Plasma Levels Of Von Willebrand Factor Research Articles

Von Willebrand disease and von Willebrand factor.

In the past several decades, knowledge of the underlying pathogenesis of von Willebrand disease (VWD) has increased tremendously, thanks in no small part to detailed genetic mapping of the von Willebrand Factor (VWF) gene and advances in genetic and bioinformatic technology. However, these advances do not always easily translate into improved management for patients with VWD and low-VWF levels. For example, the treatment of pregnant women with VWD both pre- and postpartum can be complicated. While knowledge of the VWF genotype at some amino acid positions can aid in knowledge of who may be at increased risk of thrombocytopenia or insufficient increase in VWF levels during pregnancy, in many cases, VWF levels and bleeding severity is highly heterogeneous, making monitoring recommended during pregnancy to optimize treatment strategies. VWF AND COVID-19: New challenges related to the consequences of dysregulation of hemostasis continue to be discovered. The ongoing COVID-19 pandemic has highlighted that VWF has additional biological roles in the regulation of inflammatory disorders and angiogenesis, disruption of which may contribute to COVID-19 induced vasculopathy. Increased endothelial cell activation and Weibel-Palade body exocytosis in severe COVID-19 lead to markedly increased plasma VWF levels. Coupled with impairment of normal ADAMTS13 multimer regulation, these data suggest a role for VWF in the pathogenesis underlying pulmonary microvascular angiopathy in severe COVID-19. With the increased affordability and availability of next-generation sequencing techniques, as well as a push towards a multi-omic approach and personalized medicine in human genetics, there is hope that translational research will improve VWD patient outcomes.

Dispatch and delivery at the ER-Golgi interface: how endothelial cells tune their hemostatic response.

Von Willebrand factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels.

Splenectomy ameliorates portal pressure and anemia in animal models of cirrhotic and non-cirrhotic portal hypertension

PurposePortal hypertension (PH)-associated splenomegaly is caused by portal venous congestion and splanchnic hyperemia. This can trigger hypersplenism, which favors the development of cytopenia. We investigated the time-dependent impact of splenectomy on portal pressure and blood cell counts in animal models of non-cirrhotic and cirrhotic PH. Materials and methodsNinety-six rats underwent either partial portal vein ligation (PPVL), bile duct ligation (BDL), or sham operation (SO), with subgroups undergoing additional splenectomy. Portal pressure, mean arterial pressure, heart rate, blood cell counts and hemoglobin concentrations were evaluated throughout 5 weeks following surgery. ResultsFollowing PPVL or BDL surgery, the animals presented a progressive rise in portal pressure, paralleled by decreased mean arterial pressure and accelerated heart rate. Splenectomy curbed the development of PH in both models (PPVL: 16.25 vs. 17.93 ​mmHg, p ​= ​0.083; BDL: 13.55 vs. 15.23 ​mmHg, p ​= ​0.028), increased mean arterial pressure (PPVL: +7%; BDL: +9%), and reduced heart rate (PPVL: −10%; BDL: −13%). Accordingly, splenectomized rats had lower von Willebrand factor plasma levels (PPVL: −22%; BDL: −25%). Splenectomy resulted in higher hemoglobin levels in PPVL (14.15 vs. 13.08 ​g/dL, p ​< ​0.001) and BDL (13.20 vs. 12.39 ​g/dL, p ​= ​0.097) animals, and significantly increased mean corpuscular hemoglobin concentrations (PPVL: +9%; BDL: +15%). Thrombocytopenia only developed in the PPVL model and was alleviated in the splenectomized subgroup. Conversely, BDL rats presented with thrombocytosis, which was not affected by splenectomy. ConclusionsSplenectomy improves both cirrhotic and non-cirrhotic PH, and ameliorates the hyperdynamic circulation. Hypersplenism related anemia and thrombocytopenia were only significantly improved in the non-cirrhotic PH model.

Treatment with ddAVP improves platelet-based coagulation in a rat model of traumatic hemorrhagic shock

ObjectivesTrauma-induced hemorrhagic shock is characterized by increased endothelial permeability and coagulopathy. Vasopressin analog ddAVP (desmopressin) acts by reorganizing and redistributing adhesive and tight junction molecules, enhancing endothelial barrier function. Furthermore,...

Osteoprotegerin modulates platelet adhesion to von Willebrand factor during release from endothelial cells

BackgroundPlatelet‐binding Von Willebrand Factor (VWF) strings assemble upon stimulated secretion from endothelial cells. ObjectivesTo investigate the efficiency of platelet binding to multi‐molecular VWF bundles secreted from endothelial cells and to investigate the role of osteoprotegerin, a protein located in Weibel‐Palade bodies that interacts with the VWF platelet binding domain. MethodsThe nanobody VWF/AU‐a11 that specifically binds to VWF in its active platelet‐binding conformation was used to investigate the conformation of VWF. ResultsUpon stimulated secretion from endothelial cells, VWF strings were only partially covered with platelets, while a VWD‐type 2B mutation or ristocetin enhanced platelet binding by 2–3‐fold. Osteoprotegrin, reduces platelet adhesion to VWF by 40% ± 18% in perfusion assays. siRNA‐mediated down‐regulation of endothelial osteoprotegerin expression resulted in a 1.8‐fold increase in platelet adhesion to VWF strings.Upon viral infection, there is a concordant rise in VWF and osteoprotegerin plasma levels. Unexpectedly, no such increase was observed in plasma of desmopressin‐treated hemophilia A‐patients. In a mouse model, osteoprotegerin expression was low in liver endothelial cells of vehicle‐treated mice, and concanavalin A‐treatment increased VWF and osteoprotegerin expression 4‐ and 40‐fold, respectively. This increase was translated in a 30‐fold increased osteoprotegerin/VWF ratio in plasma. ConclusionsRelease of VWF from endothelial cells opens the platelet‐binding site, irrespective of the presence of flow. However, not all available platelet‐binding sites are being occupied, suggesting some extent of regulation. Part of this regulation involves endothelial proteins that are co‐secreted with VWF, like osteoprotegerin. This regulatory mechanism may be of more relevance under inflammatory conditions.

A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD

AbstractType 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willebrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. We developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167 ± 257 mU/mL), but the VWF was completely incapable of binding FVIII, resulting in 53 ± 23 mU/mL of plasma FVIII levels that were similar to those in VWF-deficient (VWF−/−) mice. When wild-type human or mouse VWF was infused into VWF2N/2N mice, endogenous plasma FVIII was restored, peaking at 4 to 6 hours post-infusion, demonstrating that FVIII expressed in VWF2N mice is viable but short-lived unprotected in plasma due to dysfunctional 2N VWF. The whole blood clotting time and thrombin generation were impaired in VWF2N/2N but not in VWF−/− mice. Bleeding time and blood loss in VWF2N/2N mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF2N/2N mice, but not VWF−/− mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that alternative pathways can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.

Examining international practices in the management of pregnant women with von Willebrand disease

BackgroundThe management of pregnant women with von Willebrand disease (VWD) is complex as physiological pregnancy‐induced increases in plasma von Willebrand factor (VWF) may be blunted or absent. Women with VWD experience a heightened risk of postpartum hemorrhage (PPH) and special consideration must be given regarding neuraxial anesthesia (NA) and the need for prophylaxis at time of delivery. These challenges are compounded by a lack of robust evidence to guide clinical decision‐making. Objectives and MethodsTo determine the current international clinical practices in the management of pregnancy for women with VWD, the International Society on Thrombosis and Haemostasis (ISTH) conducted an international survey of health‐care providers (HCP). ResultsOne hundred thirty‐two respondents from 39 countries were included in the final analysis. Variations in clinical practice were identified in antenatal (monitoring of plasma VWF and ferritin levels), peripartum (optimal plasma VWF target at delivery) and postpartum management (definitions used for PPH and postpartum monitoring). A key area of divergence was suitability for NA for women with type 2 and type 3 VWD, with many respondents advising against the use of NA even with VWF supplementation (29% type 2 VWD, 37% type 3 VWD) but others advising use once plasma VWF activity was >50 IU/dL (57% type 2 VWD; 50% type 3 VWD). ConclusionsThis survey highlighted areas of uncertainty surrounding common management issues for pregnant women with VWD. These data underscore the need for international collaborative research efforts focused on peripartum management to improve care for pregnant women with VWD.

Abstract 6931: Epigenetic Modification of the Von Willebrand Factor Promotor Drives in situ Thrombosis in Chronic Thromboembolic Pulmonary Hypertension

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by thromboembolic obstructions of the pulmonary circulation; disease progression may involve in situ thrombosis. CTEPH patients have elevated Von Willebrand Factor (VWF) plasma levels and we hypothesized that endothelial secretion of VWF in CTEPH recruits platelets to the pulmonary vascular wall, thereby driving in situ thrombosis. Methods: CTEPH specific primary pulmonary artery endothelial cells (PAEC) were isolated from pulmonary endarterectomy material and control PAECs from patients undergoing lobectomy. Endothelial-platelet interaction was studied by an human in vitro model of thrombosis. We used an antibody and knockdown mediated approach to inhibit VWF and validate its functional relevance. Cell culture findings were validated in human tissue with immunofluorescence in situ hybridization, and proteomics and chromatin immunoprecipitation were used to investigate the underlying mechanism of CTEPH. Results: CTEPH-PAEC showed increased platelet adhesion compared to control. This was mediated by a 5-fold enhanced VWF gene expression, resulting in higher release of VWF protein upon endothelial activation. The increase in VWF mRNA was confirmed in human pulmonary artery tissue and was associated with elevated Nuclear Factor κB 2 (NFκB2) protein expression. Epigenetic histone modifications of reduced histone tri-methylation (H3K27Me3) and increased histone acetylation (H3K27Ac) of the VWF promotor in CTEPH-PAEC, support NFκB2 binding that drives VWF transcription. Knockdown of NFκB2 normalized VWF RNA expression levels and reversed the pro-thrombotic phenotype observed in CTEPH-PAEC. Conclusions: We show experimental evidence of VWF-mediated in situ thrombosis in CTEPH. Epigenetic modifications of the VWF promotor in CTEPH-PAEC, enhances NFκB2 binding and subsequently VWF transcription and platelet adhesion. We demonstrate a direct molecular link between inflammatory pathways and thrombosis in the pulmonary vascular wall that emphasizes the central role of in situ thrombosis in CTEPH.

Bleeding Outcomes in People with Von Willebrand Disease Receiving Antithrombotic Therapy

▪Aging brings additional challenges in the management of people with von Willebrand Disease (VWD). Plasma von Willebrand Factor (VWF) levels may increase but the impact on bleeding phenotype is unclear. With the development of age-related comorbidities, the use of antiplatelet (AP) or anticoagulant (AC) therapies may be warranted. As highlighted in the 2021 international VWD guidelines, limited evidence exists regarding the bleeding risk and safety of AP/AC use in people with VWD. 1 We sought to address this knowledge gap through a retrospective review of a large cohort of people with VWD attending a tertiary referral center.The records of all patients aged >50 years (y) attending our center registered with VWD were retrospectively reviewed. We identified all individuals treated with AP and/or AC, recording the indication for and duration of therapy. We also recorded disease subtype, baseline and most recent plasma VWF levels and bleeding on AP and/or AC. Bleeding episodes were stratified according to the World Health Organization (WHO) Bleeding Scale.From 255 eligible patients, 18 patients (7 male, 11 female) were identified who received AP and/or AC over a period of 22y. The median age at commencement was 60.2 years (range 40.0-74.5). 15 patients had Type 1 with baseline levels 30-50 IU/dL, (median VWF antigen, VWF:Ag, 57.5, range 39-87 IU/dL; median VWF ristocetin cofactor levels, VWF:RCo, 43, range 35-54 IU/dL). 3 patients had type 2 VWD (median VWF:Ag 64, range 27-90 IU/dL; median VWF:RCo of 16, range 10-66 IU/dL). The type of AP/AC used and indications for treatment are outlined in Table 1.Overall, 12 patients were treated with AP and 7 with AC therapy (19 therapies in total as one patient received first aspirin then warfarin therapy). The cumulative exposure to AP therapy was 61.9y with a median exposure time of 3.2y/patient (range 0.3-14.1y). Duration of AC therapy was shorter, with a cumulative exposure of 17.6y and a median of 1.5 y/patient (range 0.3-6.3y).Overall, 85.7% of patients on AC therapy had at least one episode of bleeding (6/7; 10 episodes total) in contrast to 58.3% of patients on AP (7/12; 12 episodes total). Of these 22 episodes, 5 (22.7%) were grade 1 bleeding. Grade 2 bleeding (iron deficiency or gastrointestinal (GI), gynecological or genitourinary bleeding) occurred in 5 patients (41.7%) treated with AP and 4 patients (57.1%) on AC (total of 14 episodes). 1 episode of grade 3 bleeding occurred in both the AP (8.3%) and AC (14.3%) group (GI bleeding requiring transfusion and abdominal hematoma respectively). The single grade 4 bleed was an intracranial hemorrhage (ICH) and occurred in a patient with type 2 VWD (VWF:RCo 10 IU/dL) on warfarin for atrial fibrillation; this required prothrombin complex concentrate, VWF concentrate, neurosurgical intervention and cessation of AC.Bleeding complications resulted in discontinuation of therapy in 2 patients (11.1%) treated; the individual with ICH and a patient with type 1 VWD on warfarin (baseline VWF:RCo 43 IU/dL) due to recurrent GI bleeding. No patients treated with AP therapy required discontinuation of use. The overall rate of major bleeding (WHO grade >/=3) in our study was 11.4 events/100 patient-years in VWD patients receiving AC therapy, in comparison to the rate of bleeding in the general population using AC of 7.2 events per 100 patient-years. 2For patients with type 1 VWD, plasma VWF levels were seen to increase during follow up (median 8.5y, VWF:Ag median +13 IU/dL, VWF:RCo +18 IU/dL), resulting in plasma VWF levels >50 IU/dL for 66.6% of patients in this cohort. Despite this, bleeding while on AP and/or AC was still experienced in 8/10 patients whose levels had normalized, necessitating cessation in one instance.In conclusion, this study provides important insights into the use of AP and/or AC in patients with VWD. Bleeding rates were higher in patients treated with AC therapy than AP resulting in cessation of therapy in 28.6% of those on AC. Bleeding events still occurred despite normalization of plasma VWF levels in patients with type 1 VWD. These data highlight the need for close follow up of patients with VWD whilst on antithrombotic therapy, particularly AC.1. Connell NT et al. ASH ISTH NHF WFH 2021 guidelines on the management of von Willebrand disease. Blood Adv 2021;5(1):301-325.2. Shoeb M, Fang M. Assessing Bleeding Risk in Patients Taking Anticoagulants. J Thromb Thrombolysis 2013;35(3):312-319. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Elevated Plasma von Willebrand Factor Levels Are Associated With Subsequent Ischemic Stroke in Persons With Treated HIV Infection.

BackgroundWe assessed whether key biomarkers of endothelial activation and hemostasis/thrombosis were elevated in individuals receiving effective antiretroviral therapy (ART) in the year before ischemic stroke.MethodsWe conducted a case–control study nested in the CFAR Network of Integrated Clinical Systems cohort, comparing 42 adjudicated cases with ischemic stroke with 83 controls matched for ART regimen. Angiopoietin-1, angiopoietin-2, C-reactive protein, interleukin-6, plasminogen activation inhibitor–1, P-selectin, serum amyloid–A, soluble CD14, ICAM-1, VCAM-1, apolipoprotein A1, ADAMTS13, and von Willebrand factor (VWF) were measured in stored plasma collected before the stroke event. We used conditional logistic regression to identify associations with ischemic stroke, with and without adjustment for Atherosclerotic Cardiovascular Disease (ASCVD) and Veterans Aging Cohort Study (VACS) scores.ResultsAfter adjustment for age and sex, higher plasma viral load and higher angiopoeitin-2, soluble CD14, and VWF were associated with increased odds of ischemic stroke; higher nadir CD4 count was associated with decreased odds of ischemic stroke. VWF remained associated with subsequent ischemic stroke after adjustment for ASCVD score (adjusted odds, 1.74; 95% CI, 1.01–2.98 per log2 increment). In a separate model adjusting for VACS score, only VWF (adjusted odds, 1.80; 95% CI, 1.04–3.12 per log2 increment) was associated with subsequent ischemic stroke. In a sensitivity analysis excluding participants with viral load ≥400 copies/mL, associations between VWF and ischemic stroke were attenuated, with risk estimates ranging from 1.59 to 1.64 per log2 increment.ConclusionsEndothelial activation and related release and attachment of VWF may play an important role in ischemic stroke among persons with treated HIV infection.

High plasma D-dimer and von Willebrand factor levels are associated with risk of upper gastrointestinal bleeding in patients with chronic coronary syndromes receiving long-term antiplatelet therapy

Abstract Purpose Plasma von Willebrand factor (VWF) and D-dimer levels have been proposed as markers of endothelial damage, coagulation activation and subsequent cardiovascular events. Thrombotic and hemorrhagic burdens are closely related. However, the association of coagulation markers with bleeding complications in pts with atherosclerosis remains unclear. We aimed to assess the predictive value of D-dimer and VWF levels for upper gastrointestinal bleeding (UGIB) in patients with chronic coronary syndromes (CCS) receiving long-term antithrombotic therapy. Patients and methods Single center prospective Registry of Long-term AnTithrombotic TherApy (REGATTA-1 NCT04347200) included 934 pts with CCS (78.6% males, age 61±10.7 yrs, 76% after elective PCI). The UGIB annual incidence was 1.9 events per 100 patient-years. VWF was determined in 28 pts with UGIB and 141 controls, matched for age, sex and main clinical risk factors. D-dimer was determined from 28 and 380 pts respectively Results The median for VWF was 139 [interquartile range 107–168]%. The median for D-dimer was 443,8 [interquartile range 272.5–702.4] ng/ml. ROC analysis revealed acceptable discriminatory ability for VWF (cut off &amp;gt;105%; AUC=0.67, 95% CI: 0.59–0.74, p=0.001) and for D-dimer (cut off &amp;gt;928 ng/ml; AUC=0.63, 95% CI: 0.59–0.68, p=0.002). High levels of coagulation markers remained significant in regression model adjuster for ESC panel of UGIB risk factors. OR for D-dimer = 3.4, 95% CI: 1.09–10.66; p=0.03). OR for VWF = 11.74, 95% CI: 1.19–116.18; p=0.03). Conclusion VWF and D-dimer should be considered as valuable prognostic biomarkers to improve the prediction of UGIB in addition to well-known scoring systems in CCS patients receiving long-term antithrombotic therapy. Funding Acknowledgement Type of funding sources: None.

The Relationship Between ADAMTS13 Activity and Overall Cerebral Small Vessel Disease Burden: A Cross-Sectional Study Based on CSVD.

Objectives: This study aimed to investigate the association between plasma von Willebrand factor (VWF) level, ADAMTS13 activity, and neuroimaging features of cerebral small vessel disease (CSVD), including the CSVD neuroimaging markers and the overall CSVD burden.Methods: CSVD patients admitted to our hospital from 2016 to 2020 were recruited. Plasma VWF level and ADAMTS13 activity were measured. The overall effect of CSVD on the brain was described as a validated CSVD score. We evaluated the association between VWF levels, ADAMTS13 activity, and the increasing severity of CSVD score by the logistic regression model.Results: We enrolled 296 patients into this study. The mean age of the sample was 69.0 years (SD 7.0). The mean VWF level was 1.31 IU/mL, and the ADAMTS13 activity was 88.01 (SD 10.57). In multivariate regression analysis, lower ADAMTS13 activity and higher VWF level was related to white matter hyperintensity (WMH) [β = −7.31; 95% confidence interval (CI) (−9.40, −4.93); p<0.01; β = 0.17; 95% confidence interval (0.11, 0.23); p<0.01], subcortical infarction (SI) [(β = −9.22; 95% CI (−11.37, −7.06); p<0.01); β = 0.21; 95% confidence interval (0.15, 0.27); p<0.01] independently, but not cerebral microbleed (CMB) [(β = −2.3; 95% CI (−4.95, 0.05); p = 0.22); β = 0.02; 95% confidence interval (−0.05, 0.08); p = 0.63]. Furthermore, ADAMTS13 activity was independently negatively correlated with the overall CSVD burden (odd ratio = 21.33; 95% CI (17.46, 54.60); p < 0.01) after adjustment for age, history of hypertension, and current smoking.Conclusions: Reducing ADAMTS13 activity change is related to white matter hyperintensity, subcortical infarction, but not with cerebral microhemorrhage. In addition, ADAMTS13 may have played an essential role in the progression of CSVD.

Pathology and Mechanisms Underlying Thrombus Formation in Stroke

The mechanisms underlying atherothrombus and cardiac thrombus formation are complex and multifactorial. Ischemic stroke is characterized by large thrombus formation and vascular occlusion. The degree of plaque disruption and plaque thrombogenicity determine the size of the atherothrombus. Tissue factor-driven thrombin generation contributes to fibrin formation and platelet aggregation. As opposed to collagen, the von Willebrand factor (VWF) and fibrin may provide the scaffolds required for initial platelet adhesion on ruptured plaques. VWF also contributes to occlusive thrombus formation, following plaque disruption. Acute changes in shear stress and oscillatory shear stress can induce superficial erosive injury of plaques and thrombus formation. Atrial fibrillation causes atrial intimal thickening and thrombogenic factor expression, promotes stasis of blood in the left atrium and the left atrial appendage, and can contribute to thrombogenesis and elevated plasma VWF and fibrinogen levels. Coagulation Factor XI is a target for next-generation anticoagulants because it plays an important role in thrombus growth but not in hemostasis.

Genotypes of European and Iranian patients with type 3 von Willebrand disease enrolled in 3WINTERS-IPS

AbstractType 3 von Willebrand disease (VWD3) is a rare and severe bleeding disorder characterized by often undetectable von Willebrand factor (VWF) plasma levels, a recessive inheritance pattern, and heterogeneous genotype. The objective of this study was to identify the VWF defects in 265 European and Iranian patients with VWD3 enrolled in 3WINTERS-IPS (Type 3 Von Willebrand International Registries Inhibitor Prospective Study). All analyses were performed in centralized laboratories. The VWF genotype was studied in 231 patients with available DNA (121 [115 families] from Europe [EU], and 110 [91 families] from Iran [IR]). Among 206 unrelated patients, 134 were homozygous (EU/IR = 57/77) and 50 were compound heterozygous (EU/IR = 43/7) for VWF variants. In 22 patients, no or only one variant was found. A total of 154 different VWF variants (EU/IR = 101/58 [5 shared]) were identified among the 379 affected alleles (EU/IR = 210/169), of which 48 (EU/IR = 18/30) were novel. The variants p.Arg1659*, p.Arg1853*, p.Arg2535*, p.Cys275Ser, and delEx1_Ex5 were found in both European and Iranian VWD3 patients. Sixty variants were identified only in a single allele (EU/IR = 50/10), whereas 18 were recurrent (≥3 patients) within 144 affected alleles. Nine large deletions and one large insertion were found. Although most variants predicted null alleles, 21% of patients carried at least 1 missense variant. VWD3 genotype was more heterogeneous in the European population than in the Iranian population, with nearly twice as many different variants. A higher number of novel variants were found in the Iranian VWD3 patients.

ADAMTS13 regulation of VWF multimer distribution in severe COVID‐19

BackgroundConsistent with fulminant endothelial cell activation, elevated plasma von Willebrand factor (VWF) antigen levels have been reported in patients with COVID‐19. The multimeric size and function of VWF are normally regulated through A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13 (ADAMTS‐13)‐‐mediated proteolysis. ObjectivesThis study investigated the hypothesis that ADAMTS‐13 regulation of VWF multimer distribution may be impaired in severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) infection contributing to the observed microvascular thrombosis. Patients and MethodsPatients with COVID‐19 (n = 23) were recruited from the Beaumont Hospital Intensive Care Unit (ICU) in Dublin. Plasma VWF antigen, multimer distribution, ADAMTS‐13 activity, and known inhibitors thereof were assessed. ResultsWe observed markedly increased VWF collagen‐binding activity in patients with severe COVID‐19 compared to controls (median 509.1 versus 94.3 IU/dl). Conversely, plasma ADAMTS‐13 activity was significantly reduced (median 68.2 IU/dl). In keeping with an increase in VWF:ADAMTS‐13 ratio, abnormalities in VWF multimer distribution were common in patients with COVID‐19, with reductions in high molecular weight VWF multimers. Terminal sialylation regulates VWF susceptibility to proteolysis by ADAMTS‐13 and other proteases. We observed that both N‐ and O‐linked sialylation were altered in severe COVID‐19. Furthermore, plasma levels of the ADAMTS‐13 inhibitors interleukin‐6, thrombospondin‐1, and platelet factor 4 were significantly elevated. ConclusionsThese findings support the hypothesis that SARS‐CoV‐2 is associated with profound quantitative and qualitative increases in plasma VWF levels, and a multifactorial down‐regulation in ADAMTS‐13 function. Further studies will be required to determine whether therapeutic interventions to correct ADAMTS‐13‐VWF multimer dysfunction may be useful in COVID‐microvascular thrombosis and angiopathy.

Severe COVID‐19 is associated with endothelial activation and abnormal glycosylation of von Willebrand factor in patients undergoing hemodialysis

BackgroundA major clinical feature of severe coronavirus diease 2019 (COVID‐19) is microvascular thrombosis linked to endothelial cell activation. Consistent with this, a number of studies have shown that patients with severe COVID‐19 have highly elevated plasma levels of von Willebrand Factor (VWF) that may contribute to the prothrombotic phenotype. In the current study, we investigated the extent of endothelial activation in patients receiving hemodialysis who had either mild or severe COVID‐19. MethodsPlasma VWF, ADAMTS‐13, angiopoietin‐2 (Ang2), and syndecan‐1 levels were determined by ELISA. The sialic acid content of VWF was investigated using a modified ELISA to measure elderberry bark lectin, specific for sialic acid residues, binding to VWF. ResultsPatients receiving hemodialysis with severe COVID‐19 had significantly higher plasma levels of VWF and lower ADAMTS‐13. VWF levels peaked and were sustained during the first 10 days after positive confirmation of infection. While Ang2 trended toward being higher in severely ill patients, this did not reach significance; however, severely ill patients had significantly higher soluble syndecan‐1 levels, with high levels related to risk of death. Finally, higher VWF levels in severely ill patients were correlated with lower VWF sialic acid content. ConclusionsSevere COVID‐19 in patients undergoing hemodialysis is associated with both acute and sustained activation of the endothelium, leading to alteration of the VWF/ADAMTS‐13 axis. Lower VWF sialic acid content represents altered VWF processing and further confirms the disturbance caused to the endothelium in COVID‐19.

Exome Sequencing Studies Identify Mutations in STAB2 As a Genetic Risk for Venous Thromboembolic Disease

Deep vein thrombosis and pulmonary embolism, collectively referred to as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Genetic factors account for 50-60% of VTE risk and a recent meta-analysis of genome-wide association studies confirmed that common variants in F5, ABO, F2 and seven other very low effect size loci are associated with VTE. Rare mutations in the anticoagulant genes PROC (protein C), PROS1 (protein S) and SERPINC1 (antithrombin III) have been linked to highly penetrant VTE risk in family studies but no genome-wide study of rare variants has been reported to date.We performed whole exome sequencing (WES) in 373 unrelated individuals of European ancestry with well characterized unprovoked VTE and compared results to a previously genome sequenced control cohort of 5784 unrelated Europeans. We then identified single nucleotide variants, small insertion or deletions that were present at allele frequencies less than 0.005% in ExAC and had equal sequencing coverage between groups. Using a dominant model and a collapsing association analysis, we looked for genes that were more likely to harbor “knockout” (frame shift, nonsense, splice site and damaging missense) mutations in cases versus controls. Strikingly, ranked by p-value, among 17,395 genes tested, the top 4 genes were PROS1 (P= 2.01E-09, odds ratio: 11.8), STAB2 (P=2.70E-7, odds ratio 3.37), PROC (P=3.24E-05, odds ratio 11.0) and SERPINC1 (P=1.10E-04, odds ratio 8.5), Figure. These findings suggest that heterozygous deficiency for the STAB2 encoded protein, stabilin-2, confers VTE susceptibility similar to the previously well described phenotypes of heterozygous protein C, protein S and antithrombin III deficiency.Stabilin-2 is a single transmembrane glycoprotein scavenger receptor expressed on venous endothelium of liver sinusoids and spleen. We detected damaging STAB2 mutations in 29 (7%) VTE cases versus 141 (2%) of controls. Common and uncommon variants in STAB2 have been associated with elevated plasma von Willebrand factor (VWF) and coagulation factor VIII levels, which are known risk factors for VTE. Stabilin-2 is implicated as a clearance receptor for VWF, so that haploinsufficiency of stabilin-2 may increase plasma VWF half-life. To test this hypothesis, we examined 1162 individuals in the Twins UK Study (VTE status unknown) with available whole genome sequencing data and measured their plasma VWF levels by AlphaLISA. VWF levels were adjusted by log transformation and ABO blood type tagging SNPs. We identified 38 individuals (3.2%, all heterozygous) with rare damaging variants in STAB2. VWF levels were significantly higher in individuals (N=30) with rare STAB2 variants compared to those without (N=1016) (Mann-Whitney, P= 0.0046).To further examine stabilin-2 function in vivo, we compared Stab2 deficient mice (Stab2 -/-) to wild type (Stab2 +/+) controls. Stab2 -/- mice had elevated plasma levels of factor VIII:C (118%, p=0.0006), but no differences in VWF (p=0.58). Next, we examined thrombosis in a mouse model where venous blood clotting was induced by inferior vena cava stenosis. Thrombi were weighed and longitudinal sections were analyzed by quantitative immunohistochemistry 24 hours post-stenosis. Stab2 -/- mice developed significantly larger thrombi than Stab2 +/+ mice (194%, n=19, p=0.003) but no difference in DVT incidence (64% vs 61%, P= 0.85). Despite equivalent hematocrit (P= 0.91) and white type thrombus (P> 0.05), thrombi from Stab2 -/- mice were comprised of more red type thrombus (185%, P= 0.004).Through an unbiased analysis of whole exome sequencing in 6,157 individuals, this studyconfirms the role of haploinsufficiency of PROC, PROS1 and SERPINC1 for VTE susceptibility and identifies STAB2 deficiency as a potential new risk factor. Analysis of an independent human cohort of 1162 Europeans demonstrates that STAB2 haploinsufficiency is associated with elevated VWF levels, a known risk factor for VTE. Finally, a mouse model of Stab2 deficiency demonstrates enhanced thrombosis, though without an association with elevated VWF levels. Taken together, these results suggest that rare genetic variants in STAB2 collectively play a role in individuals with unprovoked VTE by prolonging the plasma half-life of procoagulant factors such as VWF, adding an important new gene to the short list of known risk factors for this common disease. [Display omitted] DisclosuresReitsma:Leiden University Medical Center; VarmX B.V.: Equity Ownership, Patents & Royalties: co-inventor on a patent application related to this abstract.

Ancestral Sequence Reconstruction of Von Willebrand Factor Reveals Highly Conserved Structure/Function

Von Willebrand factor (VWF) is a large multimeric glycoprotein of critical importance for both primary and secondary hemostasis due to its role in platelet activation and stabilization of coagulation factor VIII (FVIII), respectively. Mutations affecting VWF synthesis, secretion, or function result in one of 6 distinct subtypes of von Willebrand disease (VWD) with mutations inhibiting FVIII binding classified as type 2N. Due to low circulating levels of plasma FVIII, type 2N patients can be misdiagnosed with hemophilia A. Because the clearance of FVIII is dominated by its association with VWF, and given recent evidence demonstrating an immuno-protective role of VWF for FVIII, understanding the molecular interaction between FVIII and VWF remains critically important for both hemophilia A and type 2N VWD therapeutic development. However, limited structural information remains a major limitation for rational design studies. Recently, ancestral sequence reconstruction (ASR) was performed to infer the sequences of FVIII predicted to have existed throughout mammalian evolution. ASR provides a platform for high-resolution mapping of sequential differences within a phylogeny while maintaining a high probability of retaining function. Sequential changes in the amino acid sequence of FVIII appears to have resulted in altered biochemical properties including biosynthesis and stability. In this study, we demonstrate that a previously characterized ancestral (An) FVIII molecule with 95% identity to human FVIII, termed An53-FVIII, exhibits a 2.5 to 5-fold increase in binding to human VWF over commercial recombinant FVIII products, suggesting a dynamic association throughout evolution. We sought to further explore the FVIII-VWF interaction through ASR of VWF and biochemical analysis.A phylogenetic tree was constructed for VWF and the ancestral amino acid sequences predicted through Bayesian inference. A compiled list of 59 extant sequences allowed accurate prediction of the sequences extending beyond the class Mammalia. Of the resulting 50, 5 An-VWF sequences representing significant branch points in primate, rodent, and ungulate evolution were selected for synthesis de novo and subsequent biochemical characterization. All sequences, including an additional human (h) VWF cDNA, were human codon optimized (co). These sequences are predicted to span 105 million years of evolution and contain 81 to 364 amino acid substitutions (Table 1). Heterologous expression in HEK293 cells demonstrated that all An-VWF sequences resulted in secreted antigen as determined by ELISA using anti-human VWF polyclonal antibodies. Furthermore, immunohistochemistry of HEK293 cells expressing An-VWF revealed the formation of pseudo Weibel-Palade bodies by all VWF proteins. Stably transected, clonal producer cell lines were generated and utilized for recombinant VWF production and purification using heparin-sepharose affinity chromatography. Consistent with our IHC studies, multimer analysis of the purified proteins revealed the presence of high molecular weight multimers equivalent to human plasma VWF. To test the functionality of An-VWF, the VWF:GPIbM assay was performed. Compared to an observed specific activity of 45 IU/mg VWF for cohVWF, An-VWF molecules displayed specific activities ranging from 6 to 94 IU/mg. These data suggest that VWF and GPIb may have co-evolved to regulate platelet adhesion and activation. Hydrodynamic injection of An63-, An88-, and An101-VWF plasmid DNA into VWF deficient mice revealed plasma VWF levels of 1.44 ± 0.48, 0.72 ± 0.16, and 2.9 μg/mL at 24 hours after administration, respectively (n = 4, 3, and 1). Additionally, endogenous mouse (m) FVIII levels in these mice were elevated to 61 ± 9, 75 ± 16, and 67% of normal levels from a basal 12.9 ± 2.4%. This data demonstrates that liver synthesized An-VWF retains FVIII association and stabilization. Molar ratios of An88-VWF:mFVIII are 6:1 compared to 14:1 and 25:1 for An63- and An101-VWF, respectively, supporting the coevolution hypothesis for VWF and FVIII. Cumulatively, this study further validates the utility of ASR for the generation of functional sequences for complex proteins with a high propensity for inactivating mutations. Further analysis of ancestral sequences may provide greater resolution of amino acid sequences responsible for biochemical properties including clearance and FVIII association. [Display omitted] DisclosuresGaucher:General Genomics: Equity Ownership.

Plasma Von Willebrand Factor Levels Predict Survival in COVID-19 Patients Across the Entire Spectrum of Disease Severity.

Background: Characterization of reticulo-endothelial activation in COVID-19 may guide treatment. Objectives: To assess reticulo-endothelial activation and its correlation with disease severity and death in patients across the entire spectrum of COVID-19 severity. Methods: Consecutive hospitalized COVID-19 patients were studied, with similar number of patients in each disease severity category. Baseline serum ferritin, sCD163 (macrophage activation markers) and plasma von Willebrand factor (VWF) antigen (endothelial activation marker) levels were studied. Clinical parameters and plasma D-dimer levels were also studied. The study parameters were correlated with COVID-19 severity and survival. Results: The 143 patients (104 males [80%], age 54 [42 – 65] years, median [inter-quartile range]) presented 4 (3—7) days after symptom onset. Thirty-four patients had mild disease, 36 had moderate disease, 36 had severe disease and 37 had critical disease at baseline. With increasing COVID-19 severity, ferritin, sCD163, VWF and D-dimer levels significantly increased at baseline, however, 139 patients had normal sCD163 levels. Of the reticulo-endothelial markers, VWF level independently correlated with COVID-19 severity and with survival. VWF level > 332.6 units/dl correlated with COVID-19 severity (odds ratio [OR]: 2.77 [95% confidence interval (C.I): 1.1 – 6.99], p value: 0.031) and in-hospital death (OR [95% CI]: 29.28 [5.2 – 165], p value < 0.001). Conclusions: Reticulo-endothelial activation markers increased incrementally with worsening COVID-19 severity. Baseline endothelial activation marker (VWF), and not macrophage activation markers, independently correlated with COVID-19 severity and death.

VWF, Platelets and the Antiphospholipid Syndrome.

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPLs). Laboratory criteria for the classification of APS include the detection of lupus anticoagulant (LAC), anti-cardiolipin (aCL) antibodies and anti-β2glycoprotein I (aβ2GPI) antibodies. Clinical criteria for the classification of thrombotic APS include venous and arterial thrombosis, along with microvascular thrombosis. Several aPLs, including LAC, aβ2GPI and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) have been associated with arterial thrombosis. The Von Willebrand Factor (VWF) plays an important role in arterial thrombosis by mediating platelet adhesion and aggregation. Studies have shown that aPLs antibodies present in APS patients are able to increase the risk of arterial thrombosis by upregulating the plasma levels of active VWF and by promoting platelet activation. Inflammatory reactions induced by APS may also provide a suitable condition for arterial thrombosis, mostly ischemic stroke and myocardial infarction. The presence of other cardiovascular risk factors can enhance the effect of aPLs and increase the risk for thrombosis even more. These factors should therefore be taken into account when investigating APS-related arterial thrombosis. Nevertheless, the exact mechanism by which aPLs can cause thrombosis remains to be elucidated.