Summary

Introduction

As of June 29, 2020, the global pandemic caused by severe acute respiratory syndrome coronavirus 2 has afflicted more than 10 million individuals worldwide, leading to more than 500 000 deaths.1 The disease consists of an early infectious phase in which the virus enters pulmonary epithelial cells via surface angiotensin-converting enzyme 2 (ACE2) receptors, causing viral pneumonia, followed by a systemic inflammatory phase characterised by respiratory failure and multiorgan dysfunction.2 Derangements in cytokine concentrations including interleukin-6 (IL-6) are believed to play a role in the disease, providing the rationale for the use of tocilizumab in patients with COVID-19.3

Among the more concerning features of COVID-19 infection is a coagulopathy characterised by high D-dimer and fibrinogen concentrations with minor changes in prothrombin time and platelet count.4, 5 This COVID-19-associated coagulopathy leads to a prothrombotic state, with venous thromboembolism prevalence of up to 69% in critically ill patients, irrespective of the use of pharmacological thromboprophylaxis, as well as reports of arterial thrombosis.6, 7 In autopsy series, microvascular thrombosis of the pulmonary vasculature has frequently been observed.8, 9

A single-centre study from China found a strong association of disseminated intravascular coagulation and mortality in patients with COVID-19.4 However, other studies have suggested that the pathophysiology of COVID-19-associated coagulopathy is distinct from disseminated intravascular coagulation.5 The presence of endotheliitis and viral inclusions in endothelial cells, as reported in small autopsy series of patients with COVID-19, coupled with identification of the ACE2 receptor on vascular endothelial cells, has raised suspicion for endothelial cell injury or activation as a central feature of the pathophysiology of COVID-19, particularly during the inflammatory phase of the disease.9, 10 In an effort to better understand the pathological mechanisms underlying COVID-19-associated coagulopathy, we performed a biochemical study that, to our knowledge, for the first time assessed markers of endothelial cell and platelet activation in non-critically ill and critically ill patients with COVID-19. The primary objective of our study was to do exploratory analyses of haemostatic factors and markers of endothelial cell and platelet activation in patients with COVID-19 admitted to the intensive care unit (ICU) and those with COVID-19 not admitted to ICU. The secondary objective was to assess the relationship between these markers and clinical outcomes, including in-hospital mortality and discharges from the hospital.

Methods

Results

68 adult patients (48 ICU, 20 non-ICU) were included in the final study cohort. In addition, 13 non-hospitalised, asymptomatic controls (five men and eight women, with a mean age of 48 years [SD 10; range 30–65]) were included as a comparator group for measurements of specific endothelial cell and platelet biomarkers that did not have standard reference ranges. Except where noted, all coagulation parameters were measured in all 68 patients, whereas endothelial cell and platelet biomarkers were measured in 50 patients (40 ICU and ten non-ICU) for whom blood remained after coagulation testing. Laboratory studies were done from April 13 to April 24, 2020. Obesity, hypertension, hyperlipidaemia, and diabetes were common, affecting up to a half of all patients (table 1 ). Three (4%) patients had active malignancy and three (4%) had cirrhosis, while one (1%) patient had a history of cirrhosis that resolved after liver transplant; all such patients were in the ICU cohort. 41 (60%) patients were on prophylactic-dose anticoagulation, 13 (19%) on intermediate-dose anticoagulation (enoxaparin 0·5 mg/kg twice daily), and 11 (16%) on therapeutic anticoagulation; three (4%) patients were not anticoagulated (appendix). 48 (71%) patients, including all but two ICU patients, had received tocilizumab before their coagulation factors were measured. Among all patient characteristics analysed, only sex showed a significant difference in distribution between ICU and non-ICU subgroups (table 1).

In terms of the measurement of coagulation factors, endogenous anticoagulants, and fibrinolytic enzymes (figure 1 ; table 2 ), there were near-universal elevations in PAI-1, with preserved α₂-antiplasmin activity among both ICU and non-ICU patients. Non-ICU patients had elevated VWF antigen, VWF activity, and factor VIII activity, with 16 (80%) patients, 15 (75%) patients, and 18 (90%) patients above the normal range, respectively. These levels were further elevated in ICU patients, with 35 (73%) ICU patients having VWF activities above the limit of detection (figure 1). For endogenous anticoagulants, antithrombin activity, protein C activity, and protein S activity in ICU and non-ICU patients were generally preserved (figure 1; table 2). Five (7%) patients had an antithrombin activity below 70%, all of whom were in the ICU and had cirrhosis or bacterial or fungal sepsis. D-dimer and TAT concentrations were elevated in the entire study cohort, with significantly higher values in ICU patients compared with non-ICU patients (figure 1; table 2). D-dimer and TAT were also significantly correlated in the entire study cohort (r=0·49; p=0·0001; appendix).

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Figure 1

Comparisons of select haemostatic factors in ICU vs non-ICU patients

Datapoints indicate individual measurements, whereas horizontal bars show mean (SD) for α₂-antiplasmin, VWF antigen, and factor VIII and median (IQR) for PAI-1, D-dimer, TAT, and VWF activity. Green shaded areas indicate the normal range of values. ICU=intensive care unit. ULN=upper limit of normal. LLN=lower limit of normal. FEU=fibrinogen equivalent units. PAI-1=plasminogen activator inhibitor-1. TAT=thrombin-antithrombin complexes. VWF=von Willebrand factor. *Thick horizontal bar denotes patients who had measurements above the limit of detection of the assay.

Following the observation of elevated VWF parameters in patients with COVID-19, together with an even greater increase in these levels associated with critical illness, we further explored the role of endothelial cell injury and platelet activation using additional plasma biomarkers. We measured soluble P-selectin (a marker of endothelial cell and platelet activation), sCD40L (a marker of platelet and T-cell activation), and soluble thrombomodulin (a marker of endothelial cell activation) in 40 ICU and 10 non-ICU patients and in 13 non-COVID-19, non-hospitalised controls (table 2; figure 2 ). Both soluble P-selectin and sCD40L were significantly elevated in ICU patients compared with controls (figure 2). Soluble P-selectin concentrations were also significantly higher in ICU patients than in non-ICU patients (figure 2). The increase in soluble thrombomodulin in ICU patients compared with controls was not significant (figure 2). Concentrations of both soluble P-selectin (r=0·36; p=0·012) and soluble thrombomodulin (r=0·48; p=0·0005) were significantly correlated with VWF antigen (appendix), consistent with their common source from endothelial cells. Both VWF antigen and soluble thrombomodulin were significantly correlated with mortality among all patients (r=0·38 for both; p=0·0022 and p=0·0078, respectively), and soluble thrombomodulin remained significantly correlated with mortality when the analysis was restricted to ICU patients (r=0·37; p=0·024; appendix).

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Figure 2

Comparisons of endothelial cell and platelet activation markers in ICU patients, non-ICU patients, and controls

Datapoints indicate individual measurements, whereas horizontal bars show mean (SD) for soluble P-selectin and median (IQR) for sCD40L and soluble thrombomodulin. ICU=intensive care unit. sCD40L=soluble CD40 ligand.

Of the 50 patients in whom soluble thrombomodulin was measured, the median value was 3·26 ng/mL (IQR 2·6–12·4), which was used to define two groups of 25 patients each: low soluble thrombomodulin (<3·26 ng/mL) and high soluble thrombomodulin (>3·26 ng/mL). 22 (88%) patients with low soluble thrombomodulin were discharged from the hospital compared with 13 (52%) patients with high soluble thrombomodulin (χ² test p=0·0050). Among the 40 ICU patients in whom soluble thrombomodulin was measured, patients with low soluble thrombomodulin were also more likely than patients with high soluble thrombomodulin to be discharged from the hospital (14 [82%] of 17 patients vs 11 [48%] of 23 patients; χ² test p=0·026). In a survival analysis, in-hospital mortality was significantly lower among patients with low soluble thrombomodulin compared with patients with high soluble thrombomodulin in the entire cohort (hazard ratio 5·9, 95% CI 1·9–18·4) and among ICU patients (4·5, 1·5–14·0; figure 3 ).

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Figure 3

Kaplan–Meier curve of survival and soluble thrombomodulin concentration

Data are shown for patients with low soluble thrombomodulin (<3·26 ng/mL) and high soluble thrombomodulin (>3·26 ng/mL). Shaded areas represent 95% CIs. ICU=intensive care unit.

Discussion

In this study of laboratory features of COVID-19-associated coagulopathy, we report several findings that are novel, to our knowledge, and suggest that endotheliopathy and platelet activation are important features of COVID-19 in hospitalised patients. Although previous studies have examined VWF elevations exclusively in the ICU setting,5, 11, 12 we report that VWF is also elevated in non-critically ill patients with COVID-19. We also show that critical illness is associated with further elevations in VWF, as well as increases in soluble P-selectin and sCD40L when compared with controls. Together, these results provide biochemical evidence that endotheliopathy and platelet activation are ubiquitous in COVID-19-associated coagulopathy and might play key roles in the progression of disease.

To our knowledge, our study is the first to suggest that a specific marker of endothelial cell injury, soluble thrombomodulin, is associated with hospital discharge status and segregates with survival in patients with COVID-19. This finding requires further validation. Soluble thrombomodulin is an endothelial transmembrane glycoprotein that is released upon endothelial disruption or injury.13 Among healthy individuals, circulating thrombomodulin is thought to be produced by physiological cleavage and shedding of membrane-bound thrombomodulin. By contrast, in hyperinflammatory states, elevated soluble thrombomodulin concentrations are thought to be secondary to direct endothelial cell damage.14 Additional effects of soluble thrombomodulin on the coagulation cascade and fibrinolysis in diseases such as diabetes have also been described.15 Although the relative contribution of thrombomodulin shedding and pathological release of thrombomodulin due to endothelial cell injury or death in COVID-19 will require further investigation, our observation that soluble thrombomodulin is a predictive marker of mortality in COVID-19, coupled with the observed increases in VWF and soluble P-selectin specifically in critically ill patients, support a model in which endotheliopathy is an important event in the transition to critical illness and death in patients with COVID-19.

Our finding of preserved antifibrinolytic α₂-antiplasmin activity in patients with COVID-19, together with the preserved endogenous anticoagulant activity (antithrombin activity, protein C activity, and protein S activity) observed both here and in other studies, supports the notion that COVID-19-associated coagulopathy is mechanistically distinct from disseminated intravascular coagulation.5, 11, 16 PAI-1 has been linked to elevated IL-6 in other contexts and has been suggested to play a role in severe acute respiratory distress syndrome.17, 18 The finding of elevated PAI-1 in critically ill and non-critically ill patients with COVID-19 further supports a picture of endotheliopathy, given the role of endothelial cells as the primary source of PAI-1.19 Elevation of PAI-1 also suggests the possibility that classical fibrinolysis might be inhibited in COVID-19-associated coagulopathy. Measurements of tissue and urokinase-type plasminogen activator and plasminogen levels might help to further assess this, although the interpretation of such levels might not be relevant if the observed elevation in PAI-1 is enough to overcome a possible increase in plasminogen activator.

We therefore propose that COVID-19-associated coagulopathy is an endotheliopathy that results in augmented VWF release, platelet activation, and hypercoagulability, leading to the clinical prothrombotic manifestations of COVID-19-associated coagulopathy, which can include venous, arterial, and microvascular thrombosis. The factors responsible for this endotheliopathy and platelet activation are uncertain but could include direct viral infection of endothelial cells, collateral damage to the tissue as a result of immune infiltration and activation, complement activation, or any number of inflammatory cytokines believed to play a role in COVID-19 disease.3, 9, 10

A central role for endothelial cells and platelets has been characterised in other forms of critical illnesses, including septic shock, acute respiratory distress syndrome, and veno-occlusive disease following haematopoietic stem-cell transplantation, where increased markers of endotheliopathy and platelet activation are observed.20, 21, 22, 23, 24 Disseminated intravascular coagulation also involves substantial endothelial cell and platelet activation but is mechanistically distinct from COVID-19-associated coagulopathy, in part as a result of unmitigated activation of the coagulation cascade and resultant endogenous anticoagulant consumption in disseminated intravascular coagulation.25 In models of septic shock, elevated VWF antigen and activity can be accompanied by reductions in the ADAMTS13 metalloproteinase responsible for cleaving ultra-large VWF multimers into smaller VWF forms, leading to thrombotic microangiopathy via a mechanism distinct from disseminated intravascular coagulation.26 We did not measure ADAMTS13 concentrations in our ICU patients but would predict that they might be similarly reduced.

Our findings of endotheliopathy and platelet activation point to antiplatelet therapy or endothelial cell modification as potential therapeutic targets in addition to traditional anticoagulation targeting thrombin generation. Thus far, no retrospective or prospective studies have identified a beneficial effect of aspirin on COVID-19 disease outcomes. However, dipyridamole, defibrotide, eculizumab, and other agents with endothelial cell-modifying effects might have therapeutic potential.27, 28, 29 In light of our preliminary finding that soluble thrombomodulin might predict mortality in patients with COVID-19, measurements of soluble thrombomodulin could be useful in identifying patients who might benefit the most from these therapies. However, the prognostic potential of this marker should be further validated.

Limitations of our study include the small sample size; the heterogeneity of patient groups, with a wide range of ages, comorbidities, length of hospitalisation; and the fact that each patient's laboratory measures for the purposes of this study were assessed once during their hospital stay, as well as limited generalisability of the findings in a single-centre study. Further studies incorporating serial sampling of relevant biomarkers over time will yield additional mechanistic insights into the biology of endotheliopathy in COVID-19. In addition, the widespread use of tocilizumab, particularly among ICU patients, might have led to an underestimation of the degree of endotheliopathy in ICU patients in this study, as previous literature has reported an association of IL-6 signalling with endothelial cell activation and VWF release, and other studies have shown that IL-6 blockade reduces TAT and PAI-1 concentrations.17, 30 In addition, five patients had blood samples drawn outside of the 0300–0700 h window, which could potentially affect PAI-1 concentrations given the circadian fluctuations of this enzyme; however, in all but one of these instances, PAI-1 was high, indicating that the time of PAI-1 measurement did not have an impact on PAI-1 concentration. Lastly, further evidence of platelet activation, beyond increased circulating concentrations of soluble P-selectin and sCD40L, will be needed to characterise the extent of platelet activation in COVID-19.

In summary, we did an expanded analysis of global haemostatic assays that, to our knowledge, have not been previously reported in both critically ill and non-critically ill patients with COVID-19. Through measurements of VWF, PAI-1, soluble thrombomodulin, soluble P-selectin, and sCD40L, we found that endotheliopathy and platelet activation might be important factors in the pathophysiology of COVID-19-associated coagulopathy. Additional studies are required to elucidate the factors that drive endotheliopathy in critical illness and to explore the possible therapeutic effects of adding antiplatelet or endothelial cell-modifying therapy.

Acknowledgments

Contributors

Declaration of interests

References