic marker [36]. D-dimers are fibrin degradation product which can be measured in the blood after a clot is broken down through fibrinolysis [37]. As a result, elevated D-dimer levels are suggestive of venous thromboembolism (VTE), pulmonary embolism (PE) or DIC; however, it is also elevated in the context of systemic inflammation, which has been demonstrated in numerous clinical settings [20,38]. With COVID-19 stimulating a systemic inflammatory response, it is likely that D-dimers are elevated whether thromboembolic disease is present or not.

Despite the increased inflammatory response associated with COVID-19, patients are less likely to develop a reactive thrombocytosis and often have a mild thrombocytopenia [39]. Platelets in patients with COVID-19 have been found to have increased activity compared to healthy patients, with increased aggregation, thromboxane generation and platelet activation [40]. It is unclear whether these changes in platelet function are associated with an increased risk of thrombosis [39,41]. One study in hospitalized patients with COVID-19 found that a platelet count >450 × 109/L on admission was associated with an increased risk of VTE (adjusted OR of 3.56 [95% CI, 1.27–9.97]) [20]. However, another study of 1476 patients hospitalized with COVID-19 found that the platelet count was inversely associated with risk of in-hospital mortality, although, it is unclear if this increased risk in mortality was a result of consumption in the context of DIC [42]. The appropriate workup of a decreasing platelet count and thrombocytopenia should be pursued in the COVID-19 patient as it would be for non-COVID patients. Given that the majority of patients with COVID-19 receive some form of heparin, a diagnosis of heparin-induced thrombocytopenia (HIT) may be considered, and the subsequent use of Platelet Factor 4 (PF4) and platelet serotonin release assay (SRA) can be used to diagnose this potentially life-threatening condition.

Upon initial screening, many patients with COVID-19 have an elevated PTT in the context of minimal clinical bleeding. This finding raises the question of whether the thrombotic related biomarker, lupus anticoagulant is affected by COVID-19 disease [43,44]. The lupus anticoagulant is an immunoglobulin which binds to phospholipids and proteins associated with the cell membrane. When evaluated in vitro, these immunoglobulins interfere with the phospholipids which induce coagulation, leading to a prolonged PTT. In vivo, however, these antibodies are often prothrombotic [45]. Two studies of patients with COVID-19 and prolonged PTT on initial presentation have demonstrated high rates of lupus anticoagulant positivity [15,43]. However, it has been noted that upon repeat testing, many patients become negative, suggesting that the lupus anticoagulant positivity may be transient and associated with severe viral illness [46]. Overall, more studies are needed to determine if lupus anticoagulant is truly associated with COVID-19 and related to an increase in VTE, and if so, whether the use of anticoagulation should be changed based on the presence of this abnormality [47].

Antithrombin deficiency, a marker of thrombophilia, can be acquired or inherited and may place patients at increased risk of thrombosis. Antithrombin is a natural anticoagulant that inhibits thrombin and factor Xa, thereby helping to prevent thrombosis. There have been case reports of AT deficiency in COVID-19 patients which may place such patients are at higher risk of thrombosis [48]. However, as with the other biochemical testing, more studies are needed to determine if this knowledge is related to adverse outcomes such as thrombosis and if these results should be used to help guide anticoagulation practices.

Manifestations

Since the emergence of COVID-19, the associated increased risk of thrombotic complications such as deep vein thrombosis (DVT), pulmonary embolism (PE) and microvascular thrombosis have been well described [17]. The spectrum of CAC is broad and may present with either or both arterial and venous thromboembolic disease [22]. In general, the majority of the thrombotic events when present are DVT or PE, however catheter associated thrombosis, other venous thrombosis and arterial events, including stroke, acute limb ischemia, bowel ischemia and myocardial infarction, have been reported [14]. Patients also appear to be at risk for microvascular thrombosis. Multiple autopsy studies have demonstrated microvascular thrombosis in the lungs of patients who have died from COVID-19’s, suggesting the multisystem organ failure often seen in COVID-19 may be a result of microvascular thrombosis [23,49].

Bleeding was initially believed to be much less common than thrombosis in patients with COVID-19, however, further data has emerged suggesting the rates of bleeding may be similar to the rates of thrombosis [20]. One autopsy study involving 82 patients found that, 6% died from hemorrhage and that over 80% of patients had some kind of hemorrhagic complication [50]. Another study of 400 hospitalized COVID-19 patients found a radiographically confirmed VTE rate of 4.8% (95% confidence interval [CI], 2.9–7.3), which was identical to the overall bleeding rate of 4.8% (95% CI, 2.9–7.3) [20].

Although clinically significant DIC is uncommon in COVID-19 patients, and if suspected, it is important to identify and aggressively treat the underlying etiology, including any superimposed bacterial infections in addition to managing the coagulopathy [51].

Diagnosis

Currently, there are no diagnostic criteria for CAC. At present, there is no evidence or guideline to support routine screening for VTE, PE or other thrombotic complications in patients with COVID-19. However, the threshold to investigate for DVT or PE should be low given the frequency with which these complications may occur in patients with COVID-19. If thromboembolic disease is suspected, appropriate investigations should depend on the clinical context, acuity of disease and resources available. A position paper from the National Pulmonary Embolism Response Team (PERT) Consortium provides a step wise approach to a suspected PE, which includes ordering a computed tomography pulmonary angiogram (CTPA) if available [52]. If the computed tomography (CT) is not available or the patient is too unstable, a lower limb ultrasound to assess for a proximal DVT, or an echocardiography to assess right heart strain may be pursued. However, it should be noted that neither of these investigations are sensitive. If none of those modalities are available, nor do they rule in a PE/DVT, and there is a high clinical suspicion for a PE, therapeutic anticoagulation should be considered pending no absolute contraindications [52].

In patients with COVID-19, many routine biomarkers of the coagulation cascade have been found to fall outside the normal range, including PTT/PT, platelet count, and fibrinogen [53]. Derangement of these tests may suggest increased disease severity. A D-dimer elevated by three- to fourfold, a prolonged prothrombin time and a platelet count <100 × 109 are all predictors of a poorer prognosis [13,54]. Despite this evidence, most of the major societal guidelines (ACF, ACCP, SCC-ISTH, CDC) have either not recommended nor commented on the routine monitoring of laboratory values to guide management, risk stratification, or triage of patients with COVID-19. For instance, the SCC-ISTH guidelines state that further study is required before using laboratory testing for risk stratification and triage of CAC. The CDC guidelines state that there is a lack of prospective data demonstrating laboratory testing as a way to risk stratify patients, and that there are insufficient data to recommend for or against using laboratory values to guide management. If a patient is bleeding or has a confirmed, or is highly suspected of having a VTE; then, the patient should be treated based on clinical context. Repeat testing of CBC, coagulation studies, fibrinogen, and d-dimer should also be performed based on the clinical setting.

When considering the use of D-dimer assays, a negative D-dimer can be useful in excluding a VTE in patients with COVID-19. However, a positive D-dimer does not necessarily equate to a diagnosis of VTE as this test is not specific and can be elevated in many other pathological and non-pathological processes [55]. Moreover, studies have demonstrated that D-dimer levels tend to be higher in severe COVID-19 cases and may be used as a potential prognostic marker [56]. Despite this connection, none of the major societal guidelines recommend routine monitoring of D-dimer nor using it to guide anticoagulation practices.

Treatment

At least seven major societal guidelines have been published to address prevention and treatment of CAC in the critical care settings, with all authors of these guidelines having prior expertise in the management of VTE [57]. Some of the societies with published guidelines include Centers for Disease Control and Prevention (CDC), International Society on Thrombosis and Hemostasis interim guidance (ISTH-IG), American Society of Hematology (ASH), American College of Chest Physicians (ACCP), Scientific and Standardization Committee of ISTH (SCC-ISTH), Anticoagulation Forum (ACF), and American College of Cardiology (ACC) [9–13]. Currently, there are no separate recommendations for prevention or management of arterial thrombosis in CAC; therefore, this patient population should follow the recommendations of the clinical syndrome in question (e.g., acute myocardial infarction requires dual antiplatelets).

Here, we review the recommendations for prevention, treatment, and monitoring of anticoagulation for CAC in the critical care setting by reviewing the common questions. A summary of the recommendations can be seen in Table 1Table 2 and Table 3:

  1. How should biomarkers be used to guide management?
  2. What are the preferred prophylactic anticoagulation regimens?
  3. When should the intensity of anticoagulation be increased?
  4. What are the preferred therapeutic anticoagulation regimens?
  5. When are thrombolytics recommended?
  6. When should anticoagulation be held?
  7. What is the utility of mechanical thromboprophylaxis?
  8. What is the appropriate method of monitoring anticoagulation?
  9. What is the recommended approach for correction of active bleeding?
  10. Should patients receive post-discharge prophylactic anticoagulation and if so, what regimens are recommended?

Table 1.

Major societal recommendations regarding using biomarkers to guide anticoagulation, choice of prophylactic anticoagulation and when to consider increasing intensity of anticoagulation

 How should biomarkers be used to guide management?What are the preferred prophylactic anticoagulation regimens?When should the intensity of anticoagulation be increased?
CDCInsufficient data to recommend for or against using hematologic and coagulation parameters to guide management decisions.LMWH or UFH (standard dosing). Insufficient data to recommend for or against the increase of anticoagulation intensity outside of a
clinical trial.
Consider when a clinically suspected thromboembolic event is present or highly suspected despite imaging confirmation. Insufficient data to recommend for or against the increase of anticoagulation intensity outside the context of a clinical trial. Mentions patients who have thrombosis of catheters or extracorporeal filters should be treated accordingly to standard institutional protocols for patients without COVID-19.
ISTH-IGNot mentionedLMWH (standard dosing)No specific recommendations
ACFBiomarker thresholds such as D-dimer for guiding anticoagulation management should not be done outside the setting of a clinical trial.Suggests an increased intensity of venous thromboprophylaxis be considered for critically ill patients# (i.e. LMWH 40 mg SC twice daily, LMWH 0.5 mg/kg subcutaneous twice daily, heparin 7500 SC three times daily, or low-intensity heparin infusion) that they state is based largely on expert opinion.Consider when a clinically suspected thromboembolic event is present or highly suspected despite imaging confirmation.
ASHNo particular change to regimen recommended for patients with lupus like inhibitors. TEG and ROTEM should not be used routinely to guide management.LMWH over UFH (standard dosing) to reduce exposure unless risk of bleeding outweighs risk of thrombosis.Consider increasing the intensity of anticoagulation regimen (i.e. from standard to intermediate intensity, from intermediate to therapeutic intensity) or change anticoagulants in patients who have recurrent thrombosis of catheters and extracorporeal circuits (i.e. ECMO, CRRT) on prophylactic anticoagulation regimens.
ACCPNot mentionedLMWH (standard dosing)Patients with PE or proximal DVT.
SCC-ISTHD-dimer levels should not be used solely to guide anticoagulation regimens.LMWH or UFH. Intermediate intensity LMWH can be considered in high risk critically ill patients (50% of responders) and may be considered in non-critically ill hospitalized patients (30% of respondents). Mentions that there are several advantages of LMWH over UFH including once vs twice or more injections and less heparin-induced thrombocytopenia. Regimens may be modified based on extremes of body weight (50% increase in dose if obese), severe thrombocytopenia*, or worsening renal function.Therapeutic anticoagulation should not be considered for
primary prevention until randomized controlled trials are available. Increased intensity of anticoagulation regimen (i.e. from standard or intermediate intensity to therapeutic intensity) can be considered in patients without confirmed VTE or PE but have deteriorating pulmonary status or ARDS.
ACCFurther investigation is required to determine the role of antiphospholipid antibodies in pathophysiology of COVID-19- associated thrombosis. D-dimer > 2 times the upper limit may suggest that patient is at high risk for VTE and consideration of extended prophylaxis (up to 45 days) in patients at low risk of bleeding. Mentions that therapeutic anticoagulation is the key to VTE treatment. Does not make distinction between confirmed or suspected VTE. Hemodynamically stable patients with submassive PE should receive anticoagulation rather than thrombolytics

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Table 2.

Major societal recommendations regarding therapeutic anticoagulation regimens, when thrombolytics should be used and when anticoagulation should be held

 What are the preferred therapeutic anticoagulation regimens?When should anticoagulation be held?When are thrombolytics recommended?
CDCStandard regimens for non-COVID-19 patients.Active hemorrhage or severe thrombocytopenia (Platelet count not defined)Insufficient data to recommend for or against thrombolytic therapy outside the context of a clinical trial. In pregnant patients, thrombolytic therapy should only be used for acute PE with life-threatening hemodynamic instability due to risk for maternal hemorrhage.
ISTH-IGNot mentionedHold when signs of active bleeding or platelet count < 25 x 109/L. Abnormal PT or PTT is not a contraindication to thromboprophylaxis.Not mentioned
ACFLMWH over UFH whenever possible to avoid additional laboratory monitoring, exposure, and personal protective equipment. In patients with AKI or creatinine clearance < 15–30 mL/min, UFH is recommended over LMWH.Active bleeding or profound thrombocytopenia (Platelet count not defined)Consider if clinical indication such as STEMI, acute ischemic stroke, or high-risk massive PE with hemodynamic instability. Otherwise, it is not recommended outside context of a clinical trial.
ASHLMWH or UFH over direct oral anticoagulants due to reduced drug-drug interactions and shorter half-life.Thromboprophylaxis is recommended even with abnormal coagulation tests in the absence of active bleeding and held only if platelet count < 25 x 109/L or fibrinogen < 0.5 g/L. Abnormal PT or PTT is not a contraindication to thromboprophylaxis. Therapeutic anticoagulation may need to be held if platelet count < 30–50 x 109/L or fibrinogen < 1.0 g/L.Not mentioned
ACCPLMWH or fondaparinux over UFH. UFH preferred in patients at high bleeding risk and in renal failure or needing imminent procedures. Recommend increasing dose of LMWH by 25–30% in patients with recurrent VTE despite therapeutic LMWH anticoagulation.Not mentionedThrombolytics over no such therapy in patients with objectively confirmed PE with hemodynamic instability or signs of obstructive shock who are not at high risk of bleeding. Peripheral thrombolysis recommended over catheter-directed thrombolysis
SCC-ISTHNot mentionedNo specific recommendations. Reports that 50% of respondents report holding if platelet count < 25 x 109/L.Not mentioned
ACCMedication regimen likely to change depending on comorbidities (i.e. renal or hepatic dysfunction, gastrointestinal function, thrombocytopenia). Parenteral anticoagulation (i.e. UFH) may be preferred in many ill patients given it may be withheld temporarily and has no known drug-drug interactions with COVID-19 therapies. LMWH may be preferred in patients who are unlikely to need procedures as there are concerns with UFH regarding the time to achieve therapeutic PTT and increased exposure to healthcare workers. DOACs have advantages including lack of monitoring that is ideal for outpatient management but may have risks in settings of organ dysfunction related to clinical deterioration and lack of timely reversal at some centers.In patients with moderate or severe COVID-19 on chronic therapeutic anticoagulation who develop suspected or confirmed DIC without overt bleeding,
it is reasonable to consider the indication of anticoagulation and risk of bleeding for adjusting dose or discontinuation of anticoagulation. The majority of authors recommended reducing the intensity of anticoagulation unless there was an exceedingly high risk of thrombosis.
A multidisciplinary PERT may be helpful for intermediate and high-risk patient with VTE. For hemodynamically high-risk PE, systemic fibrinolysis is indicated with catheter-based therapies reserved for situations that are not amenable to systemic fibrinolysis. Patients with hemodynamically stable intermediate-low or intermediate-high risk PE should receive anticoagulation and rescue systemic fibrinolysis should be considered in cases of further deterioration with catheter-directed therapies as an alternative. Catheter directed therapies should be limited to most critical situations given minimal data showing mortality benefit. When considering fibrinolysis vs percutaneous coronary intervention for STEMI, clinicians should weigh risks and severity of STEMI presentation, severity of COVID-19 in patient, risk of COVID-19 to individual clinicians and healthcare system.

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Table 3.

Major societal recommendations regarding monitoring of anticoagulation, correction of active bleeding and prophylactic anticoagulation post-discharge

 What is the appropriate method of monitoring anticoagulation?What is the recommended approach for correction of active bleeding?Should patients receive post-discharge prophylactic anticoagulation
CDCPer standard of care for patients without COVID-19Not mentionedRoutine venous thromboprophylaxis post-discharge is not recommended. FDA-approved prophylactic anticoagulation regimen (rivaroxaban and betrixaban) can be considered if high risk for VTE and low risk for bleeding using criteria from clinical trials.
ISTH-IGNot mentionedTransfuse to keep platelet count > 50 x 109/L, fibrinogen > 1.5 g/L, PT ratio < 1.5No specific recommendations
ACFRecommend monitoring anti-Xa levels to monitor UFH due to potential baseline PTT abnormalities. Reasonable to monitor anti-Xa or PTT in patients with normal baseline PTT levels and do not exhibit heparin resistance (> 35,000 u heparin over 24 h).Not mentionedNo evidence for anticoagulation beyond hospitalization, but reasonable to consider if low risk for bleeding and high risk for VTE including intubated, sedated, and paralyzed for multiple days.
ASHMay necessitate anti-Xa monitoring of UFH given artefactual increases in PTT.Transfuse one adult unit of platelets if platelets < 50 x 109/L, give 4 units of plasma if INR > 1.8, and fibrinogen concentrate (4 g) or cryoprecipitate (10 u) if fibrinogen < 1.5 g/L. In patients with severe coagulopathy and bleeding can consider 4 F-PCC (25 u/kg) instead of plasma.Reasonable to consider FDA-approved post-discharge prophylactic anticoagulation regimen (rivaroxaban and betrixaban) or aspirin if criteria from trials for post-discharge thromboprophylaxis are met.
ACCPMonitor anti-Xa levels in all patients receiving UFH given potential of heparin resistance.Not mentionedCan be considered in patients who are at low risk of bleeding if emerging data suggests a clinical benefit.
SCC-ISTHNo specific recommendations. Mentions that expert clinical guidance statements and clinical pathways from large academic healthcare systems target an anti-factor Xa level of 0.3–0.7 IU/mL for UFH.Not mentionedEither LMWH or FDA-approved post-discharge prophylactic anticoagulation regimen (rivaroxaban and betrixaban) should be considered in patients with high VTE risk criteria. Duration is 14 days at least and up to 30 days. Of note, they report that none of the respondents recommended aspirin for post-discharge thromboprophylaxis.
ACCNot mentionedTransfuse platelets to maintain platelets > 50 x 109/L in DIC and active bleeding or if platelets < 20 x 109/L in patients at high risk of bleeding or requiring invasive procedures. FFP (15 to 25 mL/kg) in patients with active bleeding with either prolonged PT or PTT ratios (> 1.5 times normal) or decreased fibrinogen (< 1.5 g/L). Fibrinogen concentrate or cryoprecipitate in patients with persisting severe hypofibrinogenemia (< 1.5 g/L). Prothrombin complex concentrate if FFP is not possible. Tranexamic acid should not be used routinely in patients with COVID-19-associated DIC given the existing data.Reasonable to consider extended prophylaxis with LMWH or DOACs for up to 45 days in patients at high risk for VTE (i.e. D-dimer > 2 times the upper limit, reduced mobility, active cancer) and low risk of bleeding.

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How should biomarkers be used to guide management?

Despite CAC being associated with biomarker abnormalities, none of the major societies to date recommend the use of biomarkers to help guide inpatient anticoagulation decisions. Most notably, D-dimer elevation has been associated with severity of COVID-19, and may be useful as a prognostic marker [36]. With COVID-19 triggering a systemic inflammatory response, it is likely D-dimers will be elevated whether thromboembolic disease is present or not. Therefore, none of the major societies recommend any role for routine monitoring of D-dimer, with its use limited to risk stratification as per ISTH-IG and ASH. Furthermore, the CDC guidelines specifically state that there is insufficient data to recommend for or against using hematologic and coagulation parameters to guide management decisions in CAC. The ACF guidelines also states that using biomarkers such as D-dimer for guiding anticoagulation management should only be done in the setting of a clinical trial. One society’s guidelines, the ACC, mentions a potential role for the use of biomarkers in decision-making: in patients with a D-dimer >2 times the upper limit may be considered for extended prophylaxis (up to 45 days) if patients are at low risk of bleeding. Currently, a multicenter randomized controlled trial is underway to evaluate the efficacy and safety of antithrombotic strategies in COVID-19 adults not requiring hospitalization at time of diagnosis. The trial is designed to compare aspirin, low dose and regular dose apixaban prophylaxis and placebo, with the results of VTE compared across increasing D-dimer levels [58]. This study will help ascertain the value of baseline D-dimer levels in this population. Another clinical trial, ATTACC, was performed to determine whether therapeutic anticoagulation improved organ support-free days [59]. This study also assessed the efficacy of therapeutic anticoagulation across subgroups based on initial D-dimer level. The D-dimer level did not appear to be useful in risk stratification.

When considering other biomarkers, none of the major societal guidelines recommend a change to the anticoagulation regimen in patients with COVID19 who have positive antiphospholipid antibodies or any other biomarker abnormality. Finally, although viscoelastic hemostasis assays such as TEG and ROTEM may suggest hypercoagulability, the ASH guidelines specifically recommend against the routine use of these tests to guide management.

What are the preferred prophylactic anticoagulation regimens?

VTE prophylaxis should be provided to all hospitalized patients with COVID-19 unless contraindicated. The majority of societal guidelines have recommended once daily administration using low molecular weight heparin (LMWH) to reduce healthcare worker exposure given the lower frequency of administration compared to unfractionated heparin (UFH), to conserve personal protective equipment and because it has a lower risk for heparin-induced thrombocytopenia. LMWH may not be preferred over UFH when the risk of bleeding outweighs the risk of thrombosis and in patients with renal dysfunction (i.e. creatinine clearance <30 mL/min). An additional benefit of heparin is its possible anti-inflammatory effects in both the vasculature and the airway [59]. With COVID-19 stimulating a proinflammatory state in both the airways and vasculature, heparin not only provides value as an anticoagulant but also may exert benefit as an anti-inflammatory agent [60]. The efficacy of heparin as an anti-inflammatory agent in patients with COVID-19 warrants further investigation.

The dosing of prophylactic anticoagulation remains controversial given that some studies have demonstrated that up to one quarter of patients with COVID in the ICU develop VTE despite thromboprophylaxis [17,22,61]. As a result, it has been suggested that intermediate or therapeutic doses of LMWH could be considered [10,12,62,63]. There is emerging evidence that initiation of therapeutically dosed anticoagulation in place of prophylactically dosed anticoagulation may decrease the need for mechanical ventilation and other life supporting interventions in non-critically ill hospitalized population but this has not been published in a peer review journal [59]. With thrombosis being a prominent feature of COVID-19, three clinical trials conducted a multiplatform clinical trial (ATTACC) to determine whether therapeutic anticoagulation improved organ support-free days (ICU level care and receipt of mechanical ventilation, vasopressors, extracorporeal membrane oxygenation (ECMO) or high-flow nasal oxygen). Although full results have not been published, the pre-publication, non-peer-reviewed, interim results show that patients who are moderately ill (hospitalized but not on ICU organ-support) had improved organ support-free days with therapeutically dosed anticoagulation in comparison to standard of care [64]. However, full-dose anticoagulation when started in critically ill patients with COVID19 was not found to be beneficial and may be harmful. Current societal guidelines which do not account for these interim findings do not recommend the use of therapeutically dosed anticoagulation as a replacement for prophylactically dosed anticoagulation. Until the results of these studies are published and validated, following current guidelines seems reasonable. Of note, ASH published new guidelines in March 2021 which continue to recommend prophylactically dosed anticoagulation in the context of hospitalized patients diagnosed with COVID-19 [65].

A retrospective analysis of 4389 COVID-19 patients found that compared with no anticoagulation, therapeutic and prophylactic anticoagulation were associated with a lower in-hospital mortality and intubation. Furthermore, when anticoagulation was initiated ≤48 h from admission, there was no statistically significant difference in outcomes between the patients that received therapeutic vs. prophylactic doses [66].

Finally, aspirin has been a proposed treatment for CAC given its anti-inflammatory and anti-thrombotic effects [67–70]. A recent meta-analysis demonstrated no association between the use of aspirin and mortality in COVID-19 [71]. The RECOVERY trial conducted a multicentre randomized control trial (RCT) testing aspirin against usual care [72]. The results of this trial released in preprint showed that aspirin was not associated with a reduction in 28-day mortality or in risk of progressing to invasive mechanical ventilation or death.

When to increase intensity of anticoagulation

At this stage, there is no consensus as to when to increase the intensity of anticoagulation with the exception of documented thromboembolism. The ACF states that increased intensity of anticoagulation regimen (i.e., from standard or intermediate intensity to therapeutic intensity) can be considered in patients, without confirmed VTE or PE, who have deteriorating pulmonary function or ARDS without clear underlying cause. They also suggest an increased intensity of venous thromboprophylaxis could be considered for critically ill patients (i.e. LMWH 40 mg SC twice daily, LMWH 0.5 mg/kg subcutaneous twice daily, heparin 7500 SC three times daily, or low-intensity heparin infusion). The SCC-ISTH guidelines state that intermediate intensity LMWH may be considered in high risk critically ill patients. They also suggest anticoagulation prophylaxis regimens may be modified based on extremes of body weight. If the patient is obese (BMI >30 kg/m2), an increase of 50% in dose has been deemed reasonable.

ASH guidelines state that in patients who have recurrent thrombosis of catheters and extracorporeal circuits (i.e., ECMO, continuous renal replacement therapy (CRRT)) on prophylactic anticoagulation regimens, may have the intensity of anticoagulation increased (i.e. from standard to intermediate intensity, from intermediate to therapeutic intensity) or change the anticoagulant regimen. The CDC guidelines state that patients who have thrombosis of catheters or extracorporeal filters should be treated according to standard institutional protocols (which may include increasing anticoagulation intensity) for patients without COVID-19. Our institution, the Massachusetts General Hospital found that a low dose heparinized saline protocol is associated with improved duration of arterial line patency in critically ill COVID-19 patients [73]. Additionally, we also found that a protocol where systemic unfractionated heparin is dosed by anti-factor Xa levels lead to lower rates of CRRT filter clotting and loss [74].

What is the preferred therapeutic anticoagulation regimens?

Several of societal guidelines (ACF, ACCP, and ACC) recommend LMWH over UFH to avoid additional laboratory monitoring, minimize healthcare worker exposure, preserve personal protective equipment (PPE) utilization, benefit from the greater anti-inflammatory effects, and decrease time to achieve therapeutic anticoagulation levels. LMWH is preferred over UFH when no imminent procedures are planned, the risk of thrombosis is greater than the risk of bleeding, and patients do not have significant renal failure. In addition to LMWH, the ACCP guidelines also recommend fondaparinux over UFH with a similar rationale, and fondaparinux may be used in patients with suspected or confirmed HIT. UFH may be preferred in patients who need imminent procedures, are at high risk of bleeding or have significant renal failure. In patients with recurrent VTE despite therapeutic LMWH anticoagulation, the ACCP guidelines recommends increasing the dose of LMWH by 25–30%. While direct oral anticoagulants (DOACs) have advantages including no need for monitoring, none of the major societal guidelines recommend their use in this critical care setting given their lack of timely reversal at some hospitals. Parenteral anticoagulants also have no known drug–drug interactions with COVID-19 therapies, and this may not be true with DOACs.

When are thrombolytics recommended?

Given autopsy findings of patients with COVID-19 revealing significant pulmonary micro- and macro-thrombosis, the question has been raised as to whether there is a role for thrombolytic therapy. There are a number of case series that suggest the use of thrombolytics in patients with severe ARDS and COVID-19 may lead clinical improvement [75,76]. Given this potential benefit, there are a number of ongoing clinical trials investigating the use of parenteral and nebulized thrombolytic therapy for patients with severe COVID-19 ARDS. If a PE is suspected, consultation with a pulmonary embolism response team (PERT) is advised if available. These teams can provide expert advice regarding issues related to diagnosis and management of a PE in patients with COVID-19. If PERT consultation is unavailable, the National PERT Consortium paper has provided an algorithm to assist in decision-making for patients with a suspected PE [52]. Our institution, the Massachusetts General Hospital, has a PERT team which is a multidisciplinary team composed of experts from cardiology, cardiac surgery, emergency medicine, hematology, pulmonary and critical care, radiology, and vascular medicine and delivers immediate and evidence-based care to patients with suspected or confirmed high risk PE [77]. When major societal guidelines do recommend thrombolytic therapy, it is in the clinical context where their use would otherwise be clinically indicated such as STEMI, acute ischemic stroke, or high-risk massive PE with hemodynamic instability and when the benefits outweigh the risks of administration. In general, thrombolytic therapy is not recommended in patients who have a PE and are hemodynamically stable [57].

When to hold anticoagulation?

Most major society’s guidelines (CDC, ISTH-IG, ACF, ASH, SCC-ISTH) advise holding therapeutic and prophylactic anticoagulation in patients who have significant active bleeding and/or severe thrombocytopenia. Both the ACF and SCC-ISTH guidelines suggest holding anticoagulation if platelet count <25 x 10^9/L. ASH recommends holding prophylactic anticoagulation if platelet count is <25 x 10^9/L or fibrinogen <0.5 g/L, and holding therapeutic anticoagulation may necessary if platelet count is <30–50 x 10^9/L or fibrinogen <1.0 g/L. Of note, many patients with COVID-19 may have abnormal baseline PT or PTT, which is not a contraindication to thromboprophylaxis according to the ISTH-IG and ASH guidelines. Therefore, PT and PTT should not be used as a guide to hold prophylactic or therapeutic anticoagulation.

What is the utility of mechanical thromboprophylaxis?

Most of the major society’s guidelines (ACF, ASH, ACCP, SCC-ISTCH, and ACC) recommend or suggest mechanical thromboprophylaxis when pharmacological thromboprophylaxis is contraindicated. Intermittent pneumatic compression devices are the preferred type of mechanical thromboprophylaxis. ACCP suggests against the additional use of mechanical thromboprophylaxis in critically ill patients receiving pharmacological prophylaxis but mentions that its addition is unlikely to cause harm.

What is the appropriate method of monitoring anticoagulation?

Monitoring of patients receiving therapeutic anticoagulation with LMWH

Currently, none of the major society guidelines recommend the routine monitoring of anti-Xa levels of patients receiving LMWH. LMWH is generally preferred if there are no contraindications given the added benefit of not needing routine monitoring. However, the ISTH-IG guidelines state that monitoring of LMWH is advised in patients with severe renal impairment, a patient population generally for which LMWH is not routinely recommended. However, the ACCP guidelines state that body weight adjusted doses for LMWH do not require laboratory monitoring in majority of patients, and the ACF guidelines state that anti-Xa level monitoring is not recommended in patients with elevated PTT levels given the lack of evidence on outcomes for thrombosis or bleeding.

Monitoring of patients receiving therapeutic anticoagulation with UFH

The PTT measures the intrinsic coagulation pathway and is the most commonly used test to monitor UFH [78]. It is not uncommon for patients with COVID-19 to have baseline coagulation abnormalities of PT and PTT. Although these abnormalities are not contraindications to anticoagulation, it may lead to difficulties measuring heparin effectiveness. When in doubt, the majority of society guidelines (ACF, ASH, ACF and ACCP) advise that therapeutic anticoagulation should be monitored with an anti-Xa level rather than PTT. The SCC-ISTH guideline does not make any particular recommendations but does mention that expert clinical guidance statements and clinical pathways from large academic healthcare systems target for therapeutic anticoagulation, an anti-factor Xa level of 0.3–0.7 IU/mL for UFH. While the ACF guideline recommends monitoring of anti-Xa levels to monitor UFH due to potential baseline PTT abnormalities and heparin resistance (>35,000 U heparin over 24 hours), they also mention that it is reasonable to monitor anti-Xa or PTT in patients with normal baseline PTT levels and in those unlikely to have heparin resistance. There is evidence in the value of implementing an anticoagulation protocol using systemic unfractionated heparin, dosed by anti-factor Xa levels. At our institution, The Massachusetts General Hospital, we found that patients with COVID-19 infection on CRRT had lower rates of CRRT filter clotting and loss when using this protocol where systemic unfractionated heparin was dosed by anti-factor Xa levels [74]. However, this needs to be studied further in clinical trials.

Heparin resistance

An important consideration when making decisions about DVT prophylaxis and monitoring of anticoagulation in COVID-19 patients is the concern for heparin resistance, which has been well documented [79]. Heparin resistance should be suspected when disproportionately large doses of heparin are required to achieve therapeutic anticoagulation. This problem is usually due to low heparin concentrations, which results from binding of heparin to acute phase proteins in the context of systemic inflammation. There is also some evidence of AT deficiency in COVID-19 patients which may contribute to suspected heparin resistance [48]. Heparin functions as an anticoagulant by binding to AT, activating it, and then inhibiting clotting factors, most notably factor Xa [80]. A way to measure the capacity of the heparin-AT complex is with anti-Xa levels. It has been well documented that patients with COVID-19 may have artefactual increases in PTT, and therefore measuring anti-Xa levels may be a more accurate way to assess the level of anticoagulation in these situations. Additionally, LMWH can’t be measured with PTT, and as a result, anti-Xa levels may be used to ensure appropriate anticoagulation levels have been achieved if necessary. Unfortunately, not all centers have the capacity to monitor anti-Xa levels.

What is the recommended approach to control active bleeding?

When addressing active bleeding, the major society guidelines recommend holding both prophylactic and therapeutic anticoagulation. However, only the ISTH-IG, ASH and ACC guidelines provide specific recommendations for blood product replacement. The ISTH-IG guidelines recommend transfusing to keep platelet count >50 x 10^9/L, fibrinogen concentrate to target fibrinogen >1.5 g/L, and FFP to target PT ratio <1.5. ASH guidelines recommend transfusing one adult unit of platelets if platelet count <50 x 10^9/L, 4 units of plasma if INR > 1.8 and fibrinogen concentrate (4 g) or cryoprecipitate (10 units) if fibrinogen <1.5 g/L. The ACC guidelines recommend transfusing platelets in patients with active bleeding or requiring invasive procedures if platelet count <20 x 10^9/L, and providing FFP (15 to 25 mL/kg) in patients with active bleeding with either prolonged PT or PTT ratios (>1.5 times normal) as well as transfusing fibrinogen concentrate or cryoprecipitate in patients with persisting severe hypofibrinogenemia (<1.5 g/L).

If volume overload is a concern in patients with active bleeding and severe COVID-19, ASH and ACC guidelines recommend the use of 4 F-PCC (25 u/kg) instead of FFP. The ACC guidelines also state that tranexamic acid should not be routinely used in patients with COVID-19 associated DIC given the lack of existing data. None of the major societies mention the use of TEG to monitor coagulopathy in patients who are actively bleeding.

DIC is an uncommon but serious complication in patients with COVID-19 [81]. It’s important to note that DIC is a clinical diagnosis with exclusion of alternate explanations for coagulation dysfunction. In patients with COVID-19, a superimposed bacterial infection is the most likely precipitant, however other causes such as HIT, drug-induced DIC and malignancy may also be contributing. Treating the underlying cause is the most important component of treating DIC, with transfusion targets the same as for active bleeding. If there is no active bleeding, replacement of fibrinogen and coagulation factors remain controversial. However, if platelet count is <10 x 10^9/L, platelets should be transfused.

Should patients receive post-discharge prophylactic anticoagulation and what regimens are available?

There is no evidence to support post-discharge DVT prophylaxis in patients who were hospitalized with COVID-19 infection. A number of studies have identified very low rates of post-discharge VTE; therefore, there is no universal recommendation for VTE prophylaxis for all patients post-discharge [82–84]. However, in high-risk patients and those who are at low risk of bleeding, the majority of major societal guidelines (CDC, ACF, ASH, ACCP, SCC-ISTH and ACC) state that it is reasonable to consider post-discharge prophylactic anticoagulation. Currently, there is an ongoing randomized trial evaluating the effectiveness and safety of low-dose apixaban in reducing thrombosis in patients who have been discharged from the hospital [85].

At the point of discharge, if a patient is deemed high risk (i.e., D-dimer >2 times the upper limit, reduced mobility, active cancer) of thrombosis and low risk of bleeding, it is reasonable to use criteria from clinical trials involving FDA-approved prophylactic anticoagulation regimens such as LMWH, rivaroxaban and betrixaban for thromboprophylaxis [57]. In terms of how long to provide thromboprophylaxis once discharged, the SCC guidelines recommend following post-discharge prophylactic anticoagulation regimen for 14–30 days. The ACC guideline states that it is reasonable to consider extended prophylaxis with LMWH or DOACs for up to 45 days in patients with high risk for VTE. For those diagnosed with COVID-19, and not admitted to hospital, there is no recommendation for DVT prophylaxis as an outpatient. However, a multicenter randomized controlled trial is underway to evaluate the efficacy and safety of antithrombotic strategies (aspirin compared with low dose and regular dose apixaban, and with placebo) in adults with COVID-19 not requiring hospitalization at time of diagnosis [58]. However, this trial is evaluating outpatients, not patients admitted post-discharge.

Conclusion

CAC is associated with macro- and micro-thrombosis, which can lead to a myriad of different presentations, and may result in multiorgan injury and ultimately death. As a result, important clinical questions regarding the optimal prevention and management of thrombosis has led to many ongoing clinical trials. Whilst data continues to be collected, major hematological societies have put forth recommendations regarding diagnosis, monitoring, and treatment of CAC. Overall, decisions should be made based on the providers understanding of a patient’s medical history, clinical course and perceived risk, in conjunction with the major societal guidelines and results from emerging clinical trial results.

Acknowledgments

We thank the educational division of Roche for allowing us to incorporate this manuscript into their CoagYOUlation platform (http://www.coagYOUlation.com). This manuscript incorporates literature and guidelines available at the time of submission. It is anticipated that the guidelines and practice management provided on the platform will be updated as additional literature becomes available.

Declaration of funding

We would also like to thank Roche for providing support for the article processing fees required in publishing this article and for initial compensation related to the creation of educational content provided in this article.Go to:

Declaration of financial/other relationships

No potential conflict of interest was reported by the author.

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Authors contributions

PJL, RPR, EAB, MGC wrote and reviewed the manuscript.

Take home message

COVID-19-associated coagulopathy (CAC) is a well-recognized hematologic complication among patients with severe COVID-19 disease, where macro- and micro-thrombosis can lead to multiorgan injury and failure. In this review, we utilize current societal guidelines to provide a framework for practitioners in managing their patients with CAC.

COVID-19: Coronavirus disease 2019

WHO: World Health Organization

CAC: COVID-19 associated coagulopathy

CDC: Centers for Disease Control and Prevention (CDC),

ISTH-IG: International Society on Thrombosis and Hemostasis interim guidance (ISTH-IG)

ASH: American Society of Hematology (ASH)

ACCP: American College of Chest Physicians

SCC-ISTH: Scientific and Standardization Committee of ISTH

ACF: Anticoagulation Forum

ACC: American College of Cardiology

vWF: von Willebrand Factor

ICU: Intensive Care Unit

TEG: Thromboelastography

ROTEM: Rotational thromboelastometry

DIC: Disseminated intravascular coagulation

PT: Prothrombin time

PTT: Partial thromboplastin time

AT: Antithrombin

MA: Maximum amplitude

INR: International normalized ratio

VTE: Venous thromboembolism

HIT: Heparin-induced thrombocytopenia

SRA: Serotonin release assay

PE: Pulmonary embolism

CTPA: Computed tomography pulmonary angiogram (CTPA)

LMWH: Low molecular weight heparin

UFH: Unfractionated heparin

RCT: Randomized control trial

DOAC: Direct oral anticoagulants

PPE: Personal protective equipment

CRRT: Continuous renal replacement therapyGo to:

Declaration of interest

No potential conflict of interest was reported by the author(s).Go to:

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Factor VIII and Functional Protein C Activity in Critically Ill Patients With Coronavirus Disease 2019: A Case Series

Ali Tabatabai 1Joseph Rabin 2Jay Menaker 2Ronson Madathil 3Samuel Galvagno 4Ashley Menne 5Jonathan H Chow 4Alison Grazioli 6Daniel Herr 1Kenichi Tanaka 4Thomas Scalea 2Michael Mazzeffi 4Affiliations expand

PMID: 32539272 PMCID: PMC7242090 DOI: 10.1213/XAA.0000000000001236

Abstract

Critically ill patients with coronavirus disease 2019 (COVID-19) have been observed to be hypercoagulable, but the mechanisms for this remain poorly described. Factor VIII is a procoagulant factor that increases during inflammation and is cleaved by activated protein C. To our knowledge, there is only 1 prior study of factor VIII and functional protein C activity in critically ill patients with COVID-19. Here, we present a case series of 10 critically ill patients with COVID-19 who had severe elevations in factor VIII activity and low normal functional protein C activity, which may have contributed to hypercoagulability.

The novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has caused a global pandemic accounting for over 190,000 deaths as of April 24, 2020. In hospitalized patients, mortality is reported to be approximately 25%, and in critically ill patients, who require mechanical lung ventilation mortality is between 65% and 85%.13 Most patients who die progress to septic shock and half develop coagulopathy with progressively increasing D-dimer.1 Age, coronary artery disease, and elevated D-dimer are independent predictors of mortality.1

Several reports suggest that SARS-CoV-2 infection is associated with coagulation derangements, particularly hypercoagulability, and that anticoagulation with heparin may improve survival.46 In our own experience, we have noted marked hypercoagulability in critically ill patients with coronavirus disease 2019 (COVID-19), including thrombosis of renal replacement therapy filters and extracorporeal membrane oxygenation (ECMO) circuits.

Factor (F) VIII is a procoagulant factor that is stored in endothelial cells and is released during inflammation. It has a critical role in coagulation, because it is a cofactor in the tenase complex, which converts factor X into activated factor X. Protein C is an endogenous anticoagulant protein that, when activated, cleaves FVIII into its inactive form. When FVIII overwhelms activated protein C, prolonged thrombin generation can occur leading to hypercoagulability. To our knowledge, there have been no studies of FVIII activity and functional protein C activity in patients with COVID-19 in the United States. We describe a case series of 10 critically ill patients with COVID-19 who had both FVIII and functional protein C activity measured during their clinical course. The University of Maryland, Baltimore institutional review board approved the case review and exempted the study from written informed consent.

CASE DESCRIPTIONS

Methods

Critically ill adult patients with COVID-19 who were on mechanical lung ventilation in the R Adams Cowley Shock Trauma Center bio-containment unit between April 1, 2020 and April 5, 2020 and had FVIII and functional protein C activity measured were included. FVIII activity and functional protein C activity were measured as part of a comprehensive coagulation evaluation in patients who had clinical evidence of hypercoagulability, including thrombosis in central and peripheral access lines, purpuric skin changes, and elevated D-dimer concentration. All patients had the following coagulation factor and inflammatory markers measured: FVIII activity, functional protein C activity, fibrinogen, antithrombin activity, C-reactive protein, and ferritin. Patients also had standard plasma-based coagulation tests and complete blood counts performed.

Continuous patient variables were summarized as the mean value ± standard deviation, and categorical patient variables were summarized as the number and percentage of patients. Coagulation factor and inflammatory marker concentrations were summarized as the mean value ± standard deviation or median value and interquartile range, depending on whether data were normally distributed or skewed. Normality was checked with the Shapiro-Wilk test. The relationships between age and coagulation factor concentrations were explored using scatterplots with fitted Loess curves. A 95% confidence band was added to Loess curves. Statistical analysis was performed using SAS 9.3 (SAS Corporation, Cary, NC).Go to:

RESULTS

Ten critically ill patients were included in the case series (Table ​(Table1).1). All patients were on mechanical lung ventilation with acute respiratory distress syndrome (ARDS) related to SARS-CoV-2 infection. The mean number of mechanical lung ventilation days was 9 ± 6 at the time of patients’ coagulation evaluation. Diabetes mellitus was the most common comorbidity, occurring in 70% of patients. Eighty percent of patients were men and 70% had type O blood.

Table 1.

Patient Characteristics

VariableMean ± SD, or n (%)
Age (y)53 ± 16
Male sex8 (80)
Body mass index (kg/m2)33 ± 8
Diabetes mellitus7 (70)
Chronic arterial hypertension4 (40)
Chronic kidney disease2 (20)
Peripheral vascular disease1 (10)
Cerebral vascular disease1 (10)
Chronic obstructive pulmonary
disease or asthma
0 (0)
Coronary artery disease1 (10)
Dyslipidemia3 (30)
Blood type
O7 (70)
A2 (20)
B1 (10)
AB0 (0)
Acute respiratory distress syndrome
Severity
Mild4 (40)
Moderate3 (30)
Severe3 (30)
Days on mechanical lung ventilation9 ± 6
Extracorporeal membrane
oxygenation
2 (20)
Acute renal failure requiring renal
replacement
3 (30)

Open in a separate window

n = 10.

Abbreviation: SD, standard deviation.

Table ​Table22 shows coagulation factor and inflammatory marker concentrations. Median prothrombin time and mean activated partial thromboplastin time were normal. Both median fibrinogen concentration and FVIII activity were markedly elevated in patients with COVID-19, while median antithrombin activity and mean functional protein C activity were low normal. Seven of 10 patients had a FVIII activity above the maximum detectable range for the assay, which is 400%. The 3 patients who did not have an FVIII activity above 400% were all less than 50 years old and 2 of them were female. Median D-dimer and ferritin concentration along with mean C-reactive protein concentration were elevated for all patients in the cohort.

Table 2.

Coagulation Factors and Inflammatory Markers

VariableMean ± SD or Median [Q1, Q3]
Prothrombin time (normal = 12–15 s)15 [14, 15]
International normalized ratio1.1 [1.1, 1.2]
Activated partial thromboplastin timea (normal = 25–38 s)30 ± 5
Fibrinogen concentration (mg/dL) (normal = 216–438 mg/dL)763 [478, 1092]
Factor VIII activityb (normal = 50%–200%)400 [369, 400]
Functional protein C activity (normal = 83%–168%)104 ± 40
Antithrombin activity (normal = 75%–135%)84 [70, 90]
D-dimerc (normal < 649 ng/mL FEU)2820 [2410, 20,000]
C-reactive protein (normal < 1 mg/dL)17 ± 14
Ferritin concentration (normal = 18–464 ng/mL)1343 [748, 2116]

Open in a separate window

n = 10.

Abbreviations: FEU, fibrinogen equivalence units; SD, standard deviation.

aExcludes 3 patients who were receiving therapeutic heparin.

bUpper limit of assay is 400% and 7 of 10 patients were at upper limit.

cUpper limit of assay is 20,000 ng/mL FEU and 3 of 10 patients were at upper limit.

Older patients appeared to have higher fibrinogen concentrations and FVIII activity along with lower antithrombin activity and functional protein C activity (Figure). The 1 patient who died in the cohort at the time of our analysis was 82 years old. He had a fibrinogen concentration of 1092 mg/dL, antithrombin activity of 37%, and functional protein C activity of 37%. Among the remaining 9 patients, 7 were extubated, 4 were discharged from the hospital and 2 remained on ECMO at the time of our report. No patient was diagnosed with symptomatic deep venous thrombosis or pulmonary embolism.Figure.

Relationships between age and coagulation factor concentration and activity.Go to:

DISCUSSION

Extensive cross talk occurs between the immune and coagulation systems. Prior studies have shown that interleukin (IL)-6 and other cytokines activate coagulation.7,8 COVID-19 is associated with a profound inflammatory response in many patients that is characterized by high IL-6, fibrinogen, and ferritin concentrations.1 COVID-19 is also associated with high D-dimer, suggesting extensive thrombin generation and fibrinolysis.1 This pattern may reflect hypercoagulability related to severe inflammation.9 Our case series demonstrates that some critically ill patients with COVID-19 in the United States have a coagulation profile characterized by severely elevated fibrinogen concentration and FVIII activity, as well as low normal antithrombin and functional protein C activity. This hypercoagulable pattern appears to be accentuated with age, particularly in men. These findings have similarities with and differences from a recent study by Panigada et al9 where functional protein C activity was normal in most patients and mean FVIII activity was elevated to 297%. Mean fibrinogen concentration in their study was 680 mg/dL compared to 763 mg/dL in our study. The authors did not explore the impact of age on coagulation profile and there were few details provided about the patients’ comorbidities. We postulate that differences between the 2 studies could be related to a higher prevalence of metabolic syndrome and other comorbidities among adults in the United States leading to more severe coagulation abnormalities.

FVIII and fibrinogen are acute phase reactants that increase during infection, pregnancy, and other inflammatory states.10,11 Antithrombin and protein C are endogenous anticoagulant proteins, both of which decrease in men as they age.12 An imbalance in procoagulant and anticoagulant factor concentrations can predispose patients to thrombotic complications and perhaps microthrombosis. Hypercoagulability has been reported in critically ill patients with COVID-19 and there are recent reports of ischemic stroke and limb ischemia.13,14

Some patients with COVID-19 have been found to have antiphospholipid antibodies.13 Our data suggest that an imbalance in the FVIII-protein C system also contributes to hypercoagulability. Given this imbalance, there may be a role for systemic anticoagulation or low-dose thrombolysis in some patients. Although not commercially available in the United States, recombinant thrombomodulin could be a potential treatment for hypercoagulable patients with COVID-19. It has immunomodulatory effects and increases protein C activation in the presence of thrombin.15 There are no high-quality studies to support systemic anticoagulation at this time, but the observational study by Tang et al6 strongly suggests that patients with an elevated D-dimer concentration have better survival when treated with anticoagulation.

The main strength of our case series is that it is the first, to our knowledge, to report FVIII activity and functional protein C activity in critically ill patients with COVID-19 in the United States. An important limitation is that our cohort was comprised overwhelmingly obese men with diabetes mellitus, which may limit generalizability. Also, patients in our study had FVIII and functional protein C activity measured once, not multiple times during their stay.

In summary, in a case series of 10 critically ill patients with COVID-19, who were mostly older men, we found that fibrinogen concentration and FVIII activity were severely elevated, while antithrombin activity and functional protein C activity were low normal. These findings suggest that an imbalance in procoagulant and anticoagulant factor concentrations may contribute to hypercoagulability in some critically ill patients with COVID-19.Go to:

DISCLOSURES

Name: Ali Tabatabai, MD.

Contribution: This author helped conceive the study, review the analysis of the data, write and approve the final manuscript.

Name: Joseph Rabin, MD.

Contribution: This author conceived the study, saw the original data, reviewed the analysis of the data, wrote and approved the final manuscript.

Name: Jay Menaker, MD.

Contribution: This author helped conceive the study, review the analysis of the data, write and approve the final manuscript.

Name: Ronson Madathil, MD.

Contribution: This author helped conceive the study, review the analysis of the data, write and approve the final manuscript.

Name: Samuel Galvagno, DO, PhD.

Contribution: This author helped conceive the study, review the analysis of the data, write and approve the final manuscript.

Name: Ashley Menne, MD.

Contribution: This author helped review the analysis of the data, write and approve the final manuscript.

Name: Jonathan H. Chow.

Contribution: This author helped review the analysis of the data, write and approve the final manuscript.

Name: Alison Grazioli, MD.

Contribution: This author helped review the analysis of the data, write and approve the final manuscript.

Name: Daniel Herr, MD.

Contribution: This author helped review the analysis of the data, write and approve the final manuscript.

Name: Kenichi Tanaka, MD, MSc.

Contribution: This author helped conceive the study, review the analysis of the data, and write and approve the final manuscript.

Name: Thomas Scalea, MD.

Contribution: This author helped review the analysis of the data, write and approve the final manuscript.

Name: Michael Mazzeffi, MD, MPH, MSc.

Contribution: This author conceived the study, saw the original data, reviewed the analysis of the data, wrote and approved the final manuscript.

Whole genome sequencing reveals host factors underlying critical Covid-19

Authors: Athanasios KousathanasErola Pairo-CastineiraJ. Kenneth BaillieArticle

Published:  nature  articles  article

We are providing an unedited version of this manuscript to give early access to its findings. Before final publication, the manuscript will undergo further editing. Please note there may be errors present which affect the content, and all legal disclaimers apply.

Abstract

Critical Covid-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalisation2–4 following SARS-CoV-2 infection. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from critically-ill cases with population controls in order to find underlying disease mechanisms. Here, we use whole genome sequencing in 7,491 critically-ill cases compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical Covid-19. We identify 16 new independent associations, including variants within genes involved in interferon signalling (IL10RBPLSCR1), leucocyte differentiation (BCL11A), and blood type antigen secretor status (FUT2). Using transcriptome-wide association and colocalisation to infer the effect of gene expression on disease severity, we find evidence implicating multiple genes, including reduced expression of a membrane flippase (ATP11A), and increased mucin expression (MUC1), in critical disease. Mendelian randomisation provides evidence in support of causal roles for myeloid cell adhesion molecules (SELEICAM5CD209) and coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of Covid-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication, or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between critically-ill cases and population controls is highly efficient for detection of therapeutically-relevant mechanisms of disease.

Author information

Author notes

  1. These authors contributed equally: Athanasios Kousathanas, Erola Pairo-Castineira
  2. These authors jointly supervised this work: Sara Clohisey Hendry, Loukas Moutsianas, Andy Law, Mark J Caulfield, J. Kenneth Baillie
  3. A list of authors and their affiliations appears in the Supplementary Information

Affiliations

  1. Genomics England, London, UKAthanasios Kousathanas, Alex Stuckey, Christopher A. Odhams, Susan Walker, Daniel Rhodes, Afshan Siddiq, Peter Goddard, Sally Donovan, Tala Zainy, Fiona Maleady-Crowe, Linda Todd, Shahla Salehi, Greg Elgar, Georgia Chan, Prabhu Arumugam, Christine Patch, Augusto Rendon, Tom A. Fowler, Richard H. Scott, Loukas Moutsianas & Mark J. Caulfield
  2. Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh, UKErola Pairo-Castineira, Konrad Rawlik, Clark D. Russell, Jonathan Millar, Fiona Griffiths, Wilna Oosthuyzen, Bo Wang, Marie Zechner, Nick Parkinson, Albert Tenesa, Sara Clohisey Hendry, Andy Law & J. Kenneth Baillie
  3. MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, UKErola Pairo-Castineira, Lucija Klaric, Albert Tenesa, Chris P. Ponting, Veronique Vitart, James F. Wilson, Andrew D. Bretherick & J. Kenneth Baillie
  4. Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, UKClark D. Russell & J. Kenneth Baillie
  5. Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UKTomas Malinauskas, Katherine S. Elliott & Julian Knight
  6. Institute for Molecular Bioscience, The University of Queensland, Brisbane, AustraliaYang Wu
  7. Biostatistics Group, Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, Guangzhou, ChinaXia Shen
  8. Centre for Global Health Research, Usher Institute of Population Health Sciences and Informatics, Teviot Place, Edinburgh, UKXia Shen, Albert Tenesa & James F. Wilson
  9. Edinburgh Clinical Research Facility, Western General Hospital, University of Edinburgh, Edinburgh, UKKirstie Morrice, Angie Fawkes & Lee Murphy
  10. Intensive Care Unit, Royal Infirmary of Edinburgh, 54 Little France Drive, Edinburgh, UKSean Keating, Timothy Walsh & J. Kenneth Baillie
  11. Department of Critical Care Medicine, Queen’s University and Kingston Health Sciences Centre, Kingston, ON, CanadaDavid Maslove
  12. Clinical Research Centre at St Vincent’s University Hospital, University College Dublin, Dublin, IrelandAlistair Nichol
  13. NIHR Health Protection Research Unit for Emerging and Zoonotic Infections, Institute of Infection, Veterinary and Ecological Sciences University of Liverpool, Liverpool, UKMalcolm G. Semple
  14. Respiratory Medicine, Alder Hey Children’s Hospital, Institute in The Park, University of Liverpool, Alder Hey Children’s Hospital, Liverpool, UKMalcolm G. Semple
  15. Illumina Cambridge, 19 Granta Park, Great Abington, Cambridge, UKDavid Bentley & Clare Kingsley
  16. Regeneron Genetics Center, 777 Old Saw Mill River Rd., Tarrytown, USAJack A. Kosmicki, Julie E. Horowitz, Aris Baras, Goncalo R. Abecasis & Manuel A. R. Ferreira
  17. Geisinger, Danville, PA, USAAnne Justice, Tooraj Mirshahi & Matthew Oetjens
  18. Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USADaniel J. Rader, Marylyn D. Ritchie & Anurag Verma
  19. Test and Trace, the Health Security Agency, Department of Health and Social Care, Victoria St, London, UKTom A. Fowler
  20. Department of Intensive Care Medicine, Guy’s and St. Thomas NHS Foundation Trust, London, UKManu Shankar-Hari
  21. Department of Medicine, University of Cambridge, Cambridge, UKCharlotte Summers
  22. William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UKCharles Hinds
  23. Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford, UKPeter Horby
  24. Department of Anaesthesia and Intensive Care, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong, ChinaLowell Ling
  25. Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, Northern Ireland, UKDanny McAuley
  26. Department of Intensive Care Medicine, Royal Victoria Hospital, Belfast, Northern Ireland, UKDanny McAuley
  27. UCL Centre for Human Health and Performance, London, UKHugh Montgomery
  28. National Heart and Lung Institute, Imperial College London, London, UKPeter J. M. Openshaw
  29. Imperial College Healthcare NHS Trust: London, London, UKPeter J. M. Openshaw
  30. Imperial College, London, UKPaul Elliott
  31. Intensive Care National Audit & Research Centre, London, UKKathy Rowan
  32. School of Life Sciences, Westlake University, Hangzhou, Zhejiang, ChinaJian Yang
  33. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, ChinaJian Yang
  34. Great Ormond Street Hospital, London, UKRichard H. Scott
  35. William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London, UKMark J. Caulfield

Consortia

GenOMICC Investigators

23andMe

Covid-19 Human Genetics Initiative

Corresponding authors

Correspondence to Mark J. Caulfield or J. Kenneth Baillie.

Supplementary information

Supplementary Information

This file contains Supplementary Figures; Supplementary Tables and Supplementary References

Age- and Sex-Specific Incidence of Cerebral Venous Sinus Thrombosis Associated With Ad26.COV2.S COVID-19 Vaccination

Authors: Aneel A. Ashrani, MD, MS1Daniel J. Crusan, BS2Tanya Petterson, MS2et al

JAMA Intern Med. 2022;182(1):80-83. doi:10.1001/jamainternmed.2021.6352

Recent reports14 suggest a possible association between Ad26.COV2.S (Johnson & Johnson/Janssen) COVID-19 vaccination and cerebral venous sinus thrombosis (CVST). Estimates of postvaccination CVST risk require accurate age- and sex-specific prepandemic CVST incidence rates; however, reported rates vary widely.5 We compared the age- and sex-specific CVST rates after Ad26.COV2.S vaccination with the prepandemic CVST rate in the population.Methods

In this population-based cohort study, to estimate the risk of CVST after Ad26.COV2.S vaccination, we first identified all incident cases of CVST in Olmsted County, Minnesota from January 1, 2001, through December 31, 2015 (eMethods in the Supplement). Sex-and age-adjusted incidence rates were adjusted to the 2010 US census population. We used CDC Vaccine Adverse Event Reporting System (VAERS) data from February 28, 2021 (vaccine approval date) to May 7, 2021, to estimate the incidence of CVST after Ad26.COV2.S vaccination assuming 3 (15, 30, and 92 days) plausible postvaccination periods during which individuals were considered to be at risk of CVST. We then compared post-Ad26.COV2.S vaccination CVST rates with prepandemic rates to estimate postvaccination CVST risk. This study was approved by the Mayo Clinic institutional review board. Medical records of Olmsted County residents with CVST were reviewed only if the residents had signed an authorization for accessing their medical records for research purposes. SAS, version 9.4 (SAS Institute Inc) and R, version 4.0.3 (R Project for Statistical Computing) were used for statistical analyses. Significance was set at a 2-sided P < .05.Results

From 2001 through 2015, 39 Olmsted County residents developed acute incident CVST. A total of 29 patients (74.4%) had a predisposing venous thromboembolism risk factor (eg, infection, active cancer, or oral contraceptives [for women]) within 92 days before the event. The median age at diagnosis was 41 years (range, 22-84 years); 22 residents with CVST (56.4%) were female. The overall age- and sex-adjusted CVST incidence was 2.34 per 100 000 person-years (PY) (95% CI, 1.60-3.08 per 100 000 PY). Age-adjusted CVST rates for female and male individuals were 2.46 per 100 000 PY (95% CI, 1.43-3.49 per 100 000 PY) and 2.34 per 100 000 PY (95% CI, 1.22-3.46 per 100 000 PY), respectively. Men aged 65 years or older had the highest CVST rate (6.22 per 100 000 PY; 95% CI, 2.50-12.82 per 100 000 PY), followed by women aged 18 to 29 years (4.71 per 100 000 person-years; 95% CI, 2.26-8.66 per 100 000 PY) (Table 1).

As of May 7, 2021, 8 727 851 Ad26.COV2.S vaccine doses had been administered in the US; 46 potential CVST events occurring within 92 days after Ad26.COV2.S vaccination were reported to VAERS. Eight events were excluded because they were potentially duplicate reports (4) or were not objectively diagnosed (4). Twenty-seven of 38 objectively diagnosed cases of CVST after Ad26.COV2.S vaccination (71.1%) occurred in female individuals. The median patient age was 45 years (range, 19-75 years). The median time from vaccination to CVST was 9 days (IQR, 6-13 days; range, 1-51 days); 31 of 38 cases of CVST (81.6%) occurred within 15 days after vaccination, and 36 (94.7%) occurred within 30 days.

The overall incidence rate of post–Ad26.COV2.S vaccination CVST was 8.65 per 100 000 PY (95% CI, 5.88-12.28 per 100 000 PY) at 15 days, 5.02 per 100 000 PY (95% CI, 3.52-6.95 per 100 000 PY) at 30 days, and 1.73 per 100 000 PY (95% CI, 1.22-2.37 per 100 000 PY) at 92 days (Table 2). The 15-day postvaccination CVST incidence rates for female and male individuals were 13.01 per 100 000 PY (95% CI, 8.24-19.52 per 100 000 PY) and 4.41 per 100 000 PY (95% CI, 1.90-8.68 per 100 000 PY), respectively. The postvaccination CVST rate among females was 5.1-fold higher compared with the pre-COVID-19 pandemic rate (13.01 vs 2.53 per 100 000 PY; P < .001) (Table 2). This risk was highest among women aged 40 to 49 years (29.50 per 100 000 PY; 95% CI, 13.50-55.95 per 100 000 PY), followed by women aged 30 to 39 years (26.50 per 100 000 PY; 10.65-54.63 per 100 000 PY).Discussion

In this population-based cohort study, we found that the CVST incidence rate 15 days after Ad26.COV2.S vaccination was significantly higher than the prepandemic rate. However, the higher rate of this rare adverse effect must be considered in the context of the effectiveness of the vaccine in preventing COVID-19 (absolute reduction of severe or critical COVID-19 of 940 per 100 000 PY).6

Most CVST events occurred within 15 days after vaccination, which is likely the highest at-risk period. The postvaccination CVST rate among females was higher than the prepandemic rate among females. The highest risk was among women aged 30 to 49 years, but the absolute CVST risk was still low in this group (up to 29.5 per 100 000 PY among women aged 40-49 years). The reason that women had a higher incidence of postvaccination CVST is unclear; concomitant CVST risk factors or autoantibody production might have been involved.2 The overall prepandemic CVST incidence rate was slightly higher in our study than in other studies (0.22-1.57 per 100 000 PY)5 likely because we captured all objectively diagnosed incident CVST cases in a well-defined population, including those discovered at autopsy.

The present study avoided referral bias and included only objectively diagnosed and confirmed cases. Only cases with adequate details or imaging findings reported on VAERS were used. Study limitations include possible ascertainment bias by including only objectively diagnosed CVST cases. VAERS reporting is voluntary and subject to reporting biases. VAERS monitors vaccine adverse events but does not prove causality.Back to topArticle Information

Accepted for Publication: September 12, 2021.

Published Online: November 1, 2021. doi:10.1001/jamainternmed.2021.6352

Corresponding Author: Aneel A. Ashrani, MD, MS, Division of Hematology, Department of Internal Medicine, Mayo Clinic, 200 First St SW, Rochester, MN 55905 (ashrani.aneel@mayo.edu).

Author Contributions: Dr Ashrani and Mr Crusan had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: Ashrani, Petterson, Bailey, Heit.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Ashrani, Crusan, Petterson.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Crusan, Petterson, Bailey.

Obtained funding: Ashrani, Heit.

Administrative, technical, or material support: Ashrani, Heit.

Supervision: Ashrani, Petterson, Bailey, Heit.

Conflict of Interest Disclosures: Dr Ashrani reported receiving grants from the National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH) during the conduct of the study. Mr Crusan reported receiving grants from the NIH during the conduct of the study. Dr Heit reported receiving grants from the NHLBI, NIH during the conduct of the study. No other disclosures were reported.

Funding/Support: This study was supported in part by grant R01HL66216 from the NHLBI, NIH (Drs Ashrani and Bailey), the Rochester Epidemiology Project (grant R01AG034676 from the National Institute on Aging, NIH), and the Mayo Foundation.

Role of the Funder/Sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

References

1.Centers for Disease Control and Prevention. Cases of cerebral venous sinus thrombosis with thrombocytopenia after receipt of the Johnson & Johnson COVID-19 vaccine New release. April 13, 2021. Accessed April 21, 2021.  https://emergency.cdc.gov/han/2021/han00442.asp

2.See  I, Su  JR, Lale  A,  et al.  US case reports of cerebral venous sinus thrombosis with thrombocytopenia after Ad26.COV2.S vaccination, March 2 to April 21, 2021.   JAMA. 2021;325(24):2448-2456. doi:10.1001/jama.2021.7517
ArticlePubMedGoogle ScholarCrossref

3.Shay  DK, Gee  J, Su  JR,  et al.  Safety monitoring of the Janssen (Johnson & Johnson) COVID-19 vaccine—United States, March-April 2021.   MMWR Morb Mortal Wkly Rep. 2021;70(18):680-684. doi:10.15585/mmwr.mm7018e2PubMedGoogle ScholarCrossref

4.Shimabukuro  T. Update: thrombosis with thrombocytopenia syndrome (TTS) following COVID-19 vaccination. Paper presented at: Advisory Committee on Immunization Practices; May 12, 2021.

5.Devasagayam  S, Wyatt  B, Leyden  J, Kleinig  T.  Cerebral venous sinus thrombosis incidence is higher than previously thought: a retrospective population-based study.   Stroke. 2016;47(9):2180-2182. doi:10.1161/STROKEAHA.116.013617PubMedGoogle ScholarCrossref

6.Sadoff  J, Gray  G, Vandebosch  A,  et al; ENSEMBLE Study Group.  Safety and efficacy of single-dose Ad26.COV2.S vaccine against COVID-19.   N Engl J Med. 2021;384(23):2187-2201. doi:10.1056/NEJMoa2101544PubMedGoogle ScholarCrossref

COVID‐19‐induced endotheliitis: emerging evidence and possible therapeutic strategies

Authors: Eleonora Calabretta, 1 , 12 Jose M. Moraleda, 2 Massimo Iacobelli, 3 Ruben Jara, 4 Israel Vlodavsky, 5 Peter O’Gorman, 6 Antonio Pagliuca, 7 Clifton Mo, 8 Rebecca M. Baron, 9 Alessio Aghemo, 10 , 12 Robert Soiffer, 8 Jawed Fareed, 11 Carmelo Carlo‐Stella, 1 , 12 , * and Paul Richardson 8 , *

Br J Haematol. 2021 Apr; 193(1): 43–51.Published online 2021 Feb 4.  doi:  10.1111/bjh.17240 PMCID:  PMC8014053PMID: 33538335

Introduction

The coronavirus disease 2019 (COVID‐19) pandemic, a viral illness caused by the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), 1 has produced at the time of this writing nearly 33 million cases of infection, with over a million deaths in 235 countries, 2 causing an unprecedented burden on healthcare systems and a severe global socioeconomic crisis. As the pandemic spreads, knowledge on the disease course, as well as potential risk factors and predictors of severity is increasing daily, and initial data from randomised controlled studies have allowed care providers to refine therapeutic strategies. Nonetheless, mortality is markedly elevated among those presenting with severe disease, long‐term sequelae among survivors are unknown, and vaccine‐based therapies currently remain at early stages of development.

Most reported cases are asymptomatic or present with mild symptoms; however, 7–26% of hospitalised patients experience severe disease, often requiring admission to intensive care units (ICUs), with progressive multiple organ dysfunction and high mortality. 3 Such differences in clinical outcomes have led physicians to initiate diverse pharmacological therapies at various stages of the disease, generating challenges as to the most appropriate therapeutic choice for COVID‐19. In this context, the use of dexamethasone has significantly reduced mortality rates in critically ill patients requiring supplemental oxygen or mechanical ventilation, 6 and remdesivir has demonstrated clinical benefit in hospitalised patients, but with unknown survival benefit to date 7 ; additional effective treatment options are therefore urgently needed.

In an initial attempt to provide a uniform and widely reproducible methodology to guide systematic treatment strategies, a three‐stage classification of COVID‐19 has been proposed. 8 The Stage I or ‘early infection’ occurs at the initial establishment of disease with high viral replication, and commonly presents with a range of complaints that can include mild and often non‐specific influenza‐like signs and symptoms. Stage II is the ‘pulmonary phase’, with preferential viral‐mediated injury of the lung parenchyma and this is characterised by shortness of breath, hypoxia and pulmonary infiltrates with some degree of lung inflammation. Stage III is characterised by an exaggerated host immune‐inflammatory response to the virus, leading to acute respiratory distress syndrome (ARDS) and multi‐organ failure (MOF).Go to:

Endothelial cells are a preferential target of COVID‐19 resulting in widespread endotheliitis

Emerging evidence suggests that endothelial damage and subsequent morphological and functional changes in the endothelium play important roles in COVID‐19‐induced hyperinflammation. The virus, which binds to the angiotensin‐converting enzyme 2 (ACE2) receptor, 9 displays a profound tropism for human lung and small intestine epithelium, as well as the vascular endothelium. 10 In an important case series from Varga et al., 11 postmortem histology from three patients affected by late‐stage COVID‐19, revealed viral inclusions in endothelial apoptotic cells and microvascular lymphocytic endotheliitis, with infiltration of inflammatory cells around the vessels and endothelial cells (ECs), as well as evidence of endothelial apoptotic cell death in the lung, kidney, small bowel and heart. Additionally, autopsy findings of 27 patients in another series confirmed the detection of the SARS‐CoV‐2 in multiple organs, including the respiratory tract, pharynx, heart, liver, brain, and kidneys. 12 Immunofluorescence of kidney specimens from six of the 27 patients showed the presence of SARS‐CoV‐2 protein in all renal compartments, and in three of the patients preferentially in the endothelium of the glomerulus. Similar microscopic findings were also noted in lung specimens from seven patients with COVID‐19, which displayed small vessel endotheliitis, microvascular thrombosis and angiogenesis, along with the presence of SARS‐CoV‐2 in pulmonary ECs, an observation strongly supporting the vascular tropism of the virus. 13 Lastly, Stahl et al. 14 have identified in the plasma and serum of 19 critically ill patients with COVID‐19 evidence of disruption of the endothelial glycocalyx, reflected by increased levels of the Tie‐2 receptor and syndecan‐1 (SDC‐1), a heparan sulphate (HS) proteoglycan. This particular observation is of interest as the endothelial glycocalyx covers the luminal surface of ECs, and its integrity is vital for the maintenance of vascular homeostasis.

Such findings suggest that virus‐mediated apoptosis may promote endothelial barrier disruption with interstitial oedema and increased recruitment of circulating activated immune cells, thus causing widespread endothelial dysfunction, as well as activation of platelets and the coagulation cascade leading to venous and arterial thrombosis. 15 Altered pro‐inflammatory and pro‐thrombotic status is confirmed by the presence of elevated inflammation‐related indices (e.g. C‐reactive protein and serum ferritin), humoral biomarkers [interleukin (IL)‐2, IL‐6, IL‐7, granulocyte‐colony stimulating factor (G‐CSF), tumour necrosis factor‐alpha (TNF‐α)], and indicators of an increased pro‐coagulant‐fibrinolytic state [e.g. von Willebrand factor (VWF), D‐dimer, fibrinogen]. Further, factor VIII (FVIII), a potent and key factor in the coagulation process, is greatly increased in ICU patients with COVID‐19. 16

In this setting, it has been proposed that the systemic hyperinflammation observed in severe COVID‐19 is comparable to a cytokine release syndrome (CRS), or cytokine storm. IL‐6 has a central role in the generation of the cytokine storm, and is commonly elevated in the serum of severely ill patients with COVID‐19. 17 High levels of IL‐6 activate ECs, thus resulting in vascular leakage, further cytokine secretion and activation of the complement and coagulation cascades. 18 Interestingly, a population of IL‐6 producing monocytes was found to be expanded in the peripheral blood of ICU patients 19 and an aberrant macrophage response exhibiting increased levels of pro‐inflammatory cytokines has been detected in bronchoalveolar fluid, especially in severely ill patients. 20 Although the exact driver of monocyte activation remains unclear, such cells are attracted to the endothelium, where the release of highly noxious molecules, such as reactive oxygen species (ROS), contributes to endothelial dysfunction and promotes hyperinflammation. 21 Further, activated monocytes enhance tissue factor expression and form aggregates with platelets through P‐selectin interaction and hence augmenting the pro‐coagulant response. 22 Indeed, significantly increased levels of VWF and FVIII and thrombomodulin, 16 aberrant coagulation, thrombosis and microangiopathy are very common in critically ill patients with COVID‐19, resulting in a disseminated intravascular coagulation (DIC)‐like syndrome characterised by massive fibrin formation and organ dysfunction. 23 Likewise, adaptive immunity actively participates in the establishment of the inflammatory response; specifically, activated and proliferating CD8+ T cells are prevalent in mild COVID‐19, whereas critically ill patients display higher levels of hyperactive IL‐6‐producing CD4+ T cells, which may contribute to disease severity, even after viral clearance. 17 Interestingly, T cells show phenotypical signs of an exhausted, functionally unresponsive state, thus allowing viral escape from immune surveillance. 22 Once initiated, the endotheliitis and resultant cytokine storm become self‐sustaining, leading to widespread organ damage. Some patients may also display features of haemophagocytic lymphohistiocytosis, such as cytopenias, hyperferritinaemia and rapid onset of MOF.

Overall, once hyperinflammation and CRS develop, rates of mortality significantly increase. 24 25 26 As direct viral activation of the vascular endothelium has an important role in initiating and maintaining the hyperinflammatory response, attempting to blunt such a response with endothelial‐protective agents is a very rational strategy. Controlled clinical trials focussing on the use of anti‐cytokine antibodies, including tocilizumab (IL‐6 inhibitor), have failed to show significant activity in this stage of the disease. 27 However, increasing evidence suggests that the altered homeostasis of the endothelium may be a key initiating event in the pathogenesis of the disease, therefore representing a potentially more promising target. 28Go to:

Endothelial cell‐related disorders in haematology: post‐bone marrow transplantation syndromes and sickle cell disease and the overlap with the pathobiology of COVID‐19

Clinically and histopathologically, COVID‐19‐associated endotheliitis resembles a spectrum of post‐bone marrow and stem cell transplantation (BMT) syndromes characterised by disruption of endothelial homeostasis and consequently dysregulation of coagulation, vascular tone, endothelial permeability and vascular inflammation. 29 These disorders include hepatic veno‐occlusive disease (VOD)/sinusoidal obstruction syndrome (SOS), idiopathic pneumonia syndrome (IPS), transplant‐associated thrombotic microangiopathy and graft‐versus‐host disease (GvHD).

Hepatic VOD/SOS develops as a result of endothelial damage to hepatic sinusoids and subsequent hepatocyte necrosis 30 . Damage to the ECs leads to a hypercoagulable state, production of inflammatory mediators, and the upregulation as well as release of heparanase. 31 Heparanase degrades the heparan sulphate scaffold of the subendothelial basement membrane, consequently allowing the extravasation of blood‐borne cells, including activated T lymphocytes, neutrophils and macrophages. 32 This cascade of events leads to postsinusoidal hypertension, hyperinflammation and ultimately MOF. Severe VOD/SOS associated with MOF without effective therapy is fatal in >80% of cases. 33 Interestingly, the histopathological examination of lung lesions in VOD/SOS shows early alveolar epithelial and lung endothelial injury, resulting in accumulation of protein‐ and fibrin‐rich inflammatory oedematous fluid in the alveolar space and progression to interstitial fibrosis, 34 35 as is also seen in fatal COVID‐19 cases.

Similarly, IPS, a widespread alveolar injury in the absence of identifiable infectious or non‐infectious causes, is characterised by histological evidence of EC injury with fibrin accumulation, luminal thrombosis and fibrotic processes. Adhesion molecules, such as intercellular adhesion molecule 1 (ICAM‐1) and/or vascular cell adhesion molecule 1 (VCAM‐1), are commonly upregulated, thus reflecting profound endothelial activation. 36 It has been suggested that TNF‐α directly causes endothelial injury, and increased levels of angiopoietin‐2 (Ang‐2), have been recently reported in cases of acute exacerbations of IPS, 37 similar to that seen in severely ill patients with COVID‐19. 38

Likewise, multifactorial endothelial damage has been also implicated in the development of transplant‐associated thrombotic microangiopathy, where micro‐vessel intimal swelling and necrosis lead to the formation of luminal microthrombi and subsequent microangiopathic haemolytic anaemia. Plasma levels of markers of EC injury and inflammation, such as thrombomodulin, plasminogen activator inhibitor‐1 (PAI‐1), ICAM‐1, VCAM‐1, IL‐1, TNF‐α, interferon gamma and IL‐8 are commonly elevated. 39 40 Endothelial dysfunction predominantly affects the kidneys and the brain, but may become widespread and progress to MOF, which in turn is associated with high mortality.

Lastly, acute GvHD (aGvHD) develops as a consequence of the activation of the immune system. Antigen‐presenting cells become activated by endothelial and tissue damage derived from direct toxicity of the conditioning regimen, thus initiating an alloreactive T‐cell response directed against recipient tissues. 41 As a result, SDC‐1 is commonly elevated in the serum of patients with GvHD and correlates with disease severity. 42 In addition to cell‐mediated cytotoxic damage, the cytokine storm generated in response to T‐cell activation and proliferation causes targeted organ damage involving mainly the skin, liver and gut. 43 It has recently been suggested that endothelial vulnerability and pro‐thrombotic shift precedes clinically evident aGvHD and that angiogenesis driven by early endothelial activation is an initiating event. 44 Indeed, increased plasma levels of VWF, 45 Ang‐2 46 and TNF receptor 1 47 have been detected in patients prior to development of aGvHD, correlating with response to therapy.

Similarly, markers of endothelial dysfunction and inflammatory activation have been detected also in the serum of patients with sickle cell disease (SCD), especially during vaso‐occlusive episodes. SCD is characterised by a chronic course of relapsing‐remitting episodes of ischaemia and then reperfusion. The polymerisation of defective haemoglobin S upon deoxygenation initiates many pathological processes, such as complement activation, generation of ROS and pro‐thrombotic molecules, secretion of numerous pro‐inflammatory cytokines and chemokines and ultimately leucocyte recruitment. 48 Oxidative stress and endothelial dysregulation plays a key role in vaso‐occlusion; ECs activated by substances released by the haemolytic process and by red blood cell adhesion initiate production and release of soluble mediators such as IL‐1β, IL‐8, IL‐6, IL‐1α and PAI‐1, 49 50 and increase the expression of adhesion molecules such as VCAM‐1, ICAM‐1, E‐selectin and P‐selectin, 51 52 reflecting a pro‐inflammatory and pro‐thrombotic shift. Vaso‐occlusive phenomena commonly affect the lung vasculature, provoking acute chest syndrome (ACS), a spectrum of diseases ranging from mild pneumonia to ARDS and MOF, which is the leading cause of morbidity and mortality in SCD. 53 Lung specimens from ACS cases showed micro‐thrombotic occlusion, endothelial VWF deposition and arterial vessel re‐modelling with initial fibrotic processes, 54 fascinatingly all comparable to the histopathological findings in COVID‐19. 55 Importantly, heme‐mediated endothelial damage to alveolar cells is regulated by the p38 mitogen‐activated protein kinase (MAPK) pathway, which plays a crucial role in the biosynthesis of pro‐inflammatory cytokines and collagen production. 56 This key pathway is also upregulated in COVID‐19 as a result of decreased ACE2 tissue functionality consequent to viral binding, and may consequently promote endotheliitis, hypercoagulation and end‐stage fibrosis. 57 58

In summary, post‐BMT syndromes, vaso‐occlusive organ dysfunction in SCD and COVID‐19‐associated endotheliitis share common pathological mechanisms including: i) dysregulation of the homeostasis of the endothelial milieu toward a pro‐inflammatory and pro‐thrombotic phenotype with thrombotic microangiopathy; ii) hyperproduction of inflammatory cytokines such as IL‐6, IL‐8 and TNF‐α; 59 60 61 62 and iii) small vessel endotheliitis and endothelial barrier dysfunction, leading to oedema of the microvascular bed, protein and fibrin accumulation and subsequent fibrotic shift. 34 36 All these conditions if untreated irremediably lead to MOF and display similar microscopic and macroscopic features in target organs upon pathological examination. At the molecular level, the p38 MAPK pathway may also be critical in promoting vasoconstrictive and inflammatory phenomena; its activation is described in SCD, COVID‐19 and also as a result of conditioning regimen‐induced endothelial damage in BMT. 63 Together, these findings support the notion that the pleiotropic character of the endothelium as a key regulator of the internal homeostasis, vascular tone, blood coagulation and the inflammatory process and therefore of so called ‘immune‐thrombosis’ events, make it an intriguing therapeutic target for post‐BMT disorders, SCD, and COVID‐19.Go to:

Agents targeting EC‐related disorders

Heparins

Classically, heparins have been the most widely used drugs for the treatment and prevention of endothelial cell disorders. Several animal studies and clinical trials have suggested that, in addition to its well‐known anticoagulant effects, heparin also possesses anti‐inflammatory properties, mainly mediated by inhibition of IL‐6 release and its activity, 64 a phenomena also demonstrated in patients with COVID‐19 treated with low‐molecular‐weight heparin. 65 Further, heparin is structurally related to HS, 66 a negatively charged glycosaminoglycan as described earlier, which serves as binding sites for growth factors, cytokines, selectins, extracellular‐matrix molecules, and a large number of human viruses, 67 including the SARS‐CoV‐2 virus. 68 69 Indeed, Clausen et al. 70 have recently demonstrated that the SARS‐CoV‐2 spike protein must bind both the ACE2 receptor and HS to enter human cells. The structural analogies between heparin and HS may result in competitive inhibition, where heparin and related compounds compete with the cell surface HS for viral binding to target cells, 68 69 thus potentially blocking or at least attenuating viral entry. The beneficial effects of heparin‐based therapies are also linked to their inhibition of circulating heparanase enzymatic activity. 32 Heparanase, an endo‐β‐glucuronidase, physiologically cleaves HS chains located in extracellular matrices and on cell surfaces. 32 It is often overexpressed during viral infections and act as a regulator of virus release after replication has occurred, promoting its dissemination. 71 72 73 74 Additionally, it may be upregulated by pro‐inflammatory molecules such as IL‐1 and TNF‐α. Once activated, heparanase stimulates the expression and release of pro‐inflammatory cytokines, including TNF‐α, IL‐1 and IL‐6. 75 The enzyme has been implicated in cancer progression, inflammation, 76 VOD/SOS development 77 and other vascular pathologies. 71

Currently, numerous clinical trials are underway to investigate the therapeutic potential of intravenous and subcutaneous heparin, as well as the appropriate dose regimen in COVID‐19. Further, nebulised heparin delivered directly to the airways may be effective in preventing infection and mitigating lung disease (clinicaltrials.gov; NCT04545541NCT04511923). Notwithstanding their anticoagulant, anti‐inflammatory and anti‐viral properties, the use of heparins is associated with a substantially increased risk of systemic bleeding, and other challenging ‘off‐target’ effects, making its use potentially part of the standard of care, but not without qualification, as well as highlighting the need for combination approaches.

Defibrotide

The use of defibrotide (DF), which has both comparable but distinct properties from heparins and negligible haemorrhagic risk 78 may therefore be warranted, especially given the established propensity for the development of DIC later in the COVID‐19 clinical course. DF is a naturally derived, complex mixture of poly‐deoxyribonuleotides extracted originally from bovine lung and now exclusively from porcine gut mucosa. 79 80 Since its original isolation >30 years ago, DF has demonstrated locally acting pro‐fibrinolytic, 81 82 83 84 anti‐thrombotic, 85 86 anti‐ischaemic and anti‐inflammatory activities, which exert protective effects on small vessel endothelia. It is currently approved for the treatment of paediatric and adult hepatic VOD/SOS with MOF. 87 88 89 In this setting, DF has demonstrated efficacy and safety in critically ill patients with MOF, as well as a significant reduction in PAI‐1 and other markers of endothelial stress in patients with VOD/SOS and MOF successfully treated with DF. 89 90 91 Furthermore, in a pivotal Phase III trial, DF prophylaxis reduced the incidence and severity of VOD/SOS in high‐risk children undergoing BMT. 92 In a more recent study, Palomo et al. 93 demonstrated that DF directly interacts with the cell membrane and becomes internalised by ECs, thus providing physical evidence of its endothelial‐protective properties. In particular, DF appears to decrease levels of pro‐inflammatory proteins, such as TNF‐α, 94 IL‐6, vascular endothelial growth factor (VEGF) 95 and to downregulate major histocompatibility complex (MHC) Class I and Class II molecules, 96 97 therefore attenuating both the inflammatory and immune responses. Furthermore, it appears to decrease interaction between leucocytes and ECs by downregulating P‐selectin, 98 ICAM‐1 95 and VCAM‐1. 99 Lastly, DF displays potent adenosine agonism. 100 Such activity may be clinically relevant, not least based on substantial improvement observed in an animal model of acute lung injury upon treatment with adenosine receptor agonists. 101

Based on such properties, the use of DF can be reasonably extended to other post‐BMT syndromes and other microangiopathies involving CRS complicating a variety of disease states and treatment modalities, such as chimeric antigen receptor (CAR) T‐cell therapy. 102 Indeed, paediatric and adult patients receiving DF as VOD/SOS prophylaxis also exhibited a reduced incidence of aGvHD, 92 103 a finding that is strongly supported by a preclinical model of aGvHD. 99 Additionally, a retrospective survey from paediatric patients treated with DF for transplant‐associated thrombotic microangiopathy showed resolution of clinical disease in 77% of patients. 104 Currently, Phase II studies investigating the use of DF for prevention of transplant‐associated thrombotic microangiopathy and VOD, and in the same context the treatment of ACS are ongoing (clinicaltrials.gov; NCT03384693NCT03805581NCT02675959). Notably, DF suppresses the expression of heparanase transcripts, cell surface expression and enzymatic activity, 95 suggesting that DF may have anti‐viral properties, although this remains to be confirmed. 71 72 73 Heparanase is putatively upregulated by the cytokine storm of advanced COVID‐19 and may contribute to further inflammation, oedema of the microvascular bed and coagulopathy. 28 75 105 106 DF is a potent inhibitor of heparanase in terms of both cell surface and gene expression, and therefore is especially attractive. Furthermore, the therapeutic use of DF in a murine model of IPS significantly improved survival compared to untreated controls by reducing, among other biomarkers, the levels of Ang‐2, 107 which is known to correlate with ARDS and is markedly elevated in critically ill patients with COVID‐19. 38

In addition, ICU‐admitted patients with COVID‐19 may display increased platelet activation and subsequent formation of platelet‐monocyte aggregates upon interaction with P‐selectin, thus stimulating monocyte‐induced inflammation and thrombosis. 22 By reducing P‐selectin and other adhesion molecules expression, DF may inhibit monocyte‐derived inflammatory and pro‐coagulant signals. Lastly and most importantly, DF has also been shown to decrease the activity of p38 MAPK and its pathway, 93 the importance of which is increasingly recognised in the pathogenesis of the COVID‐19 hyperinflammation syndrome and this may be a key therapeutic target in this process. 58 108

In summary, the multitargeted endothelial‐based therapeutic properties of DF and its relative safety, as well as its regulatory approval, make it an ideal potential therapeutic candidate for the treatment of COVID‐19 vascular complications. 28 In contrast to heparin, DF also exhibits broader anti‐cytokine, anti‐inflammatory and endothelial‐stabilising properties. Importantly, by acting on the heparanase‐HS axis, 74 102 DF may limit viral infectivity given its capacity to i) compete with HS and thereby possibly inhibit virus–cell adhesion and entry, ii) inhibit heparanase enzymatic activity and thereby attenuate virus detachment/release and spread 74 and iii) inhibit heparanase‐mediated activation of immune cells and thereby upregulation of pro‐inflammatory cytokines and the associated self‐sustaining systemic inflammatory host response (Fig 1). Actively accruing, international Phase II clinical trials are now underway and should shed critical light on DF’s therapeutic potential in patients with COVID‐19 (examples include clinicaltrials.gov; NCT04348383NCT04335201). Strikingly, two critically ill paediatric patients treated with DF for a SARS‐CoV‐2‐associated multisystem inflammatory syndrome experienced complete resolution and no attributable toxicity, with correlative studies supporting the mechanistic effects described above, as well as favourable effects seen on complement activation. 109 Similarly, preliminary results from the current studies as part of the international DEFACOVID (Defibrotide as Prevention and Treatment of Respiratory Distress and Cytokine Release Syndrome of COVID‐19) study group support both safety and promising potential efficacy to date.Fig 1

Potential mechanisms of action of defibrotide in the treatment of COVID‐19. Left, defibrotide limits viral attachment by interfering with Syndecan‐1, the primary cell surface heparan sulfate on ECs, and reduces viral dissemination, by inhibiting HPSE‐mediated viral release. Right, effects of defibrotide on endothelial‐mediated pathological processes. Viral infection of ECs promotes apoptosis with breakdown of endothelial barrier and exposure of the subendothelium, with subsequent platelet activation and thrombotic phenomena. Defibrotide inhibits platelet activation and leukocyte recruitment and blocks the generation of the cytokine storm; specifically, HPSE‐mediated activation of immune cells is suppressed, thus limiting the development of cytokine release syndrome. Sars‐Cov‐2, severe acute respiratory syndrome coronavirus‐2; ACE2, angiotensin‐converting enzyme 2; Ang‐2, angiopoietin‐2; GI, gastrointestinal; IL, interleukin; NFKB, nuclear factor kappa‐light chain‐enhancer of activated B cells; TNF‐α, tumor necrosis factor‐alpha; VWF, von Willebrand Factor.

Other heparanase inhibitors

Given the heparanase‐inhibiting activity of heparin, effort has been directed towards modifications of its structure to endow candidate molecules with potentiated anti‐heparanase activity while limiting anticoagulant effects. Specifically, N‐acetylated and glycol‐split heparins are promising agents presenting such characteristics. Indeed, administration of N‐acetylheparin (NAH) in murine models of sepsis ameliorated lung and intestinal injury and subsequent oedema by reducing tissue neutrophilic infiltration and suppressing IL‐6, IL‐1β and TNF‐α production. 110 111 Furthermore, roneparstat, the most developed glycol‐split NAH, restored pathological renal cellular damage caused by ischaemia‐reperfusion by reducing release of pro‐inflammatory cytokines and reverted established fibrotic processes, thus restoring normal tissue histology in preclinical models. 112 This aspect is especially relevant, considering the extensive formation of fibrosis and irreversible end‐organ damage in post‐BMT syndromes, SCD and advanced COVID‐19.

Additionally, much interest has been directed towards the novel heparanase‐inhibiting agent pixatimod, a modified oligosaccharide glycoside with heparan sulphate‐mimetic properties. Pixatimod is a potent inhibitor of Type 1 T‐helper cells (Th1)/Th17 effector functions, 113 IL‐6 expression, 114 M2 macrophage activation, 115 angiogenesis and tumour progression in vivo. Furthermore, it exhibits mild anticoagulant activity and despite transient infusion reactions is otherwise generally well tolerated. Guimond et al. 116 have recently demonstrated that pixatimod interacts with the SARS‐CoV‐2 spike protein binding site, and this is coherent with its heparan sulphate‐mimetic activity. Moreover, pixatimod was found to markedly inhibit SARS‐CoV‐2 infectivity, 116 supporting its clinical application as a novel therapeutic intervention for prophylaxis and treatment of COVID‐19. Taken together, heparanase emerges as a host‐encoded virulence factor that once activated enhances viral spread and triggers downstream inflammatory cascades. These preliminary data indicate that heparanase inhibitors currently under development are possible candidates for multisystem inflammatory conditions, such as COVID‐19, sepsis, thrombotic microangiopathies and cancer, but as of now studies remain preclinical with clinical application pending.

Conclusions

In conclusion, increasing evidence suggests that the SARS‐CoV‐2 directly targets ECs, promoting the release of pro‐inflammatory and pro‐thrombotic molecules. Endothelial dysfunction appears to be a crucial initiating step in the pathogenesis of the disease and its ensuing morbidity and mortality. Endotheliitis with the hyperproduction of cytokines leading to CRS, hypercoagulability and thrombotic microangiopathy are hallmarks shared by COVID‐19, VOD/SOS and other endothelial injury syndromes, underpinned by inflammation and including the vaso‐occlusive crises of SCD, so providing a common pathobiology across these respective syndromes. Most importantly, endothelial‐protective agents, such as DF, represent a promising and rational therapeutic strategy in COVID‐19, with DF currently under investigation in a variety of settings and combinations. As a unifying concept, heparanase inhibition, with the modulation of related pathways and other effects on endothelial stress responses may thus be crucial in mediating anti‐viral and anti‐inflammatory activity. In particular, as this relates to endotheliitis, it may directly abrogate CRS and its sequelae, which in turn may lead to improved patient outcome.

Author Contributions

Eleonora Calabretta, Jose M. Moraleda, Israel Vlodavsky, Ruben Jara, Carmelo Carlo‐Stella and Paul Richardson drafted the manuscript; all authors participated in the critical revision and approval of the final report.Go to:

Conflict of interest

Jose M. Moraleda declares Advisory Board fees from Jazz Pharmaceuticals; Antonio Pagliuca has received Advisory Board and Speaker fees from Jazz Pharmaceuticals; Rebecca M. Baron is on a Merck Advisory Board and a Consultant for Genentech. Robert Soiffer serves on the Board of Directors for Kiadis and Be The Match/National Marrow Donor Program; provided consulting for Gilead, Rheos Therapeutics, Cugene, Precision Bioscience, Mana Therapeutics, VOR Biopharma, and Novartis; and Data Safety Monitoring Board for Juno/Celgene; Paul Richardson is an Advisory Committee Member for Jazz Pharmaceuticals; Carmelo Carlo‐Stella is a Consultant/Advisory Board Member for Genente Science srl, ADC Therapeutics, Novartis, Roche, Karyopharm, Sanofi, Boehringer Igelheim and Servier. The remaining authors declare nothing to disclose.

Acknowledgments

This work was supported in part by a grant from the Italian Association for Cancer Research (AIRC, grant #20575 to CC‐S).Go to:

Contributor Information

Carmelo Carlo‐Stella, Email: ue.deminuh@alletsolrac.olemrac.

Paul Richardson, Email: ude.dravrah.icfd@nosdrahcir_luap.Go to:

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The coagulopathy, endotheliopathy, and vasculitis of COVID-19

Authors: Toshiaki Iba 1Jean Marie Connors 2Jerrold H Levy 3

Abstract

Background

COVID-19-associated coagulopathy (CAC) characterized by the elevated D-dimer without remarkable changes of other global coagulation markers is associated with various thrombotic complications and disease severity. The purpose of this review is to elucidate the pathophysiology of this unique coagulopathy.

Methods

The authors performed online search of published medical literature through PubMed using the MeSH (Medical Subject Headings) term “COVID-19,” “SARS-CoV-2,” “coronavirus,” “coagulopathy,” and “thrombus.” Then, selected 51 articles that closely relevant to coagulopathy in COVID-19.

Results

The primary targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the pneumocytes, immune cells, and vascular endothelial cells. The alveolar damage and the pulmonary microvascular thrombosis are the major causes of acute lung injury in COVID-19. The endotheliopathy that occurs is due to direct SARS-CoV-2 infection and activation of other pathways that include the immune system and thromboinflammatory responses leading to what is termed CAC. As a result, both microvascular and macrovascular thrombotic events occur in arterial, capillary, venule, and large vein vascular beds to produce multiorgan dysfunction and thrombotic complications. In addition to the endothelial damage, SARS-CoV-2 also can cause vasculitis and presents as a systemic inflammatory vascular disease. Clinical management of COVID-19 includes anticoagulation but novel therapies for endotheliopathy, hypercoagulability, and vasculitis are needed.

Conclusion

The endotheliopathy due to direct endothelial infection with SARS-COV-2 and the indirect damage caused by inflammation play the predominant role in the development of CAC. The intensive control of thromboinflammation is necessary to improve the outcome of this highly detrimental contagious disease.

Introduction

Ongoing reports have described the hypercoagulability and thrombotic tendency in COVID-19 [1]. The high incidence of deep vein thrombosis and pulmonary embolism has focused on the critical role of routine antithrombotic prophylaxis for COVID-19 management, especially in critically ill patients and/or elevated D-dimer levels [2,3,4]. Current reports of venous and arterial thrombotic events in the patients treated in ICU is up to 30% even with pharmacological thromboprophylaxis, and thrombotic events are associated with 5.4 times higher risk of mortality [5]. Recent postmortem evaluation of COVID-19 patients has demonstrated severe endothelial injury with cellular death/apoptosis, and the presence of intracellular virus in the autopsy lung with thrombosis and small to middle-size pulmonary vessels. The clotting and vascular damage were also confirmed in the alveolar capillary and these changes are more remarkable in COVID-19 compared to influenza induced lung injury [6]. In this summary, we will review the pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endotheliopathy, COVID-19-associated coagulopathy (CAC), and vasculitis.

Coagulopathy in COVID-19

The mechanism of coagulopathy in COVID-19

Thromboembolic complications are the hallmark of COVID-19 that can cause death even in asymptomatic COVID-19 [7]. The new coronavirus SARS-CoV-2 elicits an acute inflammatory effect with hypercoagulability, platelet activation, and endothelial dysfunction [8]. Although, this presentation has similarities with sepsis-induced coagulopathy (SIC) due to bacterial infections and disseminated intravascular coagulation (DIC), there are several important differences [8]. In CAC, patients often initially present with increased fibrinogen levels, increased D-dimers, but minor changes in prothrombin time and platelet count compared to acute bacterial sepsis that can produce thrombocytopenia, prolonged prothrombin times, and decreased antithrombin levels [49]. It is also known that inflammatory cytokine levels are elevated in COVID-19 and excess production of inflammatory cytokines can induce hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) that can result in a thrombotic coagulation disorder [10]. Although the pathophysiology of HLH/MAS seems similar to COVID-19, the reported cytokine level is much lower in COVID-19 [11]. Conversely, the endothelial derangement, detailed in the next section, is predominant in COVID-19. Other than HLH/MAS, various thrombotic diseases such as thrombotic microangiopathy, and antiphospholipid syndrome can occur, and the characteristics of these diseases look similar to CAC [12]. Even though the pathogeneses of these thrombotic diseases partially overlap with CAC, it is important to delineate the unique statue of CAC to plan a therapeutic strategy.

The evaluation of COVID-19-associated coagulopathy

D-Dimer monitoring is important in COVID-19 coagulopathy. Although D-dimer is initially elevated, other conventional coagulation laboratory tests including prothrombin time (PT), activated partial thromboplastin time (aPTT), and platelet count are often normal, and are not useful indicators of the thrombotic risk. The increase of factor VIII and von Willebrand factor (VWF) [13], potentially the presence of antiphospholipid antibodies [14], and increased activity of complement system are also reported, however, monitoring these biomarkers is not practical. The pathogenesis of coagulopathy in COVID-19 is complex but the typical CAC can be diagnosed by increased D-dimer, elevated fibrinogen and VWF levels, but relatively normal PT, aPTT, and platelet count. A pathway for diagnosing CAC versus other coagulopathies is illustrated in Fig. 1.

figure 1
Fig. 1

Thrombin generation testing (TGT) measures ex vivo thrombin formation in plasma upon activation with tissue factor. TGT allows calculation of peak and total thrombin generation, as well as time to initial and peak thrombin generation. This assay can identify both reduced and increased thrombin generation. Nougier et al. [15] revealed increased thrombin generation in COVID-19 patients despite undergone anticoagulation. The major drawbacks of TGT are the lack of standardization and the requirement of technical training.

Other potential assays to assess global coagulation status include viscoelastic testing, especially in ICU patients, as a point of care test. An increasing number of studies report hypercoagulability as indicated by decreased R or clot times, and increased maximal amplitude/maximal clot firmness by viscoelastic monitoring [16,17,18]. However, these changes are consistent with high fibrinogen levels that affect both maximal amplitude on thromboelastography (TEG), and maximum clot firmness on rotational thromboelastometry (ROTEM). Ranucci et al. [17] reported the median fibrinogen level was nearly 800 mg/dL in the COVID-19 patients treated in ICU, and such a high fibrinogen level affects TEG and ROTEM parameters considerably by itself [19].

Endotheliopathy in COVID-19

The endothelial damage and thrombosis

An important feature of CAC is the microcirculatory endothelial damage in pulmonary circulation and other vascular beds. Since SARS-CoV-2 directly infects the vascular endothelial cell causing cellular damage and apoptosis, the antithrombotic activity of the luminal surface is remarkably decreased [20]. In COVID-19, both alveolar damage and microcirculatory disturbance associated with thrombus formation contribute to respiratory dysfunction. At autopsy, findings reported include clot formation in pulmonary arterioles with diffuse alveolar damage and hyaline membranes [21]. Normal endothelial function refers to the ability of regulating vascular tonus, permeability, cell adhesion, and anticoagulation. Healthy endothelial cells synthesize nitric oxide (NO) by conversion of L-arginine to L-citrulline by nitric oxide synthase. NO released by endothelium prevents leukocyte and platelet adhesion, inflammatory cell migration into the vessel wall, smooth muscle cell proliferation, and suppresses apoptosis and inflammation. SARS-Cov-2 enters endothelial cells through endocytosis and is mediated by an interplay of Angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease serine 2 (TMPRSS-2) which sheds a part of spike protein and helps SARS-Cov-2 to enter into endothelial cell. The infected endothelial cells lose their ability to maintain aforementioned physiological functions. Subsequently, the damage of the endothelium leads to the procoagulant change of the vascular lumen, formation of immunothrombosis, and organ malcirculation.

Both systemic pulmonary microthrombosis and thromboembolism are commonly seen in COVID-19. This typical figure is thought to be the result of hypercoagulability due to the dysregulated endothelial function of the pulmonary vessels and systemic inflammation. In addition to the deep vein thrombus that results in an embolic event, in situ formation in the pulmonary arteries can be the main reason of pulmonary dysfunction. Lax et al. [22] performed autopsies in 11 patients and reported that despite the absence of clinical presentations of thromboembolism, thrombus formation in small and mid-sized pulmonary arteries was found in all of the examined cases. In these cases, thrombus is suspected to form at the peripheral arteriole and elongate proximally. According to another series of autopsy findings, the incidence of thrombus formation in the pulmonary microvasculature is approximately nine times higher than that seen in influenza [6].

The endothelial damage-derived hypercoagulability

ACE2, the host cellular receptor of SARS-CoV-2, has been identified on the vascular endothelial surface. SARS-CoV-2 uses ACE2 to invade into the cell through the fusion of its membrane to the host cell membrane. As a result, the host cell loses ACE2 activity which subsequently leads to reduced angiotensin II inactivation and decreased conversion to antiotensin1-7. Increased angiotensin II stimulates vascular constriction and decreased antiotensin1-7 suppresses nitric oxide production which triggers increased thrombogenicity due to leucocyte and platelet adhesion and vasoconstriction [23].

The vascular endothelium is coated by a gel-like component known as the glycocalyx, that regulates vascular blood flow by providing an antithrombotic surface via antithrombin binding to the heparan sulfate constituents, a major component of the glycocalyx. Although the circulating antithrombin level has been reported to be in a normal range on presentation in COVID-19 cases [13], if the glycocalyx is disrupted, the local antithrombogenicity of the endothelial surface may be altered. However, little information on the glycocalyx status in COVID-19 is available.

One of the unique features of CAC is the increase in VWF and factor VIII [1324] and it is suggested to be the result of vascular response to SARS-CoV-2 infection. VWF and factor VIII are stored in the Weibel-Palade body of endothelial cells and released in response to infectious stimuli [25] (Fig. 2). The increase in VWF suggests a possible similarity to thrombotic thrombocytopenic purpura, however, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) levels in COVID-19 although reported to be decreased, may not be severely depleted as in thrombotic thrombocytopenic purpura [26]. The increased VWF levels to 3–4 times normal values seen in patients with COVID-19 may overwhelm the ADAMTS13 activity to degrade the ultra large VWF multimers. The importance of circulating VWF and multimer size in CAC is not established, however, one of the suggested methods to reduce the risk of thrombotic events due to excess ultra large VWF itself is plasma exchange [2728].

figure 2
Fig. 2

Similar to factor VIII and VWF, angiopoietin 2, also stored in Weibel-Palade bodies, is known to be released and its circulating level increases in COVID-19 [29]. Angiopoietin 2 serves as an antagonist of angiopoietin 1 and inhibits anti-inflammatory, anticoagulatory, and antiapoptotic signaling induced by angiopoietin 1 by binding to Tie2 competitively [30] (Fig. 3). Tie2 activation by angiopoietin 1 also normalizes prothrombotic responses by inhibiting endothelial tissue factor and phosphatidylserine exposure in sepsis, and therefore, Tie2 signaling is considered to play a central role in the regulation of thrombus formation in SIC/DIC [31]. Angiotensin 2 is also known to increase endothelial permeability and is considered an important factor in acute respiratory distress syndrome [32]. The role of angiopoietin 2 in CAC has not been studied well and it should be the focus in future studies.

figure 3
Fig. 3

The monitoring of endothelial damage

One of the difficulties in clinical studies of endothelial research is the limited availability of ideal biomarkers. The glycocalyx provides an interface between blood flow and endothelial cells. Since the glycocalyx is fragile, its components are used as biomarkers of endothelial damage in various diseases including infectious diseases [30]. One such biomarker is a hyaluronic acid, a major glycocalyx component. Circulating levels of hyaluronic acid, are elevated in critically ill COVID-19 patients compared with less severe cases [33]. Other than that, the proteins that are released from the Weibel-Palade body i.e., VWF, FVIII, and P-selectin are the potential biomarkers. In addition, sensitive coagulation markers such as thrombin-antithrombin complex (TAT) and prothrombin fragment 1 + 2 can be a marker for the microthrombosis. Goshua et al. [34] reported VWF antigen/antibody, FVIII activity, and TAT levels are significantly higher in the more severe cases.

As previously mentioned, angiopoietin 2 is stored in the endothelial cells and secreted along with the endothelial damage. Reportedly, angiopoietin 2 levels are associated with coagulation disorder, organ damage and death in bacterial sepsis [35]. Smadja et al. [29] measured angiopoietin 2, D-dimer, CRP, and creatinine in consecutive 40 COVID-19 patients treated in ICU, and found angiopoietin 2, cut-off of 5000 pg/mL, as the best predictor for poor outcome (sensitivity: 80.1%, specificity: 70%). It is crucial to find a good biomarker of vascular damage in COVID-19 study.

Therapeutic strategies for endothelial damage

Despite prophylactic anticoagulation in CAC, patients can still develop thrombotic sequela. A recent study reported that despite systematic use of thromboprophylaxis, 31% of the COVID-19 patients treated in ICU developed thrombotic complications [36]. In another study, the cumulative incidence of arterial and venous thromboembolism was 49% [3]. These reports suggest that despite anticoagulation, additional therapy for endothelial injury is necessary to prevent thrombosis. Potential therapies include synthetic serine protease inhibitors such as nafamostat mesylate and camostat mesylate which theoretically prevent SARS-CoV-2 infection. Coronavirus gains entry to the cell using the host TMPRSS2 which cleaves the spike protein resulting in its ability to fuse to the host cellular membrane. These agents inhibit TMPRSS2 thereby abrogating the activating proteolytic processing of virus [37]. Since nafamostat mesylate also has anticoagulatory effects, it has been used for DIC and anticoagulation for extracorporeal circuits in Japan.

Other therapeutic considerations are the physiologic anticoagulants such as protein C and antithrombin. The dual action of protein C/activated protein C to inactivate factor VIIIa and upregulate ACE2 are the advantage of this system which suppress both coagulation and inflammation. Activated protein C can also reduce pulmonary injury by suppressing the macrophage inflammatory protein family chemokine response [38]. Antithrombin is another multifaceted serine protease inhibitor of multiple coagulation factors, but also protects the glycocalyx by binding to heparan sulfate [39]. Bikdeli et al. [40] noted in their recent review of pharmacological therapy targeting thromboinflammation in COVID-19, that antithrombin suppresses excess inflammation by inhibiting nuclear factor-κB, it may be suitable for the treatment of CAC. However, the effects of these agents in COVID-19 haven’t been examined in clinical trials and future study may be warranted.

Arterial thrombosis in COVID-19

Arterial thrombosis is an uncommon event in other infection-associated coagulopathies. In contrast, stroke, ischemic coronary disease, and thrombotic limb ischemia can occur in COVID-19. Lodigiani et al. [41] reported the rate of ischemic stroke and acute coronary syndrome was 2.5% and 1.1%, respectively, in Italy. Kashi et al. [42] reported two cases of floating thrombi in thoracic aorta and such cases are extremely odd in previously described infectious diseases. Antiphospholipid syndrome is known as a disease that result in arterial thrombosis and can occur secondary to infection. Some reports have shown increased lupus anticoagulant, anticardiolipin, and anti-β2-glycoprotein I antibodies, however, the presence of high titer IgG antibody, an important responsible factor, has not proven yet [132443], and the association between antiphospholipid syndrome and CAC is still unclear. The presence of unusually large VWF multimers and subsequent activation of platelets and microthrombi can explain the occurrence of arterial macrothrombosis [44], and Williams et al. [45] reported elevated VWF levels were associated with the increased risk for recurrent stroke. However, a definitive cause and effect relation has not been proven yet. The occurrence of arterial thrombosis is difficult to predict and there are no good prophylactic strategies. Oxley et al. [7] reported five cases of large-vessel stroke in patients younger than 50 years of age. The mechanism of arterial thrombosis in COVID-19 remains a mystery and prediction of the events was not possible in any of the cases; demographic factors, laboratory data, and severity of COVID-19 did not appear to be related to arterial events.

Clot formation in extracorporeal circuits

The high incidence of clot formation during extracorporeal circulation has been recognized. Helms et al. [13] studied 150 COVID-19 patients and reported 28 out of 29 patients (96.6%) receiving continuous renal replacement therapy (CRRT) experienced clotting of the circuit. The median lifespan of an CRRT circuit was 1.5 days which is only half of the recommendation duration. They also reported 12 patients (8%) were treated by extracorporeal membrane oxygenation (ECMO), and among them, thrombotic occlusions of centrifugal pump occurred in 2 patients. Methods to minimize extracorporeal circuit clotting include prefilter infusion of heparin and the use of citrate-based replacement fluid for dialysis are not always successful [46]. The reason for the high incidence of CRRT filter and ECMO oxygenator coagulation is not known, but factors other than endotheliopathy such as hypercoagulability, hypofibrinolysis, and platelet activation must attribute. The elevated VWF activity, increased factor VIII level, and high fibrinogen level may lead to microthrombi formation possibly occluding the filter. Suppressed fibrinolysis may also play a role [47], with excess angiotensin II enhancing the expression of PAI-1 in the endothelium in COVID-19 [48].

Vasculitis in COVID-19

A report from northern Italy observed significantly increased number of patients with Kawasaki disease, an acute self-limiting vasculitis predominantly involving the coronary arteries, with hemodynamically unstable Kawasaki disease shock syndrome (KDSS) during the COVID-19 pandemic [49]. It is also reported that children with COVID-19 are more likely to show MAS that resembles secondary HLH. Varga et al. [50] demonstrated the direct viral infection of the endothelial cell and diffuse endothelial inflammation which are followed by the induction of endothelitis, apoptosis, and pyroptosis in autopsy cases of COVID-19. Of note is the mononuclear cell infiltrations into the vascular intima along the lumen of many vessels also reported in this post-mortem analysis. This finding suggests that the virus can invade into human vasculature and cause vasculitis. Roncati et al. [51] estimated the escalation from type 2 T-helper immune response to type 3 hypersensitivity is involved in the pathophysiology of COVID-19-induced vasculitis. They reported the deposition of immune complexes inside the vascular walls causing more severe inflammatory reaction, and interleukin-6 is the key myokine in this scenario.

Conclusion

The mechanism of coagulopathy in COVID-19 continues to be investigated. However, the predominant role of endotheliopathy due to direct endothelial infection with SARS-COV-2 and the indirect damage caused by inflammation are part of the complex thromboinflammatory process. The elevated circulating levels of clotting factors including fibrinogen, factor VIII, VWF released from the stimulated endothelial cells, and the loss of the thromboprotective function with glycocalyx damage and decreased nitric oxide production also contribute to the coagulopathy and thromboinflammation. SARS-CoV-2 damages not only the luminal surface of the vasculature but also induce vasculitis, contributing to the significant pathology associated with COVID-19.

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Funding

This work was supported in part by a Grant-in-Aid for Special Research in Subsidies for ordinary expenses of private schools from The Promotion and Mutual Aid Corporation for Private Schools of Japan.

Author information

Affiliations

  1. Department of Emergency and Disaster Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo, 113-8421, JapanToshiaki Iba
  2. Hematology Division Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USAJean Marie Connors
  3. Department of Anesthesiology, Critical Care, and Surgery, Duke University School of Medicine, Durham, NC, USAJerrold H. Levy

COVID-19-associated coagulopathy: An exploration of mechanisms

Authors: Meaghan E Colling 1Yogendra Kanthi 2 PMID: 32558620

PMCID: PMC7306998 OI: 10.1177/1358863X20932640

Abstract

An ongoing global pandemic of viral pneumonia (coronavirus disease [COVID-19]), due to the virus SARS-CoV-2, has infected millions of people and remains a threat to many more. Most critically ill patients have respiratory failure and there is an international effort to understand mechanisms and predictors of disease severity. Coagulopathy, characterized by elevations in D-dimer and fibrin(ogen) degradation products (FDPs), is associated with critical illness and mortality in patients with COVID-19. Furthermore, increasing reports of microvascular and macrovascular thrombi suggest that hemostatic imbalances may contribute to the pathophysiology of SARS-CoV-2 infection. We review the laboratory and clinical findings of patients with COVID–19-associated coagulopathy, and prior studies of hemostasis in other viral infections and acute respiratory distress syndrome. We hypothesize that an imbalance between coagulation and inflammation may result in a hypercoagulable state. Although thrombosis initiated by the innate immune system is hypothesized to limit SARS-CoV-2 dissemination, aberrant activation of this system can cause endothelial injury resulting in loss of thromboprotective mechanisms, excess thrombin generation, and dysregulation of fibrinolysis and thrombosis. The role various components including neutrophils, neutrophil extracellular traps, activated platelets, microparticles, clotting factors, inflammatory cytokines, and complement play in this process remains an area of active investigation and ongoing clinical trials target these different pathways in COVID-19.Keywords anticoagulationantiplateletCOVID-19inflammationneutrophilsthrombosisvascular endotheliumvenous thromboembolism (VTE)

Introduction

In December 2019, a new betacoronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]), thought to originate in Wuhan, China, emerged as a novel human pathogen for viral pneumonia (coronavirus disease [COVID-19]), resulting in an ongoing pandemic.1,2 The number of cases worldwide now exceeds five million, with more than 350,000 associated deaths, triggering a global effort to understand the predictors of disease severity for rapid triage, and the pathology of disease for rational therapeutic development and clinical trials. A consistent finding in early case series in China and New York City is an association between elevations in D-dimer and fibrin(ogen) degradation products (FDPs) and increasing COVID-19 severity and mortality.37 We aim to review the available data on the coagulopathy observed in COVID-19 and draw from studies of prior viral epidemics to explore possible mechanisms and therapies.

Coronaviruses are enveloped, non-segmented, positive-sense RNA viruses of the Nidovirales order within the Coronaviridae family. Different strains are infectious to a broad range of animals including humans, bats, cats, racoon dogs, rabbits, pigs, and cattle.8 In general, coronavirus infections in humans are mild; however, two recent epidemics of betacoronaviruses – SARS in 2003911 and Middle East Respiratory Syndrome (MERS) in 201212,13 – were associated with significant mortality with death rates around 10% and 35%, respectively.14,15 While the observed case fatality rate for the COVID-19 pandemic is lower,16,17 the population at risk is much higher due to the global spread of the disease and the infectivity of the virus,18 and worldwide fatalities already exceed those in the prior epidemics.

Common clinical manifestations of patients with COVID-19 include fever and cough, and less commonly fatigue, dyspnea, headache, sore throat, anosmia, nausea, vomiting, or diarrhea.6 In the largest case series to date of over 44,000 patients with COVID-19, > 75% of cases were mild, 14% were severe, and 5% were critical, with an overall case fatality rate of 2–2.5%. All deaths occurred in patients with critical disease (in which the case fatality rate was almost 50%).19 While the majority of critically ill patients with COVID-19 have isolated respiratory failure, often acute respiratory distress syndrome (ARDS), multiple organ dysfunction occurs in 20–30% of patients with critical illness and more often in fatal cases.16 Hematologic findings, such as mild to moderate thrombocytopenia and lymphopenia, are associated with COVID-19;20,21 however, the most significant and concerning vascular aspect of this disease is coagulopathy. We have attempted to summarize the data on the pathogenesis, epidemiology and outcomes related to COVID-19-coagulopathy and thrombotic disease using PubMed as well as the pre-print server https://medrxiv.org (date of last search April 23, 2020).

Coagulopathy of SARS-CoV-2 and other infections

There is particular interest in the coagulopathy in patients with COVID-19 as abnormal coagulation parameters, most consistently elevations in D-dimer and FDPs, are associated with disease severity.22,23 An elevated D-dimer, the most common coagulation abnormality in COVID-19 (found in up to 45% of patients), is an independent risk factor for death,6,22,24,25 and patients with D-dimer greater than 1000 ng/mL are almost 20 times more likely to die from their infection than patients with lower D-dimer values.25 In contrast, most patients with COVID-19 have a normal or mildly prolonged prothrombin time (PT) and a normal or shortened activated partial thromboplastin time (aPTT) on presentation and these labs are not reliably associated with disease severity.5,17,22,24,25 Both initial and longitudinal monitoring of coagulation parameters can predict disease severity, as elevated D-dimer and FDP levels on admission and decreased levels of fibrinogen and antithrombin III during the admission are associated with death.23 Although changes in plasminogen activator inhibitor-1 (PAI-1) levels and activity have not been studied, an increase in the PAI-1/tissue plasminogen activator (t-PA) ratio would not be unexpected. These findings may be due to uncontrolled activation of coagulation with ongoing consumption and widespread microvascular thrombosis.

While early descriptions of the coagulopathy identified it as disseminated intravascular coagulation (DIC), in DIC, unlike in severe COVID-19, platelet count and PT prolongation correlate with sepsis severity and mortality, while fibrinogen and FDPs levels do not.26,27 And while the majority of patients who die from COVID-19 develop some laboratory evidence of DIC during their admission, elevations in D-dimer and prolonged PT with mild thrombocytopenia and normal fibrinogen are commonly seen.23 Thromboelastography in patients with COVID-19 in the ICU shows a hypercoagulable state.28 These observations suggest the underlying pathophysiology in at least a subset of critically ill patients with COVID-19 is distinct from traditional systemic DIC and may be due to a unique coagulopathy.

Elevations in D-dimer are common in critical illness and are associated with disease severity and mortality in many severe infections.2931 Patients with influenza, SARS, HIV, hantavirus, Ebola virus, and dengue have elevations in D-dimer, prothrombin fragments, thrombin–antithrombin complexes, and/or plasmin-α2-antiplasmin complexes.32 Similar to patients with SARS-CoV-2 infections, there is an association between elevated D-dimer and mortality in patients with H1N1 and H5N1, which is not seen in SARS.3335

Additionally, in the H1N1 pandemic, patients with severe disease had high rates of venous thromboembolism (VTE) and many patients with thromboembolism did not have evidence of systemic DIC.3639 Patients with ARDS from H1N1 infection had a greater than 20-fold increase in risk of pulmonary embolism compared to patients with ARDS unrelated to H1N1.39 Empiric therapeutic anticoagulation in patients with ARDS was associated with decreased rates of VTE in patients with ARDS from H1N1, but had no effect on VTE rates in patients with ARDS unrelated to H1N1 infection. There are reports of VTE in patients with COVID-19, despite concerns regarding underdiagnosis given baseline elevations in D-dimer, as well as pragmatic challenges in diagnostic imaging while in isolation, including use of personal protective equipment and longer duration of exposure of health care workers.40,41 Although data remain scarce, there are increasing reports of arterial thrombotic events including ischemic strokes in patients with COVID-19.4143 Myocardial injury, defined by elevations in cardiac troponin levels, is common in patients hospitalized with COVID-19 and is associated with severe disease and high risk of mortality.44,45 Myocardial injury may result from systemic inflammatory response syndrome (SIRS) and inflammation as well as due to acute thrombotic events.46,47 Similar observations of myocardial injury have been found in patients with other viral infections.48,49

Pathologic findings in SARS-CoV-2 infection

Although there are only a few published pathologic reports of patients with COVID-19, histopathology of lung specimens from patients with early disease shows characteristic findings of ARDS and evidence of small vessel occlusion.50,51 There are several mechanisms by which SARS-CoV-2 infection may result in microvascular and macrovascular thrombosis, including cytokine storm with activation of leukocytes, endothelium and platelets resulting in upregulation of tissue factor, activation of coagulation, thrombin generation and fibrin formation,52 deranged coagulation with imbalances in PAI-1, tissue factor pathway inhibitor, and activated protein C that promotes fibrin generation and limits fibrinolysis,53,54 hypoxic vaso-occlusion, and direct viral effects with cell activation (Figure 1). It remains an active area of investigation whether these are specific to SARS-CoV-2 infection or a final common pathway in the thromboinflammatory response to viral infections and a marker of disease severity. Early COVID-19 autopsy reports have also identified a possible role for neutrophils as microvascular thrombi contained numerous neutrophils, which in some cases were partially degenerated, consistent with neutrophil extracellular traps (NETs).55,56 NETs are tangles of DNA released from neutrophils, and are decorated with antimicrobial and nuclear proteins that propagate intravascular thrombosis.57,58 NETs initiate both the extrinsic and contact pathways by augmenting presentation of tissue factor, activation of factor XII (FXII), as well as trapping and activating platelets.5962 Consistent with these observations, patients with severe COVID-19 have elevated serum markers of neutrophil activation and NET formation.63 In one study, neutrophil activation measured in serum correlated with, and sometimes preceded, VTE in patients with COVID-19.64

Figure 1. Immune activation and mechanisms of coagulopathy in patients with coronavirus disease 2019 (COVID-19).

Multiple processes may contribute to COVID-19-associated coagulopathy including direct infection of type II pneumocytes and endothelial cells, leading to barrier dysfunction and increased permeability; inflammatory responses characterized by activation of T cells, neutrophils, monocytes, macrophages, and platelets resulting in exuberant inflammatory cytokine release (including IL-1, IL-6, IL-10, TNF-α), monocyte-derived TF and PAI-1 expression; and culminating in the development of microvascular and macrovascular thrombi composed of fibrin, NETs, and platelets.

IL, interleukin; NETs, neutrophil extracellular traps; PAI-1, plasminogen activator inhibitor-1; TF, tissue factor; TNF-α, tumor necrosis factor-alpha.

Dysregulation of hemostasis and coagulopathy in acute respiratory distress syndrome (ARDS)

Thrombi in the pulmonary micro- and macrovasculature are observed in patients with ARDS with or without overt DIC, and changes consistent with a prothrombotic state have been found both in blood and in alveolar fluid studies of these patients.65,66 Higher levels of FDPs and D-dimer are seen in patients who developed ARDS as compared to patients with similar predisposing conditions that did not develop ARDS.67 Lower levels of protein C and higher levels of soluble thrombomodulin and PAI-1 are also associated with multiple organ failure, disease severity, and mortality in ARDS in some studies.53,6872 Finally, plasma and alveolar levels of tissue factor are higher in patients with ARDS than patients with pulmonary edema.73 Mechanistically, there is increased thrombin generation by tissue factor coupled with an impaired fibrinolytic response due to elevations in PAI-1. Elevations in D-dimer, a breakdown product of crosslinked fibrin, may result from residual t-PA/plasmin activity, as well as from alternative fibrinolytic pathways such as human neutrophil elastase activity.74,75

As patients with COVID-19 frequently have isolated pulmonary findings, the initial hemostatic dysregulation may be localized to the lungs as a consequence of the bidirectional relationship between the innate immune system and thrombosis. Activated platelets through degranulation and coordinated interactions with monocytes, dendritic cells, and neutrophils, as well as activated T cells, NETs, tissue factor-bearing microparticles, and coagulation proteases may facilitate this crosstalk.54,76,77 In this model, immune cells, inflammatory cytokines, and pathogen-associated molecular patterns induce thrombi consisting of fibrin, monocytes, neutrophils, and platelets.57,58,78 These immunothrombi initially serve a protective purpose, promoting pathogen recognition and creating a sterile barrier against further pathogen invasion, but can become maladaptive and injurious to tissue and organ perfusion.57,79,80 During this process, there is abundant intra- and extra-vascular fibrin deposition and impaired fibrinolysis, which has been well described in ARDS.81,82 In postmortem studies, both macro- and microvascular thrombi are common in patients in ARDS (observed in up to 95% of patients).82,83 In COVID-19, the alveolar immunothrombotic response may be an attempt to limit dissemination of SARS-CoV-2 outside the alveoli.

Findings from the SARS epidemic provide possible viral-specific mechanisms for ARDS and uncontrolled coagulation. Autopsy studies of patients who died of SARS pneumonia, identified the SARS-CoV spike (S) protein in cells expressing the receptor angiotensin-converting enzyme 2 (ACE2),8487 the leading candidate receptor for SARS-CoV-2.88,89 Binding of the S protein to ACE2 induces expression of a nuclear factor kappa B (NFκB)-driven inflammatory module, resulting in production of proinflammatory cytokines including monocyte chemoattractant protein 1 (MCP-1), transforming growth factor-beta 1 (TGF-β1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, which have been implicated in thrombogenesis.90 Although inflammatory responses are important in host-defense, hyperinflammatory responses result in tissue damage, disruption of the endothelial barrier, and uncontrolled activation of coagulation.54 Overall, these findings are consistent with a model in which SARS-CoV and SARS-CoV-2 directly infect endothelial and epithelial cells, increasing levels of proinflammatory cytokines, causing immune-mediated damage to the vasculature and surrounding tissue, with exposure of tissue factor and associated thromboinflammatory changes.91 While these changes appear to be predominantly in the lungs, endotheliitis in COVID-19 has been observed in kidneys, liver, heart, and intestine.91

Additional studies in SARS-CoV and influenza found dysregulation of urokinase, coagulation, and fibrinolysis pathways contributed to the severity of lung injury, possibly through altering the hemostatic balance with subsequent coagulation-induced ischemic injury.92 Plasminogen was protective against severe influenza A, H5N1, and H1N1 infections.93 These groups hypothesized that increased fibrinolysis led to a positive feedback loop of vascular permeability, leukocyte recruitment, and fibrin generation. Interestingly, one hypothesis suggests that elevated plasminogen may be a risk factor for SARS-CoV-2 infection because plasmin may cleave the S protein of the virus and increase its infectivity.94 These findings highlight the delicate balance between corralling infection and uncontrolled inflammation and thrombosis.

Therapeutic considerations

Markers of hypercoagulability and higher inflammatory mediators are consistently associated with worse outcomes in patients with ARDS and sepsis. These observations have led to numerous clinical trials targeting various components of inflammatory and coagulation pathways in acute lung injury, ARDS or sepsis. Studies with heparin, steroids, non-steroidal anti-inflammatory drugs, and TNF-α inhibitors have been disappointing.95100

Given the laboratory and clinical findings in patients with severe COVID-19, several repurposed and novel therapies are under investigation in clinical trials to prevent the hyperinflammatory response or mitigate uncontrolled coagulation. As elevations in D-dimer and FDPs likely reflect ongoing lung injury and microvascular thrombi, possible therapeutic targets include inflammatory cytokines, activated platelets, neutrophils, or microparticles that may propagate thrombosis; or anticoagulants and fibrinolytics that could limit thrombosis. Supporting this enthusiasm was a recent retrospective study in China in which VTE prophylaxic dose heparin was associated with a survival benefit in patients with severe COVID-19 and evidence of sepsis-induced coagulopathy.101 The study found no benefit among patients with milder COVID-19 illness; however, the study did not control for other markers of disease severity nor other therapies, such as antivirals. The study raises the possibility that prophylactic or therapeutic anticoagulation may benefit patients with severe infection. Heparin may alter the biology of the disease not only through its anticoagulant properties, but also due to its anti-inflammatory effects that promote a quiescent endothelium.

Current expert recommendations, including interim guidelines from the International Society on Thrombosis and Haemostasis (ISTH) and the American College of Cardiology (ACC), recommend use of prophylactic dose LMWH or unfractionated heparin in all COVID-19 patients requiring hospital admission; for patients with a contraindication to pharmacologic prophylaxis, mechanical prophylaxis should be used.102,103 While a number of VTE risk stratification tools exist for hospitalized medical patients, these have not been validated in patients with COVID-19. Extended VTE prophylaxis with LMWH or direct oral anticoagulants after hospitalization for acute medical illness reduces the risk of VTE with an associated increased risk of bleeding.104106 There are currently no data regarding extended prophylaxis in patients with COVID-19; however, the ACC expert opinion statement recommends consideration of extended prophylaxis in patients with elevated risk of VTE, such as patients with cancer or prolonged immobility who have low bleeding risk. Given early reports and ongoing concerns of high rates of VTE, randomized trials of empiric therapeutic anticoagulation or antifibrinolytics are ongoing, and there are reports of empiric therapeutic anticoagulation in patients with significantly elevated D-dimer both in Italy and in the US. While heparin offers both anti-inflammatory and anticoagulant effects, the benefit of therapeutic anticoagulation remains uncertain, with a risk of bleeding complications in critically ill patients with respiratory failure.95,107 Clinical trials will help define the role of heparin in the treatment of hospitalized patients with COVID-19. Outside of a trial setting, we advocate universal standard-dose pharmacologic VTE prophylaxis in patients without a contraindication. In patients with a high suspicion of VTE where access to confirmatory or serial imaging is limited, clinicians may consider empiric anticoagulation, although there is a paucity of evidence to provide guidance in this context. There are currently no randomized data to recommend empiric therapeutic or intermediate-dose anticoagulation in patients without documented VTE, or an other indication for anticoagulation, or outside the context of a clinical trial. A recent retrospective, observational study in New York City showed therapeutic anticoagulation was associated with decreased mortality in patients with COVID-19 who required mechanical ventilation, but not in all hospitalized patients with COVID-19. Although these findings are provocative, interpretation is limited by their observational nature.108

There are over 300 trials ongoing for patients with COVID-19, many of which aim to simultaneously reduce inflammation and thrombosis, including cytokine-directed therapies (against IL-1, IL-6, interferon gamma), corticosteroids, Janus kinase inhibitors, TLR ligands, complement inhibitors, N-acetylcysteine, serine protease inhibitors, DNAse enzymes, and anti-viral agents. However, suppressing the cytokine storm or hypercoagulability may be insufficient once initiated, and targeting upstream pathways to prevent activation of this self-amplifying feedback loop may be more effective.

One therapeutic candidate to treat COVID-19 is dipyridamole, an adenosinergic drug indicated for use as an arterial thromboembolic prophylaxis agent in combination with aspirin or warfarin.109 Dipyridamole has recently been shown to suppress human neutrophil and T-cell activation, upstream of cytokine effectors.58,110 Dipyridamole induces a type I interferon response, which is necessary for physiologic anti-viral activity, and inhibits SARS-CoV-2 replication in vitro by inhibiting a critical viral replication complex.111,112 Administered orally, dipyridamole has a favorable safety profile, and a small clinical trial in patients with COVID-19 suggests it may improve D-dimer levels.113 Randomized clinical trials of agents active at the intersection of inflammation and coagulation in COVID-19, such as dipyridamole, t-PA, and heparin are necessary to determine if these therapeutics can restore the balance of inflammation and coagulation without dampening early or late physiologic anti-viral responses. The heterogenous response to the SARS-CoV-2 infection and the various time-dependent pathways driving pathology make universal therapies challenging. The temporal and mechanistic role each pathway plays in severe SARS-CoV-2 infection remains uncertain and requires further exploration for treatment opportunities as efforts to control this pandemic continue.

Conclusions

In conclusion, in patients with COVID-19, the presence of coagulopathy, characterized by elevations in D-dimer and FDPs, is consistently associated with more severe illness and mortality. Laboratory, clinical, and early histopathologic findings suggest this coagulopathy is distinct from sepsis-induced DIC and may reflect dysregulated hemostasis. Similar findings have been associated with several other viral infections, and it remains uncertain if this coagulopathy is specific to SARS-CoV-2 or the end common pathway of the thrombo-inflammatory response to severe viral infections. There are efforts to target numerous components of the thrombo-inflammatory pathway to improve outcomes in patients with severe COVID-19. The optimal management for these patients including strategies to diagnose VTE, appropriate anticoagulation doses and duration, and effectiveness of novel therapies are under active investigation in the current pandemic.

Acknowledgements

The authors would like to thank Charles Bolan, MD and Jason Knight, MD, PhD for guidance and review of the manuscript, and all members of the ‘NETwork to Target Neutrophils in COVID-19’ and the SVM Next Generation Committee for their helpful advice and encouragement. The authors credit Alan Hoofring for the illustration.

Declaration of conflicting interests
Yogen Kanthi has served as a consultant for Surface Oncology and has a pending patent on use of biogases in vascular disease. Meaghan E. Colling has nothing to disclose.

Funding
Meaghan E. Colling is supported by the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH). Yogen Kanthi is supported by grant funding from the NIH-NHLBI (K08HL131993, R01HL150392), A. Alfred Taubman Medical Research Institute, Michigan Medicine Frankel COVID-19 Cardiovascular Impact Research Ignitor Program, Falk Medical Research Trust Catalyst Award, American Venous Forum-JOBST Award, University of Michigan BioInterfaces Institute, and Bo Schembechler Heart of A Champion Foundation.

ORCID iD
Yogendra Kanthi  https://orcid.org/0000-0002-5660-5194

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