New research from RCSI University of Medicine and Health Sciences has provided greater insight into the causes of Long COVID syndrome.
The findings, which further investigate the link between Long COVID and blood clotting, have been published in the Journal of Thrombosis and Haemostasis.
Long COVID syndrome is a broad collection of symptoms including shortness of breath, fatigue and reduced physical fitness that can continue for many months after initial infection with COVID-19. Understanding is limited about why these symptoms persist in some patients but not others, and the novel syndrome remains a considerable clinical challenge for both doctors and patients alike.
To gain a new understanding of what causes Long COVID, researchers at RCSI studied patients in Ireland with symptoms of Long COVID, and saw that the body’s blood-clotting and immune systems can remain tipped out of balance long after the initial infection.
The team of researchers, led by Professor James O’Donnell at the RCSI School of Pharmacy and Biomolecular Sciences with Dr Helen Fogarty as Clinical Fellow, analysed blood from 50 patients with Long COVID syndrome up to 12 weeks post infection with the COVID-19 virus. They compared the samples to ‘controls’, blood from healthy people who did not have Long COVID syndrome.
The study found that the blood of patients with Long COVID syndrome had higher levels of a blood-clotting booster called von Willebrand Factor (VWF), and lower levels of a protein that normally breaks down VWF, called ADAMTS13. Their analysis also suggests that blood vessels were still being damaged long after the initial infection, and that specific cells of the immune system were at abnormal levels in patients with Long COVID.
“In this study, we examined 50 patients with symptoms of Long COVID syndrome. We saw that, in patients with Long COVID, the normally finely tuned balance of pro- and anti-clotting mechanisms were tipped in favour of blood clotting,” said Dr Helen Fogarty, Health Research Board Irish Clinical Academic Training (ICAT) Programme Fellow and lead author on the paper. “Our analysis also suggests that abnormal clotting and disturbed immunity go hand in hand in Long COVID. Together, these findings may help explain some of the symptoms of Long COVID syndrome.”
Commenting on the study, Professor James O’Donnell said: “Extensive research has been carried on the dangerous clotting observed in patients with acute severe COVID-19 infection, and we now understand a lot more about how and why these deadly clots occur. In this study, we put the focus on Long COVID syndrome, as so much less is known about this persistent illness which is affecting millions of people worldwide.”
The study was carried out by clinical colleagues at St James’s Hospital and researchers at RCSI as part of the Irish COVID-19 Vasculopathy Study (ICVS) collaboration, which includes scientific researchers in RCSI, Trinity College Dublin and University College Dublin as well as clinical partners in St James’s, St Vincent’s and Beaumont Hospitals. The ICVS is supported by a Health Research Board COVID-19 Rapid Response award (COV19-2020-086), and a philanthropic grant from the 3M Foundation to RCSI in support of COVID-19 research.
Helen Fogarty, Soracha E. Ward, Liam Townsend, Ellie Karampini, Stephanie Elliott, Niall Conlon, Jean Dunne, Rachel Kiersey, Aifric Naughton, Mary Gardiner, Mary Byrne, Colm Bergin, Jamie M. O’Sullivan, Ignacio Martin‐Loeches, Parthiban Nadarajan, Ciaran Bannan, Patrick W. Mallon, Gerard F. Curley, Roger J. S. Preston, Aisling M. Rehill, Ross I. Baker, Cliona Ni Cheallaigh, James S. O’Donnell, Niamh O’Connell, Kevin Ryan, Dermot Kenny, Judicael Fazavana. Sustained VWF‐ADAMTS‐13 axis imbalance and endotheliopathy in long COVID syndrome is related to immune dysfunction. Journal of Thrombosis and Haemostasis, 2022; DOI: 10.1111/jth.15830
Cite This Page: MLAAPAChicago RCSI. “Blood-clotting imbalance persists in Long COVID, research finds.” ScienceDaily. ScienceDaily, 23 August 2022. <www.sciencedaily.com/releases/2022/08/220823095434.htm>.
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.
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.
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.
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.
Ongoing reports have described the hypercoagulability and thrombotic tendency in COVID-19 . 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 . 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 . 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 . The new coronavirus SARS-CoV-2 elicits an acute inflammatory effect with hypercoagulability, platelet activation, and endothelial dysfunction . Although, this presentation has similarities with sepsis-induced coagulopathy (SIC) due to bacterial infections and disseminated intravascular coagulation (DIC), there are several important differences . 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 [4, 9]. 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 . Although the pathophysiology of HLH/MAS seems similar to COVID-19, the reported cytokine level is much lower in COVID-19 . 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 . 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) , potentially the presence of antiphospholipid antibodies , 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.
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.  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.  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 .
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 . 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 . 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.  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 .
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 .
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 , 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 [13, 24] 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  (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 . 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 [27, 28].
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 . 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  (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 . Angiotensin 2 is also known to increase endothelial permeability and is considered an important factor in acute respiratory distress syndrome . The role of angiopoietin 2 in CAC has not been studied well and it should be the focus in future studies.
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 . 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 . 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.  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 . Smadja et al.  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 . In another study, the cumulative incidence of arterial and venous thromboembolism was 49% . 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 . 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 . Antithrombin is another multifaceted serine protease inhibitor of multiple coagulation factors, but also protects the glycocalyx by binding to heparan sulfate . Bikdeli et al.  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.  reported the rate of ischemic stroke and acute coronary syndrome was 2.5% and 1.1%, respectively, in Italy. Kashi et al.  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 [13, 24, 43], 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 , and Williams et al.  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.  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.  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 . 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 , with excess angiotensin II enhancing the expression of PAI-1 in the endothelium in COVID-19 .
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 . It is also reported that children with COVID-19 are more likely to show MAS that resembles secondary HLH. Varga et al.  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.  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.
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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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 anticoagulation, antiplatelet, COVID-19, inflammation, neutrophils, thrombosis, vascular endothelium, venous thromboembolism (VTE)
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.3–7 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 20039–11 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.29–31 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.33–35
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.36–39 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-19.41–43 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.59–62 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.
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.53,68–72 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),84–87 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.
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.95–100
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.104–106 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.
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.
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.
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COVID-19, the disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) can present with symptoms ranging from none to severe. Thrombotic events occur in a significant number of patients with COVID-19, especially in critically ill patients. This apparent novel form of coagulopathy is termed COVID-19-associated coagulopathy (CAC), and endothelial derived von Willebrand factor (vWF) may play an important role in its pathogenesis.Content
vWF is a multimeric glycoprotein molecule that is involved in inflammation, primary and secondary hemostasis. Studies have shown that patients with COVID-19 have significantly elevated levels of vWF antigen and activity, likely contributing to an increased risk of thrombosis seen in CAC. The high levels of both vWF antigen and activity have been clinically correlated with worse outcomes. Furthermore, the severity of a COVID-19 infection appears to reduce molecules that regulate vWF level and activity such as ADAMTS-13 and high-density lipoproteins (HDL). Finally, studies have suggested that patients with group O blood (a blood group with lower baseline levels of vWF) have a lower risk of infection and disease severity compared to other ABO blood groups; however, more studies are needed to elucidate the role of vWF.Summary
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially identified in Wuhan, China in 2019. COVID-19, the disease caused by SARS-CoV-2, quickly evolved into a global pandemic. According to the Johns Hopkins COVID-19 Dashboard, there were more than 20 million confirmed cases and almost 350,000 deaths in the US alone, by the end of 2020. Although COVID-19 may present with a variety of symptoms, a large majority of infected individuals may have none to only mild symptoms (1). However, the mortality rate is dominated by a subset of patients with severe respiratory failure that meet the criteria for acute respiratory distress syndrome (ARDS) and require respiratory support (1, 2). The development of severe disease is related to interstitial viral pneumonia, systemic inflammation, respiratory failure, and multiorgan dysfunction (3).Impact Statement
COVID-19 is a global pandemic with no current effective treatment. COVID-19-associated coagulopathy contributes to patient morbidity and mortality. von Willebrand factor (vWF) may play an important role in the pathogenesis of this coagulopathy. Currently, available studies have demonstrated that patients with COVID-19 have significantly elevated levels of vWF antigen and activity as well as reduced regulatory molecules, which could contribute to an increased risk of thrombosis seen in patients who develop coagulopathy. Elucidation of vWF role in patients with COVID-19 may offer additional insights into developing novel therapies for this disease.
SARS-CoV-2 preferentially binds to host cells that express the angiotensin-converting enzyme-2 receptor (ACE2) through the viral spike protein structure. The initiation and progression of the SARS-CoV-2 infection is likely dependent on a combination of factors, including, but not limited to, host cell expression of ACE2, anatomic contiguity with the environment, inoculation dose at the time of exposure, and the host immune response to the infection. In general, the initial infection by the SARS-CoV-2 virus targets the cells of the respiratory system such as nasal or bronchial epithelial cells and pneumocytes. However, if the severity of the infection progresses to a systemic inflammatory phase, the mechanism is likely a complex combination of the virus entering the blood stream, infection of other cells expressing ACE2 receptors, tissue/organ specificity, and the inflammatory milieu. However, the extent to which each factor contributes to the systemic severity remains unclear. Additionally, in severe COVID-19 cases, endothelial cells (ECs), which also express ACE2 receptors, are activated, leading to endothelial dysfunction and possible injury that parallels clinical manifestations, such as coagulopathy and prothrombotic tendency (4).
COVID-19 Associated Coagulopathy
It is clear that a significant component of the observed morbidity and mortality is directly related to lung injury as supported by COVID-19 related autopsies (5, 6). The predominant pattern of injury was found to be diffuse alveolar damage, which includes hyaline membrane formation, capillary congestion, inflammation, and pneumocyte necrosis. In addition, the study also identified platelet-fibrin thrombi in small arterial vessels in 87% of their cases (6). A more recent, albeit small, series showed that all COVID-19 related autopsies demonstrated platelet-fibrin thrombi in multiple organs, including the liver, kidney, heart, and lungs (5). Another autopsy case series compared lung tissue from equally severe, age-matched patients with ARDS with either COVID-19 or influenza A (H1N1) and found that alveolar capillary microthrombi were more prevalent in COVID-19 than influenza (7). This study also observed that COVID-19 lung tissue showed significant EC injury associated with intracellular SARS-CoV-2 infection (7). Furthermore, there is some evidence to suggest that COVID-19 associated coagulopathy (CAC) might be different from other coagulopathic conditions, such as disseminated intravascular coagulation (DIC) and thrombotic microangiopathy (TMA), which are associated with other underlying causes such as infections, malignancy, autoimmune, and hereditary diseases (Table 1) (8). Taken together, the data indicate that a distinct coagulopathy may be occurring in COVID-19 patients, particularly those with severe symptoms.
Laboratory data in COVID-19 and other coagulopathies.
within reference range
within reference range
within reference range
within reference range
within reference range
within reference range
within reference range
within reference range
within reference range
generally, mildly elevated early and decreases as severity increases
no change to mildly elevated
increased activation, may result in lower antigen levels due to consumption
no change to decreased
no change to elevated
no change to elevated
normal to mildly increased
normal to mildly elevated
no change to elevated
no change to elevated
usually mildly increased but may be normal
normal to mildly elevated
no change to elevated
no change to elevated
moderate to severely increased
normal to moderately decreased
normal to mildly elevated
Normal values will vary among laboratories due to varying methodologies and reagents. Given that there are multiple markers for complement activation, inflammation, and acute phase reactants, reference ranges for these (patho)-physiological events are not provided. Of note, ADAMTS13 measurement is generally the reliable biomarker distinguishing TTP from HUS/atypical HUS. HUS can be distinguished from aHUS if the patient has history of Shiga-toxin or Streptococcus exposure. Other biomarkers may be overlapping in the spectrum from DIC/SIC to TTP/HUS/aHUS. DIC: disseminated intravascular coagulation, SIC: sepsis-induced coagulopathy, TMA: thrombotic microangiopathy, TTP: thrombotic thrombocytopenia purpura, aHUS: atypical hemolytic uremic syndrome, PT: prothrombin time, aPTT: activated partial thromboplastin time, vWF: von Willebrand factor. Adapted from Iba et al. (8).Open in new tab
Incidence of CAC, especially in severe COVID-19 cases, was apparent from early reports in Wuhan (9). A number of studies have shown that the development of CAC is an important prognostic indicator of poor outcomes (10–12). One study evaluated the rate of arterial and venous thrombotic events in COVID-19 pneumonia patients admitted into the intensive care unit (ICU) and found that the incidence of thrombotic events in 184 patients was 49% (after adjustment for competing risk of death) despite receiving routine pharmacologic thromboprophylaxis; not surprisingly, these thrombotic complications led to a higher risk of death (13). Additional studies have shown similar incidence rates of thrombotic events in COVID-19 ICU patients (14, 15). Collectively, clinical studies suggest that CAC leads to a prothrombotic state even with standard pharmacologic thromboprophylaxis treatment.
In general, CAC is characterized by mild thrombocytopenia, slight prolongation of the prothrombin time (PT), high levels of D-dimer, and elevated fibrinogen (8, 12, 16) (Table 1). Recent International Society for Thrombosis and Hemostasis (ISTH) interim guidance recommends monitoring these 4 parameters in the management of patients with CAC. D-dimer was designated the highest level of priority as many studies have shown that elevated levels are associated with increasing severity of disease and mortality risk (3, 10, 11, 17–20). These studies reported a range of associations of higher D-dimer levels in COVID patients, including greater risk of mortality (3, 11, 18), increased disease severity (10, 11), increased incidence of pulmonary emboli (17), and need for intensive care (20). Based on this data, clinical services can order a baseline D-dimer level to determine the current morbidity and mortality risk that a COVID-19 patient carries and can follow a D-dimer level to predict progression to more severe disease.
D-dimer is a breakdown product of mature clots (cross-linked fibrin mesh) that undergoes fibrinolysis. Though some studies reported data where the association with D-dimer and death may not be as compelling (21, 22), D-dimer levels do play a role during the follow-up and treatment of patients with CAC. There is, however, another biomarker, von Willebrand factor (vWF), which may also play an important role in the evaluation of CAC patients due to its direct relationship to hemostasis, inflammation, and EC activation/injury, which are all important aspects of COVID-19 pathogenesis. The biological role of vWF and its association with CAC will be the focus of the remainder of this review.
vWF Physiology and Laboratory Testing
vWF is a multimeric glycoprotein ranging from 2 to >60 prepropolypeptide units that are each 2138 amino acids in length. The vWF propeptide sequence serves to align 2 units together to allow proper cross-linking during the multimerization process. Further post-translational modification leads to removal of the propeptide sequence as well as glycosylation, including the addition of blood group determinants. This addition of an A or B blood group determinant only occurs during EC glycosylation. Following these processes, a heterogenous mix of ultra-large-vWF (UL-vWF) molecules are synthesized and stored in megakaryocytes and ECs, respectively, in alpha granules and Weibel–Palade bodies (WPB). Additionally, other processing components such as vWF propeptides are found in the WPB of ECs. Although platelets do play an important role in both storage and secretion of vWF, this review will focus on ECs.
When ECs are activated, UL-vWF molecules are released and can either remain free-floating in the plasma or localized on endothelial surfaces. UL-vWF have greater prothrombotic activity than smaller vWF multimers. Therefore, as UL-vWF molecules are secreted, ADAMTS-13 (a disintegrase and metalloproteinase with a thrombospondin type 1 motif, member 13), cleaves vWF into smaller multimers to mitigate unwanted thrombus formation and leads to a variation in the sizes of vWF found both in the plasma and on endothelial surfaces. Elevated vWF activity levels depend on the presence of the largest vWF multimers and activation by shear stress in the circulatory system. vWF responds to shear stress by unfolding and exposing sites for activity such as self-association, platelet binding, and ADAMTS-13 cleavage. Accordingly, the imbalance of these components may lead to a prothrombotic state.
Role in Primary Hemostasis
Primary hemostasis is the process of the platelet clot formation at the site of blood vessel injury. For proper primary hemostasis to occur, platelet adhesion and aggregation must occur. During platelet adhesion at the site of blood vessel injury, platelets can bind directly to the exposed subendothelial collagen (via GPIa-IIa or GPVI receptors) or indirectly via vWF. In the latter case, platelets bind to the vWF molecule via the platelet glycoprotein Ib-V-IX receptor (GPIb) while vWF is bound to subendothelial collagen. Additionally, vWF also promotes platelet aggregation (platelet–platelet interaction) by binding to platelet surface receptor GPIIb/IIIa. Although GPIIb/IIIa is better known as a fibrinogen receptor, it can bind to both fibrinogen and vWF. In summary, vWF plays a vital role in platelet adhesion and aggregation in clot formation.
Role in Secondary Hemostasis
vWF also performs an important role in secondary hemostasis. Secondary hemostasis involves coagulation factors and the coagulation cascade to produce a fibrin meshwork at the site of vessel injury. vWF facilitates the secondary hemostasis process in two ways. First, vWF serves as a carrier protein for Factor VIII, extending Factor VIII’s half-life in the plasma. Although this may initially seem trivial, the vWF carrier activity stabilizes Factor VIII and significantly extends its half-life 4 to 6-fold. Second, it releases and concentrates Factor VIII at the site of injury. Factor VIII is a clotting factor that, when activated, complexes with other factors to ultimately produce fibrin. To highlight the significance of vWF in this process, mutations affecting the vWF binding site for Factor VIII leads to decreased levels of Factor VIII, known as Type 2N von Willebrand disease (vWD), resulting in a clinical presentation similar to hemophilia A, which is a bleeding disorder that occurs when an individual lacks the ability to produce adequate amounts of Factor VIII for proper clotting.
vWF, Inflammation, and Endothelial Activation/Injury
During the inflammatory process, various chemical mediators are released. These inflammatory molecules activate ECs to release their WPB contents, including vWF and other molecules such as P-selectin, which has been directly linked to leukocyte recruitment (23, 24). In addition, UL-vWF molecules that remain bound to EC surface will subsequently bind platelets and may have the ability to act as a molecular surface for leukocyte interaction (25). With increased release of vWF, the inflammatory process is expected to induce a prothrombotic state. Studies show that inflammation enhances vWF self-association, which may lead to increased adhesiveness of platelets while decreasing ADAMTS-13 cleavage (24). Additionally, high-density lipoprotein (HDL) decreases during inflammation in both chronic and acute phases. HDL may play a vital role in preventing shear stress-induced vWF self-association, thus decreasing prothrombotic risk under normal circumstances (24). This concept will become a point of discussion later in the review. In summary, the data indicate that during the inflammatory process there is an increased thrombotic risk due to the imbalance of increased vWF and activity levels via EC activation and reduced ADAMTS-13 activity.
Laboratory Testing of vWF
To understand the studies that will be mentioned in connection with CAC, it is important to briefly discuss basic vWF laboratory testing. There are 3 basic tests performed to assess vWF; the exact methods may vary between manufacturers for those that are highly automated but the fundamental parameters rest on testing vWF quantity, activity, and multimer size.
The quantity of the vWF level in a specimen is commonly referred to as antigenic testing (vWF:Ag). An immunoturbidimetric method is commonly used for vWF:Ag measurement. However, the details of the assays vary by manufacturer. This allows for quantitative determination of the physical presence of the molecule without assessment of function. ABO blood typing and Factor VIII levels are also performed concurrently; it is well documented that individuals of blood group O have physiologically lower levels of vWF, and therefore Factor VIII (since vWF binds and stabilizes it) levels are also slightly lower than individuals of non-O blood groups (see the “vWF Association with Blood Type” section).
The quality of present vWF is known as functional or activity testing; this involves testing the ability of vWF to bind to platelet receptor GPIb, collagen, and Factor VIII (vWF:RCo). There are a number of assays and methods that revolve around testing the ability of vWF to bind its natural physiologic substrates (with or without ristocetin). Depending on the substrate used to assess its binding function, these tests will often carry an acronym such as vWF:Ac, vWF:RCo, vWF:Co, or vWF:VIII. It is important to note that there are important and distinct differences amongst these tests; however, this is beyond the scope of the review.
Additionally, the qualitative variation of vWF multimers is performed to visualize the presence and size distribution of vWF located in the plasma using gel electrophoresis and vWF labeling. This assessment is important since multimer presence and size is directly correlated to the function and activity level of the vWF molecule.
Finally, although not a laboratory test, the results of the activity and antigenic assays may be juxtaposed to obtain the ratio of vWF activity to antigen (RCo:Ag ratio). A ratio that is less than 0.5–0.7 would indicate that a qualitative defect in the vWF molecules is likely and this helps categorize the pattern and subtypes of vWD, if present.
Examination of vWF in COVID-19 Associated Coagulopathy
Endothelial Activation and vWF
As a molecule present in ECs that plays a fundamental role in hemostasis and thrombosis, vWF is a reasonable candidate marker to consider when monitoring clinical issues related to endothelial injury and coagulopathy in COVID-19. Early studies duly noted that D-dimer levels were an important prognostic marker in COVID-19. However, studies also began to recognize and demonstrate that significantly elevated levels of vWF were also present (14, 16, 19, 26). Further, studies then recognized that vWF activity is also increased and that ADAMTS-13 activity levels are relatively mild to moderately reduced, leading to an imbalance favoring thrombosis (27, 28). Similarly, in a well-recognized pathological entity, thrombotic thrombocytopenic purpura (TTP) is associated with reduced activity levels of ADAMTS-13. TTP is generally due to an extremely hindered or absent ADAMTS-13 activity by either an acquired inhibitor or congenital absence. The decreased activity levels of ADAMTS-13 result in an excess of overactive UL-vWF multimers that promote microthrombi formation.
However, in contrast to TTP, the mild to moderately decreased ADAMTS-13 activity levels observed in CAC may not lead to excessive UL-vWF. Thus, it is important to distinguish that activity levels of ADAMTS-13 may not be low enough in CAC cases to detect an excessive increase in UL-vWF as seen in severe deficiency such as in TTP. In line with this, a recent study showed decreased activity levels of ADAMTS-13 in patients with severe COVID-19 but found no evidence of UL-vWF multimers in the plasma (29). Further, the authors of this study emphasized the significance of the elevated vWF:Ag to ADAMTS-13 activity ratio in association with increasing severity of disease. This suggests that an increased risk of thrombosis seen in patients with COVID-19 may, in part, be due to a relative decrease of ADAMTS-13 activity rather than an absolute decrease as seen in TTP.
The high levels of both vWF antigen and activity have been correlated clinically with increased thrombotic events (14), increased likelihood for treatment in ICUs (19), and increased need for oxygen support (26), as well as correlated with other laboratory testing such as decreased clotting times, increased clot formation velocities as demonstrated by whole blood viscoelastic testing (16) and increased levels of other markers of platelet and endothelial activation, such as Factor VIII and thrombomodulin (16, 19, 26–28, 30). As new biomarkers to assess CAC severity emerge, reexamining the synthetic pathway of vWF may have some utility. One promising avenue is to examine levels of vWF propeptide; its physiologic role in the multimerization process would suggest that elevated levels of vWF propeptide indicate elevated vWF release. In addition, a greater level of increase in vWF and propeptide in comparison to an increase in Factor VIII suggest that this is due to release of vWF from pulmonary ECs involved in the COVID-19 pathophysiologic process (31). The ratio of propeptide levels to vWF levels can also examined; this ratio seems to decrease with disease progression suggesting that while the propeptide is cleared normally, levels of vWF may stay elevated due to decreased clearance (29). Further examination of propeptide levels in patients with COVID-19 are indicated to elucidate these possible relationships.
High-Density Lipoprotein and vWF
Aside from endothelial activation and injury, a more indirect mechanism may contribute to increased vWF activity levels. In general, infection leads to an inflammatory state and, as mentioned previously, this decreases HDL levels. Although most commonly known for its important role in preventing atherosclerotic disease, additional physiologic functions include activity as an antiinflammatory, antiapoptotic, and antioxidant agent. However, lesser-known roles include preventing thrombosis through binding to ECs to ramp up nitric oxide (a vasodilatory molecule) production and preventing shear stress-induced vWF self-association, thus decreasing prothrombotic risk (24, 32). Interestingly, a retrospective analysis of total cholesterol, LDL and HDL levels of patients in Changsha, China showed that HDL levels were lower in patients with COVID-19 than normal and patients with severe disease had lower HDL levels than patients with mild disease (33). Beyond the general infectious inflammatory state that may reduce HDL levels, a study showed that patients with COVID-19 had reduced apolipoprotein A1 (ApoA1) levels, which is a major protein component of HDL molecules (34). The study also showed that as patients went from nonsevere to severe disease, apolipoprotein decreased. Indeed, it has been shown, both in vivo and vitro models, that ApoA1 prevents vWF self-association and binding to vessel walls (32). Additional studies in the future could shed light on the role of HDL in CAC patients and possibly lead to novel treatment options.
vWF Association with Blood Type and COVID-19 Susceptibility
If increased levels of vWF can be monitored as a marker of endothelial damage and used to predict prognosis in patients with COVID-19, then decreased levels of vWF may be protective. One naturally existing population of patients who have baseline lower levels of vWF are patients of blood group O. Group O individuals naturally have a baseline level of vWF ∼25% less than the non-group O cohort (blood groups A and B). Although the exact molecular mechanism by which group O individuals have lower vWF levels is not fully elucidated, it has been hypothesized that perhaps theadditional glycosylation status, which occurs within ECs, by non-group O individuals prevents the activity of ADAMTS-13 to cleave vWF. This leads to reduced clearance and an increased half-life that is demonstrated by baseline higher levels of vWF when compared to group O individuals (31).
Initial data from China found a greater than expected proportion of group A and a smaller than expected proportion of group O individuals among patients with COVID-19. However, this involved a small cohort of patients with limited analysis due to lack of available clinical information (35). Following this, a genome-wide association study on patients in Italy and Spain also found group O individuals to have a lower relative risk than non-group O individuals (36). Another study showed a similar pattern of this phenomenon in a cohort of patients treated at the New York Presbyterian Hospital System (37). However, conflicting information is reported among these and other studies with some reporting no significant difference in severity and some reporting contradicting patterns in terms of need for mechanical ventilation. Preliminary data from these studies do potentially suggest that the lower vWF levels may be associated with decreased severity of disease in group O patients but more data is needed to clarify this relationship.
CAC is a significant contributor to patient morbidity and mortality. We highlight the role of vWF in CAC and compare and contrast it to the normal physiological response, mild and severe COVID-19 disease, and TTP (Fig. 1). Direct infection of ECs with SARS-CoV-2 and/or activation of ECs due to high levels of inflammatory mediators results in release of prothrombotic factors such as vWF. vWF, bound to the ECs or in plasma, promotes platelet aggregation and thrombus formation. It is likely that multiple mechanisms contribute to an imbalance of the vWF-ADAMTS-13 axis, pushing patients with CAC toward a more prothrombotic tendency. For example, in this review we discussed HDL and role it plays in reducing vWF activity, in which little discussion has been seen in other review articles of CAC and vWF. Nevertheless, the range of clinical presentation may be a reflection of the severity of this imbalance since reports show that though vWF is elevated in patients who are both critically ill and noncritically ill (19), there is a significant difference in vWF and ADAMTS-13 levels in patients who suffer thrombotic events versus those that do not (38). Multiple biomarkers, including vWF-associated proteins such as vWF propeptide and P-selectin, may help demonstrate the level of imbalance, as well as the mechanisms causing the imbalance. This would clarify the roles of therapies that would counter the actions of these prothrombotic molecules, whether by mitigating their release by reducing inflammation, such as N-acetylcysteine (39), or by inhibiting their activity once released or activated, such as caplacizumab (anti-vWF) or crizanlizumab (anti-P-selectin). Regardless, vWF has clearly demonstrated that it plays a role in the progression of CAC in patients with COVID-19, however, to what extent remains unclear. Further studies are needed to elucidate the many roles of vWF and the mechanism by which it becomes imbalanced.Fig. 1Open in new tabDownload slide
Proposed mechanism and distinguishing characteristics in mild and severe cases of COVID-19 associated coagulopathy and a comparison to a normal physiological response and thrombotic thrombocytopenic purpura. (A), Normal physiological response to stress and or injury. After endothelial activation, vWF multimers are bound to the endothelial surface, ADAMTS-13 actively cleaves large multimers and HDL assists in the regulation of vWF self-association resulting in well-controlled thrombus formation during a physiologic response. (B), COVID-19 associated coagulopathy in mild disease. Localized infection and minimal systemic inflammation lead to a higher level of endothelial cell activation. Regardless, in this scenario, infection and inflammation remains fairly well regulated. Furthermore, the HDL and ADAMTS-13 mechanisms are mostly intact, leading to only a slight increase of pathologic thrombotic events. (C), COVID-19 associated coagulopathy in severe disease. Infection and or inflammation becomes overwhelmingly dysregulated, leading to an extremely elevated level of endothelial activation. Additionally, both HDL and ADAMTS-13 levels are decreased, leading to a much higher increase risk of pathologic thrombotic events. (D), Thrombotic thrombocytopenia purpura (TTP). In TTP, ADAMTS-13 activity levels are significantly lower than observed in COVID-19 coagulopathy. TTP leads to increased levels of ultralarge and large multimers of vWF. Subsequently, there are increased levels of platelet binding, which leads to highly increased thrombotic risk.
All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and(d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.
Authors’ Disclosures or Potential Conflicts of Interest:Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:Employment or Leadership: H.P. Pham, University of Southern California. Consultant or Advisory Role: H.P. Pham, Sanofi Genzyme. Stock Ownership: None declared. Honoraria: H.P. Pham, Alexion. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared.
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Coronavirus disease 2019 (COVID-19) is mainly a respiratory tract disease and acute respiratory failure with diffuse microvascular pulmonary thrombosis are critical aspects of the morbidity and mortality of this new syndrome.
The aim of our study was to investigate, in severe COVID-19 hospitalized patients, the P-selectin plasma concentration as a biomarker of endothelial dysfunction and platelet activation.
46 patients with severe or critical SARS-CoV-2 infection were included in the study. Age-matched patients then were divided in those requiring admission to the intensive care unit (ICU, ICU cases) vs those not requiring ICU hospitalization (non-ICU cases). Blood samples of severe COVID-19 patients were collected at the time of hospital admission. The quantification of soluble P-selectin was performed by ELI, assay.
Our study showed a higher P-selectin plasma concentration in patients with Covid-19, regardless of ICU admission, compared to the normal reference values and compared to ten contextually sampled healthy donors (HD); (COVID-19): median 65.2 (IQRs: 45.1–81.1) vs. HD: 40.3 (IQRs: 24.3–48.7), p=0023. Moreover, results showed a significant reduction of P-sele din after platelets removal in HD, in contrast, both ICU and non-ICU COVID-19 patients showed similar high levels of P-selectin with and without platelets.
Elevation of P-selectin suggests a central role of platelet endothelium interaction as part of the multifaced pathogenic mechanism of COVID-19 leading to the local activation of hemostatic system forming pulmonary thrombi. Further work is necessary to determine the therapeutic role of antiplatelets agents or of the anti P-selectin antibody Crizanlizumab.Keywords: P-selectin, Covid-19, Endothelium, PlateletsGo to:
Despite a worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection approaching, in January 2021, one hundred million cases and two million deaths, this disease’s pathophysiology remains inadequately defined and largely ununderstood.
COVID-19 is mainly a respiratory tract disease, and acute respiratory failure and diffuse microvascular pulmonary thrombosis are critical aspects of the morbidity and mortality of the coronavirus disease 2019 (Covid-19).1 However, both autopsy findings and clinical observations have described vascular damages and thrombotic complications in a wide range of organs.
Available published data suggest that from one-third to one-half of patients hospitalized with COVID-19 have hemostatic laboratory parameters suggestive of a pro-thrombotic state leading to a coagulopathy. These patients also manifest a hyperinflammatory state characterized by elevated inflammatory markers, strongly associated with severe pneumonia and a high mortality rate.3
SARS-CoV-2 enters human cells by binding to the angiotensin-converting-enzyme 2 (ACE2) receptor, expressed on respiratory epithelial cells and other cell types, including endothelial cells.2
Direct infection of endothelial cells, as well as the inflammatory environment, might result in an endothelial activation that drives the expression of P-selectin and tissue factor (TF), thus promoting platelet recruitment and aggregation.4 Subsequent accumulation of mononuclear cells provides a platform for the initiation of plasma coagulation by triggering prothrombin’s cleavage to thrombin and fibrin formation.5
The molecular interaction between P-selectin expressed in platelets and endothelial cells rapidly triggers TF exposure on monocytes,6 and this may represent a mechanism by which platelets and mononuclear cells contribute to disproportionate intravascular micro-thrombosis in SARS-CoV-2.
The aim of our study was to investigate, in COVID-19 hospitalized patients compared to healthy adult human controls, the ex-vivo P-selectin plasma concentration as a biomarker of endothelial dysfunction and platelet activation. The association between this parameter at the time of hospital admission and the severity and the outcomes of the disease with subsequent admittance into the intensive care unit (ICU) was finally assessed.
A group of 46 patients with confirmed SARS-CoV-2 infection, admitted to our Institute between March and April 2020, was included in the study. All enrolled patients had severe illness (respiratory rate >30, SpO2 <93% on room air at sea level, PaO2/FiO2 <300, or lung infiltrates >50%), or critical illness (association of acute respiratory distress syndrome (ARDS), septic shock, cardiac dysfunction, cytokine storm and/or exacerbation of underlying comorbidities. Age-matched patients were then divided into those requiring admission to the intensive care unit (ICU, ICU cases) vs. those non requiring ICU hospitalization (non-ICU cases). A significant effort was made to exclude from the study population those with prior administration of anti-platelet agents or anticoagulant drugs.
A group of ten age-matched healthy donors (HD) were enrolled in the study as controls. Characteristics of enrolled patients are described in Figure 1.
The expression of P-selectin and Annexin V on platelet surface was evaluated in plasma samples by flow cytometry (A). The removal of platelets/vescicles in EV-free plasma samples was confirmed by flow cytometry (B).
Material and Methods
Blood samples of severe COVID-19 patients were collected at the time of hospital admission. Heparin peripheral blood was centrifuged at 1200 rpm for 10 minutes at room temperature to obtain plasma samples containing extracellular vesicles and platelets (Plasma). After that, 500 ul of plasma samples were further centrifuged at 5000 rpm for 5 minutes at room temperature to eliminate platelets and extracellular vesicles (EV-free plasma). To verify the removal of platelets/vesicles in EV-free plasma, we performed a flow cytometry analysis. Specifically, plasma and EV-free plasma were stained with P-selectin and Annex V for 15 minutes at room temperature and then acquired to a FACS Canto II cytometer (Figure 2). The quantification of soluble P-selectin was performed by ELISA assay (R&D system; average value in heparin plasma: mean 39 ng/ml (range: 25–53).
Clinical features of enrolled COVID-19 patients (A). Soluble P-selectin was quantified in plasma samples (B–C) and in extracellular-free plasma samples (EV-free, C) from healthy donors (HD, n=10), ICU (n=27) and in non-ICU (n=19) COVID-19 patients by ELISA assay. Data were compared by Mann-Whitney test. * p<0.05 was considered significant.
Our study showed a higher P-selectin plasma concentration in patients with Covid-19, regardless of ICU admission, compared to the normal reference values and compared to contextually sample healthy donors; (COVID-19): median 65.2 (IQRs: 45.1–81.1) vs. HD: 40.3 (IQRs: 24.3–48.7), p=0.0023). Moreover, results showed a significant reduction of P-selectin after platelet removal in HD, suggesting that most of this molecule was trapped in the platelets. In contrast, both ICU and non-ICU COVID-19 patients showed similar P-selectin levels with and without platelets, suggesting that Covid-19 induced a release of these molecules from activated platelets/cells (Figure 1C). A similar platelet count has been observed in the two groups ranging within the standard value (150–400/mmc). More significantly lower lymphocyte count was observed in ICU patients, confirming an association between lymphocytopenia and disease severity.6,7
Our results suggest a central role of platelet endothelium interaction as part of the multifaced pathogenic mechanism of COVID-19, leading to the local activation of the hemostatic system forming pulmonary thrombi. More, these interactions amplify the leukocyte recruitment, increasing chemokine expression on the endothelial surface with extensive adhesion, activation, and leukocyte trafficking across the endothelial wall.8
It will be interesting to examine whether therapies inhibiting platelet-endothelium interaction or inhibiting platelet function might improve microvascular perfusion, reduce thrombo-inflammation, and finally reduce COVID-19 morbidity and mortality.
In this perspective, we suggest studying, in the early phases of COVID-19 disease, the role of anti-platelet agents, acetylsalicylic acid, GPIIb, GPIIIa antagonists, and P2Y12 antagonists, not only in de novo therapy initiation but also in patients previously in prophylaxis or in treatment for cardiovascular disorders. The suggested mechanism to study is not only the direct P-selectin/platelet interaction but also the neutrophil extracellular trap (NET) production as described in sepsis and transfusion-related acute lung injury (TRALI).9,10,11 Further, Crizanlizumab-tmca, a selectin blocker humanized IgG2 kappa monoclonal antibody that binds to P-selectin, and approved to reduce the frequency of vaso-occlusive crises (VOCs) in adult and pediatric patients, might be evaluated in severe cases not responding or in combination to anti-platelet therapy.12,13
Supported by The Italian Ministry of Health (Ricerca Corrente Linea 1, COVID-2020-12371735 and COVID-2020-12371817). All Authors have reviewed and approved the manuscript. All authors have reviewed the authorship policy. No author has any conflicts of interest related to this work.
We gratefully acknowledge the Collaborators Members of INMI COVID-19 study group: Maria Alessandra Abbonizio, Amina Abdeddaim, Chiara Agrati, Fabrizio Albarello, Gioia Amadei, Alessandra Amendola, Mario Antonini, Tommaso Ascoli Bartoli, Francesco Baldini, Raffaella Barbaro, Bardhi Dorian, Barbara Bartolini, Rita Bellagamba, Martina Benigni, Nazario Bevilacqua, Gianlugi Biava, Michele Bibas, Licia Bordi, Veronica Bordoni, Evangelo Boumis, Marta Branca, Donatella Busso, Marta Camici, Paolo Campioni, Maria Rosaria Capobianchi, Alessandro Capone, Cinzia Caporale, Emanuela Caraffa, Ilaria Caravella, Fabrizio Carletti, Concetta Castilletti, Adriana Cataldo, Stefano Cerilli, Carlotta Cerva, Roberta Chiappini, Pierangelo Chinello, Carmine Ciaralli, Stefania Cicalini, Francesca Colavita, Angela Corpolongo, Massimo Cristofaro, Salvatore Curiale, Alessandra D’Abramo, Cristina Dantimi, Alessia De Angelis, Giada De Angelis, Maria Grazia De Palo, Federico De Zottis, Virginia Di Bari, Rachele Di Lorenzo, Federica Di Stefano, Gianpiero D’Offizi, Davide Donno, Francesca Faraglia, Federica Ferraro, Lorena Fiorentini, Andrea Frustaci, Matteo Fusetti, Vincenzo Galati, Roberta Gagliardini, Paola Gallì, Gabriele Garotto, Saba Gebremeskel Tekle, Maria Letizia Giancola, Filippo Giansante, Emanuela Giombini, Guido Granata, Maria Cristina Greci, Elisabetta Grilli, Susanna Grisetti, Gina Gualano, Fabio Iacomi, Giuseppina Iannicelli, Giuseppe Ippolito, Eleonora Lalle, Simone Lanini, Daniele Lapa, Luciana Lepore, Raffaella Libertone, Raffaella Lionetti, Giuseppina Liuzzi, Laura Loiacono, Andrea Lucia, Franco Lufrani, Manuela Macchione, Gaetano Maffongelli, Alessandra Marani, Luisa Marchioni, Raffaella Marconi, Andrea Mariano, Maria Cristina Marini, Micaela Maritti, Alessandra Mastrobattista, Giulia Matusali, Valentina Mazzotta, Paola Mencarini, Silvia Meschi, Francesco Messina, Annalisa Mondi, Marzia Montalbano, Chiara Montaldo, Silvia Mosti, Silvia Murachelli, Maria Musso, Emanuele Nicastri, Pasquale Noto, Roberto Noto, Alessandra Oliva, Sandrine Ottou, Claudia Palazzolo, Emanuele Pallini, Fabrizio Palmieri, Carlo Pareo, Virgilio Passeri, Federico Pelliccioni, Antonella Petrecchia, Ada Petrone, Nicola Petrosillo, Elisa Pianura, Carmela Pinnetti, Maria Pisciotta, Silvia Pittalis, Agostina Pontarelli, Costanza Proietti, Vincenzo Puro, Paolo Migliorisi Ramazzini, Alessia Rianda, Gabriele Rinonapoli, Silvia Rosati, Martina Rueca, Alessandra Sacchi, Alessandro Sampaolesi, Francesco Sanasi, Carmen Santagata, Alessandra Scarabello, Silvana Scarcia, Vincenzo Schininà, Paola Scognamiglio, Laura Scorzolini, Giulia Stazi, Fabrizio Taglietti, Chiara Taibi, Roberto Tonnarini, Simone Topino, Francesco Vaia, Francesco Vairo, Maria Beatrice Valli, Alessandra Vergori, Laura Vincenzi, Ubaldo Visco-Comandini, Pietro Vittozzi, Mauro Zaccarelli.
Competing interests: The authors declare no conflict of Interest.
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