key: cord-0702421-fvmnjqr1 authors: Mancini, Ilaria; Baronciani, Luciano; Artoni, Andrea; Colpani, Paola; Biganzoli, Marina; Cozzi, Giovanna; Novembrino, Cristina; Boscolo Anzoletti, Massimo; De Zan, Valentina; Pagliari, Maria Teresa; Gualtierotti, Roberta; Aliberti, Stefano; Panigada, Mauro; Grasselli, Giacomo; Blasi, Francesco; Peyvandi, Flora title: The ADAMTS13‐von Willebrand factor axis in COVID‐19 patients date: 2020-12-18 journal: J Thromb Haemost DOI: 10.1111/jth.15191 sha: d1a5df64d279f111ec64f01c87dd2dff4f9bb574 doc_id: 702421 cord_uid: fvmnjqr1 BACKGROUND: Severe coronavirus disease 2019 (COVID‐19) is characterized by an increased risk of thromboembolic events, with evidence of microthrombosis in the lungs of deceased patients. OBJECTIVES: To investigate the mechanism of microthrombosis in COVID‐19 progression. PATIENTS/METHODS: We assessed von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin‐cofactor (VWF:RCo), VWF multimers, VWF propeptide (VWFpp), and ADAMTS13 activity in a cross‐sectional study of 50 patients stratified according to their admission to three different intensity of care units: low (requiring high‐flow nasal cannula oxygenation, n = 14), intermediate (requiring continuous positive airway pressure devices, n = 17), and high (requiring mechanical ventilation, n = 19). RESULTS: Median VWF:Ag, VWF:RCo, and VWFpp levels were markedly elevated in COVID‐19 patients and increased with intensity of care, with VWF:Ag being 268, 386, and 476 IU/dL; VWF:RCo 216, 334, and 388 IU/dL; and VWFpp 156, 172, and 192 IU/dL in patients at low, intermediate, and high intensity of care, respectively. Conversely, the high‐to‐low molecular‐weight VWF multimers ratios progressively decreased with increasing intensity of care, as well as median ADAMTS13 activity levels, which ranged from 82 IU/dL for patients at low intensity of care to 62 and 55 IU/dL for those at intermediate and high intensity of care. CONCLUSIONS: We found a significant alteration of the VWF‐ADAMTS13 axis in COVID‐19 patients, with an elevated VWF:Ag to ADAMTS13 activity ratio that was strongly associated with disease severity. Such an imbalance enhances the hypercoagulable state of COVID‐19 patients and their risk of microthrombosis. Severe acute respiratory syndrome coronavirus 2 (SARS- is the cause of the ongoing pandemic of coronavirus disease 2019 , which has spread around the world, causing more than 1.1 million deaths and a global health care, social, and economic crisis of unprecedented proportions. 1 COVID-19 is characterized by a wide variety of clinical manifestations, ranging from mild symptoms such as fever and cough to severe forms of pneumonia, potentially leading to acute respiratory distress, multiorgan failure, and death. 2 The identification of biomarkers associated with variable disease severity, besides providing valuable insights into the disease mechanisms in place, are pivotal to help stratifying patients' risk and developing the most efficacious therapies. We, among others, have shown that marked hypercoagulability and perturbation of the endothelium are hallmarks of COVIDdisease, in which a high rate of venous thromboembolism has been consistently reported, particularly in patients with critical illness requiring intensive care. [3] [4] [5] [6] [7] More recently, the hypothesis of pulmonary microvascular thrombosis as a driver of disease worsening has also emerged. [8] [9] [10] Cardiopulmonary findings from 10 autopsies performed on COVID-19 patients showed diffuse alveolar damage, with CD4+ aggregates around thrombosed small vessels and significant associated hemorrhage. Interestingly, evidence of thrombotic microangiopathy in the lungs was found and the pulmonary arteries at the hilum of each lung were free of thromboemboli. In all cases, small platelet-rich thrombi were present within small vessels of the peripheral parenchyma and alveolar capillaries, highlighted by CD61 and von Willebrand factor (VWF) immunostaining. 9 The role of VWF in primary hemostasis is essential, mediating platelet adhesion and aggregation at the sites of vascular injury. 11 VWF is also a marker of endothelium activation, being massively released after inflammation-mediated vascular damage. 12 Metalloprotease with ThromboSpondin 1 repeats, number 13), the severe deficiency of which (activity below 10 IU/dL) is diagnostic for thrombotic thrombocytopenic purpura (TTP), a severe and life-threatening thrombotic microangiopathy caused by the accumulation of hyperactive ultra-large VWF multimers. 13, 14 Reduced levels of this metalloprotease have been reported to be a risk factor for thrombosis in patients with ischemic stroke and myocardial infarction. 15, 16 In line with this, TTP survivors with reduced ADAMTS13 activity during disease remission have a higher risk of TTP-unrelated stroke, 17 and congenital TTP patients treated with prophylactic ADAMTS13 replacement therapy have a reduced risk of ischemic stroke. 18 In addition, the imbalance between high molecular weight VWF multimers and ADAMTS13 could cause a prothrombotic state in inflammatory-induced conditions, as demonstrated in sepsis and overt disseminated intravascular coagulation. [19] [20] [21] Previous work from our group demonstrated an elevated level of VWF in COVID-19 patients, increasing with the severity of the disease. 4, 22 The aim of the present study was to focus on the VWF-ADAMTS13 axis to better understand the pathophysiology of microthrombosis in COVID-19. To do so, we assessed the VWF multimeric pattern, the VWF propeptide, and ADAMTS13 levels in patients with different degrees of disease severity. Peripheral blood samples of COVID-19 patients were collected into 3.2% buffered sodium citrate solution-containing evacuated tubes and centrifuged for 20 minutes at 3000g at controlled room temperature. Hematology testing of VWF antigen (VWF:Ag) and VWF ristocetin-cofactor (VWF:RCo) activity was performed on freshly centrifuged capped evacuated tubes on a fully automated coagulation analyzer. The remaining plasma was aliquoted, nitrogen-frozen, and stored at −80° until use. 24 ADAMTS13 activity was measured using the FRETS-VWF73 assay, as previously described. 25 For all of these assays, results were expressed as IU/dL with reference to a pooled normal plasma calibrated with the relative World Health Organization (WHO) international standard (NIBSC codes 07/316 and 12/252). The VWF multimeric pattern was assessed with the Hydragel 11 von Willebrand multimers kit (H11VWM) using the semiautomated HYDRASYS 2 instrumentation (Sebia, Lisses, France), as previously described. 26 Agarose gel electrophoresis was performed using precast 2% agarose gels, direct immunofixation, and visualization with peroxidase-labeled antibody and a specific sub- Categorical variables were expressed as counts and percentages and continuous variables as medians and ranges. Study groups were compared using the nonparametric Kruskal-Wallis H test. In case of statistical significance, the post hoc Dunn's test for pairwise comparisons followed, and Bonferroni-adjusted P values were reported. To further evaluate the relationship between the analyzed laboratory parameters and the degree of intensive care as an ordinal variable, a Spearman's rank-order correlation was run. Median differences with 95% confidence intervals were estimated using the Hodges-Lehmann method to compare the results of VWF:Ag and ADAMTS13 activity in COVID-19 patients and historical controls. The relationship between these markers and case-control status was also studied by logistic regression analysis before and after adjusting for age and sex. All statistical analyses were performed by SPSS, release 26.0 (IBM Corp.). The baseline characteristics of the 50 COVID-19 patients are reported in Table 1 . The majority of patients were men (64% overall). Median age was 59 years, similar in all intensity care groups. Almost one-half of the patients had at least one comorbidity among hypertension, diabetes, active cancer, or obesity. Among these, hypertension (28%) was the most prevalent, followed by obesity (22%). VWF:Ag levels were markedly increased in COVID-19 patients compared with historical controls (median difference 245 IU/dL) ( Table 2) . Conversely, ADAMTS13 activity levels were reduced (median difference −34 IU/dL), resulting in an even more remarkable increase in the VWF:Ag to ADAMTS13 activity ratio (median difference with historical controls 4.69). The association of these markers with case-control status were maintained after adjusting by age and sex, as tested by logistic regression analysis ( Table 2 ). The results of the laboratory measurements stratified by the degree of care intensity are reported in Table 3 , Figure 1 , and Figure S1 . Italy was the first European country to be hit by the COVID-19 pandemic. Our hospital, one of the major hospitals in Lombardy, became a COVID-19 hub for the management of patients. A high incidence of venous thromboembolic events in COVID-19 patients, between 20% to 50% depending on the intensity of care and the severity of the clinical manifestations, was observed. [5] [6] [7] [27] [28] [29] [30] In agreement with others, we found a state of hypercoagulability driven by high factor VIII levels, and a perturbation of the endothelium, with elevated VWF and complement activation. 3, 4, 22 We thus decided to evaluate in more depth the role of VWF in COVID-19 by assessing multiple biomarkers involved in a high shear microvessel environment, such as platelets, VWF antigen, VWF ristocetin-cofactor activity, VWF multimeric pattern, VWF propeptide, and ADAMTS13, the VWF-cleaving protease. We found a significant alteration in the platelet-VWF-ADAMTS13 axis Note: Results are expressed as median and interquartile range. Median differences with 95% confidence intervals are reported, as well as results of logistic regression analysis before and after adjusting for age and sex. Estimated ORs for 10 (VWF:Ag and ADAMTS13 activity) and 1 (VWF:Ag to ADAMTS13 ratio) unit increase of the independent variable. Median difference with 95% confidence intervals estimated with the Hodges-Lehmann method. Abbreviations: CI, confidence interval; OR, odds ratio; OR 1 : unadjusted; OR 2 : adjusted for age and sex. in COVID-19 patients, with extremely high VWF levels coexisting with normal to high platelet counts and an important increase of three-to seven-fold of the VWF antigen to ADAMTS13 activity ratio associated with the severity of disease. Under high shear stress conditions, VWF plays a key role in mediating platelet adhesion at the sites of vascular injury. 11 However, under normal conditions, VWF and platelets circulate in blood without apparent interactions. This fine balance is regulated by several elements such as the VWF concentration and the size of its molecules, the number of circulating platelets, the changes of blood flow rate (shear stress), 31 multimers with a corresponding relative increase of intermediate and low molecular weight VWF multimers, more pronounced in the most severe cases, in line with the reduced ratio between the VWF ristocetin-cofactor activity and VWF antigen. These findings may be explained by an early increase of VWF proteolysis by ADAMTS13, which must overcome the massive release of VWF multimers by the activated endothelium as a consequence of local inflammation. Hypothetically, ADAMTS13 is ultimately consumed in the frame of this process, reaching the aforementioned reduced levels in the most severe cases. The decrease of high molecular weight multimers may also be explained by the formation of VWFplatelet aggregates which results in the consumption of larger VWF multimers and platelets, as seen in TTP patients at presentation of a first acute event ( Figure 2 ). 33 In this regard, we did not see any evidence of thrombocytopenia in our patients, not even in the most severe cases in contrast to what was previously described. 34 Nevertheless, it is reasonable to speculate that platelets and VWF spontaneously interact in these patients who have extremely elevated levels of VWF antigen and platelet counts, which, despite being normal in the majority of patients, tend to exceed or exceed the normal range upper limit in the most severe cases. In severe COVID-19, microthrombi may form onto the mem- The increase of VWF antigen paralleled by decreased ADAMTS13 activity resulted in a three-to seven-fold higher VWF to ADAMTS13 ratio than in historical controls. To further investigate the central role of the endothelium in the progression into severe COVID-19, we also measured the VWF propeptide that, not being consumed by platelet aggregation or influenced by blood group, 42 represents a more accurate marker of endothelial activation. Furthermore, because of the equimolar secretion but differential half-lives of the two polypeptides, 43 the VWF propeptide to VWF antigen ratio has been used as a tool to distinguish between acute or chronic vascular perturbation (ie, increase of both markers as seen in acute thrombotic microangiopathy or sepsis vs increase in VWF antigen alone as seen in diabetes) 24 and to evaluate VWF clearance. 41 and high molecular weight multimers (HMWM, peaks 8 and above) are indicated on the right side of the blot image. The solid gray line and the pink line depict densitograms of pooled normal plasma and patient plasmas, respectively disease. Notwithstanding these limitations, we identified new potential markers of disease severity and provided further evidence supporting inflammatory microthrombogenesis in patients with severe COVID-19. In conclusion, our data do not suggest a dysfunctional VWF-ADAMTS13 axis but rather a quantitatively imbalance between the substrate and the enzyme, with a seven-fold increased VWF antigen to ADAMTS13 activity ratio associated with severe COVID-19 that required high-intensity care and mechanical ventilation. Based on these laboratory data, COVID-19 microangiopathy does not resemble TTP, but rather a microangiopathy secondary to sepsis (with functional ADAMTS13, perhaps reduced from consumption), needed to tackle huge VWF plasma levels. The VWF-ADAMTS13 imbalance further increases the hypercoagulable state promoted by COVID-19 disease and the risk of microthrombosis in these patients. The authors thank Dr. L.F. Ghilardini for his help in preparing the figures. This work was partially supported by the Italian Ministry of Health -Bando Ricerca Corrente and partially financed by Italian fiscal contribution "5x1000" 2017 devolved to Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. 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