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Online Clinical Investigations

von Willebrand Factor Multimer Formation Contributes to Immunothrombosis in Coronavirus Disease 2019

Doevelaar, Adrian A. N. MD1; Bachmann, Martin MD2; Hölzer, Bodo MD1; Seibert, Felix S. MD1; Rohn, Benjamin J. MD1; Bauer, Frederic MD1; Witzke, Oliver MD3; Dittmer, Ulf MD4; Bachmann, Michael MD5; Yilmaz, Serap MD2; Dittmer, Rita MD6; Schneppenheim, Sonja MD6; Babel, Nina MD1; Budde, Ulrich MD6; Westhoff, Timm H. MD1

Author Information
doi: 10.1097/CCM.0000000000004918

Abstract

Coronavirus disease 2019 (COVID-19) is associated with thrombotic complications affecting both the venous and arterial vasculature. This process is commonly described as “immunothrombosis.” Venous thromboembolism occurs in up to one third of patients in intensive care (1). Arterial thromboembolism events, such as stroke, limb ischemia, and myocardial infarction, have been described in COVID-19 as well (2,3). In addition to these macrovascular events, autopsy studies have demonstrated microvascular thrombosis in the lungs (4,5). The pathogenesis of severe acute respiratory syndrome severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–induced immunothrombosis is incompletely understood. On one hand, endothelial injury is supposed to play a central role (6), on the other, blood hyperviscosity and an imbalance of pro- and antithrombotic factors have been reported including elevated factor VIII and fibrinogen (7–9). The increasing number of reports on thrombotic events despite plasmatic anticoagulants in therapeutic doses indicates a pivotal mechanism beyond plasmatic coagulation.

In some of our patients with COVID-19, we observed laboratory findings of microangiopathic hemolysis. Simultaneously, first cases of thrombotic microangiopathy (TMA) were reported by other groups in COVID-19 patients (10,11). To date, the pathophysiology of TMA in COVID-19 remains elusive. Based on the mechanisms underlying TMA in general, complement mediation and a deficiency in a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) may be considered. We investigated whether the latter mechanism may play a pivotal role in SARS-CoV-2 associated immunothrombosis.

As a blood glycoprotein involved in hemostasis von Willebrand factor (vWf) is produced by endothelial cells and megakaryocytes (12). Endothelial cell activation is associated with the elevation of vWf. Accordingly, an increasing number of reports describe increased concentrations of vWf in COVID-19 (13,14). The plasma protease ADAMTS13, a zinc-containing metalloprotease, cleaves the large string-like molecules of vWf and is necessary to avoid accumulation of vWf multimers in the blood stream. In patients with ADAMTS13 deficiency, accumulated vWf multimers cause thrombotic thrombocytopenic purpura (TTP). So far, ADAMTS13 has rarely been investigated in the context of COVID-19 and, in contrast to TTP, was normal or only mildly reduced (11,15,16). We therefore hypothesized that a massive elevation of vWf with clinically relevant vWf multimer abnormalities in COVID-19 patients may exceed the enzymatic capacity of ADAMTS13, which could contribute to the SARS-CoV-2–associated immunothrombosis.

To test this, we performed a prospective controlled trial on vWf antigen (vWf:Ag), ADAMTS13 activity, and vWf multimer formation in a cohort of 75 COVID-19 patients. Thirty healthy subjects served as control.

METHODS

We enrolled 75 patients, who had tested positive for SARS-CoV-2 by reverse transcriptase-polymerase chain reaction (PCR) analysis of respiratory specimens (nasopharyngeal swab test or bronchoalveolar lavage). Patients were recruited at Ruhr-University Bochum, University of Duisburg-Essen, and Asklepios Klinikum Hamburg Harburg, Germany. The severity of COVID-19 ranged from mild to critical and was categorized according to the guidelines of the Robert Koch Institute, Germany. Patients with moderate and severe COVID-19 were recruited after the first symptoms occurred, and a positive SARS-CoV-2 PCR result, in median 4 days after the diagnostic test, was available. For patients with critical disease, the recruitment took place at the ICU, being diagnosed with COVID-19 in median 14 days before. In this first approach, vWf:Ag and ADAMTS13 activity were analyzed at one time point. The study was approved by the ethical committees of Ruhr-University Bochum (20-6886), University Hospital Essen (20-9214-BO), and the Medical Association Hamburg. Demographic and clinical characteristics of patients are summarized in Table 1.

Measurement of ADAMTS13 Activity, vWf:Ag, and vWf Multimer Analysis

ADAMTS13 activity (%) was analyzed from citrate-plasma using Technozym ADAMTS13 enzyme-linked immunosorbent assay (ELISA) (Technoclone, Vienna, Austria) (17). vWf:Ag (IU/mL) was measured using a sandwich ELISA with polyclonal antibodies (18). vWf multimer analysis was performed via sodium dodecyl sulfate agarose gel electrophoresis including a control sample containing normal vWf in each run to ensure proper conditions of the separation and blotting apparatus (19). We chose gels with intermediate resolution because they are a good compromise between low resolution gels, which only separate small (1–5), intermediate (6–10), large (10–15), and ultralarge (> 15) multimers, and high resolution gels that show the distribution of the proteolytic subbands. Our intermediate gels show the distribution of subbands as well as the whole range of multimers. Assessment of vWf multimer distribution was performed both via qualitative inspection and quantitative measurement via densitometry. The ADAMTS13/vWf:Ag ratio was calculated as (ADAMTS13 [IU/mL]/vWf:Ag [IU/mL] × 100). A ratio below 10 reflects the clinical situation of patients with acquired TTP, where the vast majority of patients show ADAMTS13 activities below 10%. It is well known that a residual activity above 10% is sufficient to cleave the potentially harmful ultralarge vWf multimers. In the clinical situation of COVID-19 patients, the imbalance between the ADAMTS13 activity in the low reference range and the massively enhanced vWf leads to a TTP-like situation, whenever the ADAMTS13/vWf ratio falls below 10.

Statistics

Data are presented as mean ± sd. Baseline differences in severity groups were compared by chi-square tests for dichotomic variables and by unpaired two-tailed t tests for continuous variables. Differences in ADAMTS13 activity, vWf:Ag, and ADAMTS13/vWf:Ag ratio between patients with COVID-19 and healthy controls and between surviving patients and patients, who ultimately died from COVID-19, were investigated by unpaired two-tailed t tests. Receiver operating characteristic (ROC) curves were built to assess the predictive value for death from COVID-19. Univariate linear regression analysis was performed to investigate the association of age and laboratory findings on the ADAMTS13/vWf:Ag ratio in COVID-19 patients. p value of less than 0.05 was regarded significant. Statistical analyses were performed using SPSS Statistics 25 (IBM, Chicago, IL) and Prism 8 (Graph Pad, San Diego, CA).

RESULTS

We enrolled 75 patients with SARS-CoV-2 infection and 30 healthy controls in the study. Measurements of vWf:Ag and ADAMTS13 were conducted successfully in all the patients. Mean age of the COVID-19 population was 66 ± 16 years. Gender distribution was homogeneous with n = 38 being female (50.7%). Three patients (4%) had mild, 26 (34.7%) moderate, 28 (37.3%) severe, and 18 (24%) critical disease. Thirteen patients (17.3%) died. The control population had a mean age of 39 ± 15 and was predominantly female. Table 1 summarizes demographic and clinical characteristics of the study population.

TABLE 1. - Clinical Characterization of the Patients With Coronavirus Disease 2019
Variables COVID-19 Population (n = 75)
Age (yr), mean ± sd 66 ± 16
Female, n (%) 38 (50.7)
Male, n (%) 37 (49.3)
Severity of COVID-19, n (%)
 Mild 3 (4)
 Moderate 26 (34.7)
 Severe 28 (37.3)
 Critical 18 (24)
Laboratory findings, mean ± sd
 Serum creatinine concentration  (mg/dL) 1.15 ± 0.78
 Glomerular filtration rate (mL/min,  Modification of Diet in Renal Disease) > 60
 C-reactive protein (mg/dL) 9.0 ± 7.6
 Lactate dehydrogenase (U/L) 356.6 ± 157.4
 Hemoglobin (g/L) 11.6 ± 2.5
 WBC count (/nL) 8.3 ± 5.3
 Platelet count (/nL) 257. ± 182.7
COVID-19 = coronavirus disease 2019.

Table 2 presents the coagulation variables of the study population and the control group. The vWf:Ag was substantially increased compared with the control group (4.03±2.18 IU/mL vs 0.99 ± 0.31 IU/mL; p < 0.0001) (Fig. 1A). Analysis of variance (ANOVA) analysis showed no significant difference between the degrees of severity. ADAMTS13 activity was comparable to healthy controls (67.8% ± 22.4% vs 73.9% ± 15.5%; p = 0.18) (Fig. 1B). There was a significant difference in ADAMTS13 activities, however, among the different severity degrees of COVID-19 (ANOVA p = 0.001). The ratio of ADAMTS13/vWf:Ag was substantially lower in COVID-19 patients than in healthy controls (24.4 ± 20.5 vs 82.0 ± 30.7; p < 0.0001) (Fig. 1C). The ratio of ADAMTS13/vWf:Ag decreased continuously with the degree of COVID-19 severity (ANOVA p = 0.026).

TABLE 2. - Coagulation Variables in Coronavirus Disease 2019 Patients and Healthy Controls
Variables Coronavirus Disease 2019 Population (n = 75), Mean ± sd Healthy Controls (n = 30), Mean ± sd P
d-dimer (mg/L) 3.35 ± 4.23 0.25 ± 0.15 0.0001
Fibrinogen (mg/dL) 536 ± 142 373 ± 52 < 0.0001
Activated partial thromboplastin time (s) 32.0 ± 13.2 29.3 ± 2.6 0.271
International normalized ratio 1.17 ± 0.37 0.97 ± 0.07 0.005
vWf:Ag (IU/mL) 4.03 ± 2.18 0.99 ± 0.31 < 0.0001
ADAMTS13 (%) 67.8 ± 22.4 73.9 ± 15.5 0.176
ADAMTS13/vWf:Ag 24.4 ± 20.5 82.0 ± 30.7 < 0.0001
ADAMTS13 = a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, vWf:Ag = von Willebrand factor antigen.

Figure 1.
Figure 1.:
von Willebrand factor antigen (vWf:Ag) (A), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity (B), and the ratio of ADAMTS13/vWf:Ag (C) in the population infected by severe acute respiratory syndrome coronavirus 2 (n = 75) in total and in dependence of disease severity compared with healthy controls (n = 30). Analysis of variance (ANOVA) was used to test for significant differences in patients with coronavirus disease 2019 (COVID-19). p < 0.05 was regarded significant.

Subgroup analyses for pre-existant diseases showed that patients with congestive heart failure had significantly higher vWf:Ag levels (6.12 ± 1.34 vs 3.90 ± 2.19 IU/mL; p = 0.036) and a lower ADAMTS13/vWf:Ag ratio (14.48 ± 4.55 vs 26.41 ± 20.73; p = 0.004). Other underlying diseases, that is, arterial occlusive diseases, hypertension, diabetes, immunosuppression, chronic lung disease, malignancy, and chronic kidney disease, showed no significant difference in ADAMTS13/vWf:Ag ratio. In binary logistic regression analyses, underlying chronic diseases mentioned above had no significant impact on the outcome in this collective.

Comparing patients who died from COVID-19 with those who survived, ADAMTS13 and ADAMTS13/vWf:Ag ratio were significantly lower in subjects who did not survive COVID-19 (72.6% ± 20.4% vs 45.2% ± 18.0%; p < 0.001 for ADAMTS13 and 26.8 ± 21.4 vs 13.0 ± 10.3; p = 0.001 for ADAMTS13/vWf ratio). vWf:Ag did not significantly differ between survivors and those who died (p = 0.181). ROC analyses for ADAMTS13/vWf:Ag and death from COVID-19 provided an area under the curve (AUC) of 0.232. The results are presented in Figure 2.

Figure 2.
Figure 2.:
Receiver operating characteristic (ROC) curve analysis for death from coronavirus disease 2019 (COVID-19) in dependence of the ratio of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13/von Willebrand factor antigen.

Univariate linear regression analysis was performed to investigate the association of age and laboratory findings on the ADAMTS13/vWf:Ag ratio in COVID-19 patients. Age (T = –3.269; p = 0.002), C-reactive protein (T = –2.777; p = 0.007), platelet count (T = 2.345; p = 0.022), hemoglobin (T = 2.401; p = 0.019), activated partial thromboplastin time (T = –2.576; p = 0.012), and interleukin 33 (T = 2.452; p = 0.017) revealed significant associations with the ADAMTS13/vWf:Ag ratio. In multivariate linear regression for these variables, a significant association remained for age (p = 0.022) and platelet count (p = 0.043). Detailed results can be found in the Supplementary Table (Supplemental Digital Content 1, http://links.lww.com/CCM/G203). Binary logistic regression analysis showed a significant impact of ADAMTS13/vWf:Ag ratio on survival (regression coefficient [B] = 0.092; se = 0.042; p = 0.028). vWf:Ag did not differ between survivors and nonsurvivors and had no significant impact on mortality in binary logistic regression analyses, neither overall nor in any subgroup.

vWf multimers were analyzed by gel analysis and categorized as large (> 10 oligomers), intermediate (6–10 oligomers), and small (1–5 oligomers). Gel analysis was successful in all but one patient. In TTP, large and ultralarge multimers accumulate in the microthrombi and therefore show reduced concentrations in the circulation. Twenty-five percent of the samples showed a normal amount of large vWf multimers (80–100% of the control sample), 50% showed a mild reduction (60–79% of the control sample), and in 25%, they were severely reduced (< 60% of the control sample). Large multimers in COVID-19 patients were significantly lower than in healthy pool samples (68.69% ± 16.16% vs 112.04% ± 13.31%; p < 0.0001). The triplet structure of the small multimers showed smear—an indicator of ADAMTS13 dysfunction—in 39% of the patients. There was a loss of largest multimers of 13–29% in COVID-19 and 18–47% in a cohort of TTP patients who were analyzed at the MEDILYS laboratory. Figure 3 presents a representative gel of a healthy control, three COVID-19 patients with severe disease, and three patients with TTP. It illustrates the similarity of plasmatic multimer composition in COVID-19 and TTP.

Figure 3.
Figure 3.:
Von Willebrand factor multimers in a medium resolution gel (1.8% low-gelling temperature-agarose) of (1) a normal person, (2–4) three patients with coronavirus disease 2019 (COVID-19), (5, 9) a normal plasma pool, and (6–8) three patients with acute thrombotic thrombocytopenic purpura (TTP) prior to initiation of treatment. The gel shows smear in the patient lanes of both COVID-19 and thrombotic TTP. There was a loss of largest multimers of 13–29% in COVID-19 and 18–47% in thrombotic TTP patients.

DISCUSSION

The present study identifies TTP-like vWf multimer patterns as a potential driver of immunothrombosis in COVID-19. In TTP, the ratio of ADAMTS13/vWf:Ag is decreased below a critical level of 10. In this situation, the amount of ADAMTS13 does not suffice to cleave the ultralarge vWf multimers. The present study shows that this scenario occurs in the context of COVID-19 as well. In TTP, the low ADAMTS13/vWf:Ag ratio results from an absolute deficiency of ADAMTS13 with activity levels less than 10%. In the vast majority of TTP patients, ADAMTS13 deficiency is acquired, for example, by antibody formation, whereas in a minority, it is inherited known as “Upshaw-Schulman syndrome” (20,21).

We observed no single case of critical absolute deficiency of ADAMTS13. ADAMTS13 has rarely been measured in COVID-19 before (11,15,16). In line with our findings, these isolated publications do not report a critical deficiency of ADAMTS13 either. Thus, the low ADAMTS13/vWf ratio occurred due to an excess of vWf and not an absolute reduction of ADAMTS13. Whereas there is no absolute deficiency, however, the massive production of vWf exceeds the processing capacity of the protease leading to a relative deficiency of ADAMTS13. Thus, in 18.7% of the study population, the ADAMTS13/vWf:Ag ratio fell below the critical threshold of 10.

The vascular endothelium plays a crucial role in the regulation of hemostaseologic homeostasis. It contributes to a multitude of regulatory functions including liberation of pro- and anticoagulant aggregatory factors, fibrinolysis, vascular permeability, inflammation, and oxidative stress. The present investigation of vWf and ADAMTS13 therefore covers only a specific part of this complex system. Endothelial damage is a consistent finding in autopsy series of patients with COVID-19. It is supposedly mediated both by direct infection of endothelial cells and secondary damage due to microvascular inflammation, for example, mediated by interleukin and other acute phase reactants (22,23). Indeed, however, the exact mechanisms remain elusive. The angiotensin-converting enzyme-2 receptor is expressed by endothelial cells, but there is still no concrete evidence that SARS-CoV-2 enters and damages endothelial cells using this receptor. Besides these mechanisms, complement mediated endothelial injury has been suggested. An in vitro study demonstrated that SARS-CoV-2 spike protein can activate the alternative complement pathway (4,24).

Endothelial injury may thereby constitute a crucial common trait in the multiple organ manifestations of COVID-19. Endothelial activation is associated with an increased production of vWf, which likely explains the massive up-regulation in the present study population. To avoid formation of ultralarge vWf multimers in states of endothelial damage with increased vWf generation, there is an evolutionary preserved large reserve of ADAMTS13. Under physiologic conditions, 10% of the available ADAMTS13 is sufficient to prevent formation of vWf multimers. Our present findings show, however, that the massive increase of vWf in COVID-19 exceeds the reserve of ADAMTS13. In this patient population, vWf levels were elevated to a mean of 4.03 IU/mL and were thus 4.1 times higher than the average vWf levels in the healthy control group population. It may be speculated that the ubiquitous endotheliitis causes the extraordinarily large increase in vWf plasma levels. The ADAMTS13/vWf:Ag ratio is therefore a valuable biomarker to detect a critical relative deficiency of ADAMTS13.

Gel analyses support the hypothesis of an exceeded protease capacity of ADAMTS13: They showed significantly reduced concentrations of large vWf multimers compared with healthy subjects with almost 40% of the COVID-19 patients having the typical TTP pattern of smear in triplet structure analysis of the small multimers. Figure 3 illustrates the similarity in multimer composition of the two diseases despite the normal absolute concentrations of ADAMTS13.

The hypothesis of a pathophysiologic role of relative ADAMTS13 deficiency is further supported by the finding that the ADAMTS13/vWf:Ag ratio predicted mortality in the study cohort. With an AUC of 0.232 in ROC analysis, the ratio demonstrated a quite high predictive value for fatal outcome. Interestingly, ADAMTS13/vWf:Ag continuously decreased with the level of COVID-19 severity. Hence, the ratio is not only a predictor of mortality but also reflects the intensity of morbidity. Furthermore, a clinical relevance is supported by the significant association of the ADAMTS13/vWf:Ag ratio with platelets in regression analysis. Platelet counts are reduced in TMA.

Based on the autopsy findings of multiple venous thromboses and thromboembolisms, plasmatic anticoagulation was recommended for COVID-19 patients (25). In the Hamburg study population, almost all patients who were treated in the ICU and died were autopsied. Interestingly, the patients had multiple thromboses and thromboembolisms even though they all had been treated with heparin or argatroban in therapeutic doses from the beginning of their stay on ICU. These findings support the hypothesis that there must be an additional pathogenic mechanism, which cannot be sufficiently addressed by plasmatic anticoagulation therapies.

The present study identifies a massive increase of vWf with relative deficiency of ADAMTS13 as a candidate mechanism of TMA in COVID-19. Besides this mechanism, there is some hypothesis generating data indicating that complement activation might contribute to TMA as well. Complement activation has been reported in mouse models of severe acute respiratory syndrome (26). More importantly, histologic studies of SARS-CoV-2 pneumonitis revealed septal capillary luminal fibrin accumulation and accumulations of terminal complement components C5b-9 and C4d in the microvessels, consistent with an activation of the alternative complement pathway (4,27). Attempts to address complement overactivation in COVID-19 with eculizumab are ongoing. Finally, tissue factor–enriched neutrophil extracellular traps were suggested as drivers in COVID-19 immunothrombosis (28).

Our findings suggest two further therapeutic approaches. First, caplacizumab has recently been approved for acquired TTP. Caplacizumab is an anti-vWf nanobody that inhibits the interactions of ultralarge vWf multimers and platelets (29). Furthermore, plasma exchange may be a promising therapeutic approach for patients with COVID-19 and the laboratory constellation of TMA. Plasma exchange eliminates excessive vWf, delivers ADAMTS13, and—additionally—is able to reduce complement activation. There are already first case series, in which plasma exchange was used in order to attenuate circulating cytokines and inflammatory mediators in critically ill patients with COVID-19. Case series from Barcelona and Heidelberg describe favorable effects on variables of inflammation and clinical outcome (30,31). In a cohort of 31 patients in Oman, 11 critically ill patients underwent plasma exchange. Plasma exchange was associated with higher extubation rates and lower 14 days and 28 days all-cause mortality.(32) In line with our findings, these reports provide a rationale to consider plasma exchange as a promising rescue therapy and to perform prospective trials that are now under way. In light of the present findings, the contributing centers in Hamburg and Herne have successfully started to offer plasma exchange as rescue therapy to individual patients.

Our study is limited by the cross-sectional assessment of vWf:Ag and ADAMTS13. Future studies should address the dynamics of vWf and ADAMTS13 during COVID-19. Furthermore, the study focused on ADAMTS13 activity and vWf antigen. Future studies should encompass ADAMTS13 antigen and VWF activity as well and will have to confirm the critical importance of the ADAMTS13/vWf:Ag ratio in larger patient cohorts.

Without therapeutic interventions, the mortality of patients with TMA is high. The present study enlarges our understanding of the mechanisms underlying TMA in COVID-19. It appears reasonable to include vWf:Ag and ADAMTS13 in the diagnostic workup of COVID-19, at least in laboratory constellations of microangiopathic hemolysis. Until data from prospective clinical trials on the use of plasma exchange in COVID-19 are available, clinical decision for its use should be made in an individualized manner.

ACKNOWLEDGMENTS

We thank the laboratory staff Kerstin Will, Claudia Fiedelschuster, Barbara Schocke, and Dr. Antje Pieconka of MEDILYS, Hamburg; Krystallenia Paniskaki of the Department of Infectiology, University Hospital Essen, Essen; and Ulrik Stervbo of the Center for Translational Medicine, University Hospital Marien-Hospital Herne, Herne, for their indefatigable efforts in this study in times of excessive routine workloads.

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Keywords:

ADAMTS13; coronavirus disease 2019; immunothrombosis; plasma exchange; severe acute respiratory syndrome coronavirus 2; Von Willebrand factor

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