Elevated Plasma Von Willebrand Factor Research Articles

Elevated Plasma Levels of Von Willebrand Factor and Syndecan-1 Predict 60-Day Mortality in Patients with Severe and Critical COVID-19

Introduction: Endotheliopathy is common pathologic findings in acute and long COVID-19, which is associated with the disease severity and predisposes to long-term complications. Previous studies have demonstrated that plasma levels of von Willebrand factor (VWF) and proteoglycan syndecan-1 are significantly elevated in patients with severe COVID-19. However, the role of these endothelial biomarkers for predicting a long-term outcome is still lacking. Patients and Methods:151 consecutive hospitalized patients between January and March 2022 at the University of Kansas Hospital with a confirmed SARS-CoV-2 infection by polymerase chain reaction were prospectively enrolled for the study. Whole blood anticoagulated with EDTA or sodium citrate was collected at least 3 times on admission (T1), 4-5 days following treatment (T2), and a few days prior to discharge or death (T3). Plasma levels of VWF antigen, ADAMTS13 antigen/activity, and syndecan-1 were determined using immunoassays or a functional assay. Kaplan-Meier survival analysis was performed to determine the predictive value of each biomarker for 60-day mortality. Results. Of 151 patients with a positive PCR test for SARS-CoV2, 124 patients were studied and analyzed. These patients were categorized into an asymptomatic control, a moderate, a severe, and a critical group according to an updated World Health Organization (WHO) guidance. The admission plasma levels (median, IQR) of VWF in patients of critical (334%,164.7-526.2%) (p=0.0049) and severe (255%, 143.1-410.9%) (p=0.0088) groups were significantly higher than in those of moderate (153%, 186.3-240.1%) (Fig. 1A). Interestingly, no statistically significant difference in the admission plasma levels of ADAMTS13 antigen among all groups (p>0.05) (Fig. 1B) despite a significantly lower plasma ADAMTS13 activity (median, IQR) in patients with severe (39.8%, 30.0-49.3%) (p=0.0067) and critical (38.6%, 26.9-51.5%) (p=0.047) COVID-19 than in those with a asymptomatic disease (49.2%, 37.5-103.4%) (Fig. 1C). Furthermore, plasma levels of syndecan-1 on admission were not different among all groups (p>0.05) (Fig. 1D). Longitudinal analysis demonstrated that plasma levels of VWF antigen and syndecan-1 were persistently elevated from baseline in patients who died but not in those who survived (Fig. 2A-D). Kaplan-Meier survival analysis revealed that an elevated level of plasma VWF (> 320%) on T2 (p<0.0001) (Fig. 2E) or an elevated level of plasma syndecan-1 on admission (T1) (> 260 ng/mL) (p=0.0002) (Fig. 2F), but not 4-5 days following therapy (T2) or before being discharged home or death (T3) predicted the 60-day mortality rate. Conclusions: Our results demonstrate significantly elevated plasma VWF antigen and reduced ADAMTS13 activity on admission in patients with severe and critical COVID-19. The dynamic change in plasma VWF or the initial plasma levels of syndecan-1 on admission predict the 60-day survival. The findings suggest that the additional therapeutic strategies may be needed to alleviate endothelial activation and damage to improve survival and long-term complications in patients with severe and critical COVID-19. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Breast cancer cells mediate endothelial cell activation, promoting von Willebrand factor release, tumor adhesion, and transendothelial migration

BackgroundBreast cancer results in a three‐ to four‐fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the mechanisms underpinning breast cancer‐associated thrombosis remain poorly defined. Tumor cells can trigger endothelial cell (EC) activation resulting in increased von Willebrand factor (VWF) secretion. Importantly, elevated plasma VWF levels constitute an independent biomarker for VTE risk. Moreover, in a model of melanoma, treatment with low molecular weight heparin (LMWH) negatively regulated VWF secretion and attenuated tumor metastasis. ObjectiveTo investigate the role of VWF in breast cancer metastasis and examine the effect of LMWH in modulating EC activation and breast tumor transmigration. Methodsvon Willebrand factor levels were measured by ELISA. Primary ECs were used to assess tumor‐induced activation, angiogenesis, tumor adhesion, and transendothelial migration. Results and ConclusionPatients with metastatic breast cancer have markedly elevated plasma VWF:Ag levels that also correlate with poorer survival. MDA‐MB‐231 and MCF‐7 breast cancer cells induce secretion of VWF, angiopoietin‐2, and osteoprotegerin from ECs, which is further enhanced by the presence of platelets. Vascular endothelial growth factor‐A (VEGF‐A) plays an important role in modulating breast cancer‐induced VWF release. Moreover, VEGF‐A from breast tumor cells also contributes to a pro‐angiogenic effect on ECs. VWF multimers secreted from ECs, in response to tumor‐VEGF‐A, mediate adhesion of breast tumor cells along the endothelium. LMWH inhibits VWF‐breast tumor adhesion and transendothelial migration. Our findings highlight the significant crosstalk between tumor cells and the endothelium including increased VWF secretion which may contribute to tumor metastasis.

PO-25: Elevated plasma von Willebrand Factor in patients with breast cancer may contribute to the early metastatic niche, promoting endothelial adhesion, invasion and angiogenic properties of circulating breast cancer cells

The publisher regrets that this article has been temporarily removed. A replacement will appear as soon as possible in which the reason for the removal of the article will be specified, or the article will be reinstated. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Primary Myeloma Cell Induced Von Willebrand Factor Release from the Endothelium Is Mediated By VEGF and Attenuated By Heparin

IntroductionMultiple Myeloma (MM) remains an incurable disease and is associated with high rates of venous thromboembolism (VTE), the biological basis for which is not fully understood. Critically, VTE is associated with increased mortality in MM. This highlights the clinical importance of understanding cancer-coagulation crosstalk in MM. Accumulating evidence demonstrates that many solid tumours trigger endothelial cell (EC) activation with resultant von Willebrand Factor (VWF) secretion proposed to contribute to both risk of VTE and cancer metastasis. However, the interplay between VWF and MM disease biology remains poorly defined.Methods/Results100 patients with plasma cell disorders were recruited to this study. Significantly elevated plasma VWF antigen (VWF:Ag) levels were observed in patients with newly diagnosed MM (NDMM) compared to those with monoclonal gammopathy of undetermined significance (MGUS) or smouldering MM (SMM) (median 292.7 IU/dL vs 133.1 IU/dL; P<0.0001). Moreover, VWF:Ag levels in those with relapsed/refractory MM (RRMM) were further elevated (median 339 IU/dL; P<0.0001]). VWF:Ag levels also appear to correlate with disease response to therapy, with lower levels seen in patients achieving a complete response/very good partial response compared with those gaining only a partial response or worse (median 154.45 IU/dL vs 338.5 IU/dL; P<0.05).We also observed increased VWF collagen binding activity in NDMM/RRMM compared with MGUS/SMM (median 455.7 IU/dL vs 189.7 IU/dL; P<0.05). This qualitative measurement is sensitive to alterations in haemostatically active high molecular weight multimers of VWF secreted upon EC activation. Supporting this, raised plasma VWF propeptide (VWFpp) levels, a marker of acute EC activation, were seen in those with NDMM/RRMM compared with MGUS/SMM (median 182 IU/dL vs 133.01 IU/dL; P<0.05). Reduced VWFpp/VWF:Ag ratios were seen in those with NDMM/RRMM, indicating decreased circulatory clearance of VWF. We hypothesise that MM may exhibit a ‘double-hit’ in regulation of plasma VWF:Ag with increased secretion of VWF but also simultaneously reduced VWF clearance leading to sustained and markedly elevated systemic plasma VWF levels.Next we measured VWF:Ag levels in bone marrow (BM) samples from 10 patients and found that VWF is released locally within the BM niche (median 190.2 IU/dL). Co-culture of primary human ECs with supernatant from freshly isolated primary MM cells or several Human Myeloma Cell Lines (HMCLs) demonstrated that MM cells stimulate release of VWF from ECs in a rapid manner, suggestive of Weibel-Palade body exocytosis (median 34.9ng/ml vs 9.2ng/ml in untreated EC; P<0.05). Elevated levels of Vascular Endothelial Growth Factor-A (VEGF-A), a potent EC activator, were observed in MM cell supernatants. Pre-treatment of primary MM cells or HMCLs with anti-VEGF antibody bevacizumab abolished EC VWF secretion in a dose dependant manner (32.3ng/ml vs anti-VEGF treated 10.1ng/ml P<0.0001). Furthermore, clinically relevant doses of low molecular weight heparin (LMWH) also attenuated VWF secretion (29.6ng/ml vs LMWH treated 15ng/ml; P<0.05). Measurement of VEGF-A in primary MM cell and HMCL supernatant treated with LMWH or bevacizumab confirmed significant reductions in VEGF-A levels. Together these data indicate that stimulation of VWF release from the endothelium by MM cells is mediated by VEGF-A and directly inhibited by LMWH treatment.Using flow cytometry, we assessed the direct interaction of MM cells and VWF in vitro. Both primary MM cells and HMCLs bound significantly to human recombinant VWF in a dose-dependent manner. Binding was reduced by 50% following treatment with LMWH (P<0.05). Given that the VWF A1 domain contains a heparin binding motif, we speculate that MM cell adhesion to VWF may be at least in part mediated by this domain and future work focuses on elucidating the receptors involved.ConclusionThese data suggest that MM is not only associated with a marked quantitative increase in plasma VWF:Ag, but also impacts VWF functional activity. Our novel data help define the biological mechanisms underpinning elevated VWF levels in MM, with a key contribution of VEGF-A secreted directly from MM cells in the BM microenvironment. Collectively, our findings provide insights into cancer-coagulation crosstalk in MM and may help identify novel therapeutic targets to reduce VTE risk and disease progression. DisclosuresGlavey: Celgene and BMS company: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding. Quinn: Takeda: Honoraria. O'Sullivan: Leo Pharma: Research Funding.

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.

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.

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.

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.

Plasma levels of von Willebrand factor and future risk of incident venous thromboembolism

Several case-control studies have reported elevated plasma von Willebrand factor (VWF) levels in patients with venous thromboembolism (VTE) compared with controls. However, because few studies have investigated the association in a prospective design, it is unclear whether elevated plasma VWF is a risk factor or a consequence of the VTE event. Therefore, we aimed to investigate the prospective association between plasma VWF levels and risk of VTE, as well as to perform subgroup analyses of deep vein thrombosis (DVT) and pulmonary embolism. We established a population-based nested case-control study of 414 VTE cases and 843 age- and sex-matched controls based on the Tromsø study cohort (1994-2007). Blood samples were collected at cohort baseline (1994-1995). Odds ratios (ORs) with 95% confidence intervals (CIs) for VTE were estimated across quartiles of VWF levels. We found that the risk of VTE increased linearly across quartiles of VWF levels (P for trend = .023). Participants with VWF in the highest quartile had an OR of 1.45 (95% CI, 1.03-2.03) for VTE compared with those in the lowest quartile. The association was strongest for unprovoked VTE (OR, 2.74; 95% CI, 1.66-4.54) and unprovoked DVT in particular (OR, 6.73; 95% CI, 3.07-14.76). Further adjustment for body mass index, C-reactive protein, hypertension, estrogen use, and smoking had a modest effect on the risk estimates. To conclude, we found a dose-dependent relationship between plasma VWF levels and future risk of incident VTE, and unprovoked events in particular. Our findings suggest that VWF may represent a promising biomarker for future risk of incident VTE.

Von Willebrand Factor Activates the Alternative Complement Pathway In Vivo in a Mouse Model of Complement Thrombotic Microangiopathy

Introduction Thrombotic microangiopathy (TMA) is a group of disorders presenting with microvascular thrombosis, microangiopathic hemolytic anemia, thrombocytopenia, and microvascular endothelial injury ultimately leading to end organ damage. Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated TMA (TA-TMA) are two types of TMA known as "complement TMA," as they are associated with dysfunction of the alternative complement pathway (AP), part of the innate immune system. Complement TMA episodes are frequently initiated during inflammation, and the role of inflammation in these TMAs may be in part related to the finding that the inflammatory cytokine TNF up-regulates complement components, down-regulates complement regulators, and causes AP activation in human glomerular microvascular endothelial cells (GMVECs) in vitro. However, the precise mechanism of AP activation in complement TMA is poorly understood. It has been demonstrated that von Willebrand factor (VWF) serves as a surface for AP activation in vitro. Inflammatory cytokines are stimulatory to endothelial cells, leading to excessive VWF secretion; in fact, patients with inflammatory disorders such as complement TMA demonstrate elevated plasma VWF levels, and this abundance of VWF may lead to excessive AP activation. We hypothesized that in complement TMA, VWF released from endothelial cells during inflammation activates the AP. The objective was to investigate whether VWF-mediated AP activation is initiating episodes of complement TMA in vivo in a complement TMA mouse model. Methods A mouse model of complement TMA was developed. To provoke complement TMA, wild-type (WT) C57BL/6J mice (purchased from Jackson Laboratories) were injected intraperitoneally with 5 mg/kg of the inflammatory endotoxin lipopolysaccharide (LPS). Twenty-four hours after injection of either LPS or saline (control), markers of complement TMA-lactate dehydrogenase (LDH), creatinine, platelet count, and complement activation by a rabbit erythrocyte (ER) lysis assay-were measured. ER specifically activate the AP in mammalian plasma in the presence of Mg/EGTA, and the resulting lysis is an indicator of the extent to which complement components have been consumed in vivo: low ER lysis in vitro indicates high complement activation in vivo. To assess the role of VWF in AP activation in vivo, VWF-/- mice (purchased from Jackson Laboratories on a C57BL/6J background) were injected with 5 mg/kg LPS to induce complement TMA. Twenty-four hours after injection, plasma ER lysis was measured and compared to ER lysis in LPS-injected WT C57BL/6J mice. Other markers of complement TMA, including LDH, creatinine, and platelet count, were also measured in both LPS-injected VWF-/- and WT C57BL/6J mice. Results Compared to saline-injected WT C57BL/6J mice, the LPS-injected mice demonstrated significantly: A) higher LDH (hemolysis); B) higher creatinine (renal dysfunction); C) lower platelet count; and D) lower ER lysis (increased AP activation) (Fig. 1). These results confirm the establishment of a complement TMA murine model. Furthermore, LPS-injected VWF-/- mice demonstrated significantly higher ER lysis values than the LPS-injected WT C57BL/6J mice, indicating less AP activation (Fig 2). Because VWF-/- mice do not produce any VWF, these data suggest that VWF contributes to AP activation in vivo in complement TMA. Finally, the LPS-injected VWF-/- mice had less severe TMA as demonstrated by lower LDH levels, lower creatinine levels, and higher platelet counts compared to the LPS-injected WT C57BL/6J mice (Fig. 3). The LPS-injected VWF-/- mice still had a mild degree of AP activation and TMA compared to saline-injected WT C57BL/6J mice, implying that there are complex mechanisms of AP activation in complement TMA that rely heavily on VWF. Conclusions In this study we demonstrated that VWF was important in complement activation and provocation of TMA in a complement TMA murine model. These data are the first step toward a better understanding of VWF's role in complement TMA and may lead to the development of therapeutics that inhibit inflammation, VWF release, or VWF-complement binding to curb AP activation in aHUS and TA-TMA, thereby preventing devastating outcomes. Further study is necessary to delineate the precise mechanisms of VWF-mediated AP activation and TMA development so that these treatment modalities may be explored. Disclosures No relevant conflicts of interest to declare.

The aptamer BT200 effectively inhibits von Willebrand factor (VWF) dependent platelet function after stimulated VWF release by desmopressin or endotoxin

Von Willebrand factor (VWF) plays a major role in arterial thrombosis. Antiplatelet drugs induce only a moderate relative risk reduction after atherothrombosis, and their inhibitory effects are compromised under high shear rates when VWF levels are increased. Therefore, we investigated the ex vivo effects of a third-generation anti-VWF aptamer (BT200) before/after stimulated VWF release. We studied the concentration-effect curves BT200 had on VWF activity, platelet plug formation under high shear rates (PFA), and ristocetin-induced platelet aggregation (Multiplate) before and after desmopressin or endotoxin infusions in healthy volunteers. VWF levels increased > 2.5-fold after desmopressin or endotoxin infusion (p < 0.001) and both agents elevated circulating VWF activity. At baseline, 0.51 µg/ml BT200 reduced VWF activity to 20% of normal, but 2.5-fold higher BT200 levels were required after desmopressin administration (p < 0.001). Similarly, twofold higher BT200 concentrations were needed after endotoxin infusion compared to baseline (p < 0.011). BT200 levels of 0.49 µg/ml prolonged collagen-ADP closure times to > 300 s at baseline, whereas 1.35 µg/ml BT200 were needed 2 h after desmopressin infusion. Similarly, twofold higher BT200 concentrations were necessary to inhibit ristocetin induced aggregation after desmopressin infusion compared to baseline (p < 0.001). Both stimuli elevated plasma VWF levels in a manner representative of thrombotic or pro-inflammatory conditions such as arterial thrombosis. Even under these conditions, BT200 potently inhibited VWF activity and VWF-dependent platelet function, but higher BT200 concentrations were required for comparable effects relative to the unstimulated state.

Endothelial cells orchestrate COVID-19 coagulopathy

Endothelial cells orchestrate COVID-19 coagulopathy

Elevated Plasma von Willebrand Factor Antigen and Activity Levels Are Associated With the Severity of Coronary Stenosis.

von Willebrand factor (VWF) acts as a bridge between platelets and the subendothelial matrix following vessel damage and plays a vital role in coronary artery disease (CAD). The aim of this study was to investigate the association between VWF and the severity of coronary stenosis quantified by the Gensini score in acute myocardial infarction (AMI), the most dangerous complication of CAD. Plasma VWF antigen (VWF: Ag) and VWF-collagen binding (VWF: CB) in normal controls (n = 123) and in patients with AMI (n = 205) were tested, and then the patients were divided based on Gensini scores. The levels of VWF: Ag and VWF: CB in patients with AMI were significantly higher than those in the control group (P < .001). Plasma levels of VWF: Ag and VWF: CB were positively correlated with both Gensini score and the number of affected vessels. Both VWF: Ag and VWF: CB were independent factors for coronary stenosis, adjusting confounding factors. Thus, the levels of VWF: Ag and VWF: CB were positively correlated with the severity of coronary stenosis. Screening of VWF at time of AMI may have prognostic value in terms of the severity of coronary stenosis.

Long-lasting prothrombotic state implied by changes of plasma von Willebrand factor parameters after radical prostatectomy for prostate malignancy

ObjectivesThromboembolic complications are present in 0.8%–16.8% of the cases after radical prostatectomy (RP). Association between elevated plasma von Willebrand factor (VWF) levels—as an endothelial activation marker—and increased risk of thrombotic events has been evidenced.We aimed to elicit new data on the VWF after RP in prostate cancer patients and explore the role of it as a thrombotic risk factor. Upon perioperative plasma VWF levels (VWF:Ag) its collagen-binding (CB) activity (VWF:CB), multimerization, and cleaving enzyme (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type repeats, motif 1, type 13]) of the VWF multimers were quantitated along with Factor VIII and routine laboratory parameters in this observational pilot study. MethodsPlasma samples of 24 prostate cancer patients were collected before (-1 day; D-1) and after RP (1 hour, 6 days, 1 month, and 10 months; H1, D6, M1, and M10). VWF:Ag, VWF:CB, ADAMTS13:Ag were measured by ELISA, and the multimer distribution by electrophoresis and quantitative densitometry. Factor VIII, fibrinogen, D-dimer, and other routine laboratory parameters were determined as well. Preoperative values served as baselines which were compared to controls (24 healthy individuals). ResultsVWF:Ag and CB elevated by 122% and 143% respectively at H1 after RP then plateaued at D6 compared to baseline values. ADAMTS13/VWF:Ag ratio reduced by 41% at H1, and by 46% at D6, meanwhile the ratio of high molecular weight multimers increased as well. Values returned to baseline at M1 and further reduced to the levels of the controls at M10. All of the 24 patients at H1 and D6 and 14 at M10 were in potential prothombotic state according ROC analysis of the VWF parameters as indicators. ConclusionsProstate malignancy and then surgical stress, and inflammatory reactions induced release of VWF from the endothelial cells, along with an increasing amount of large multimers and relative reduction of ADAMTS13 level. Because these changes mark a prothrombotic state even at M1 after RP, more than 1 month follow-up and prophylactic targeting through the thrombotic and inflammatory activity of the VWF is proposed. Evaluation of VWF parameters provides new information about the long-term disturbances of primary hemostasis after radical pelvic oncologic surgery like RP and might improve the understanding the physiological and pathological recovery.

Systematic review: role of elevated plasma von-Willebrand factor as predictor of mortality in patients with chronic liver disease.

In this systematic review, we aimed to assess role of plasma von-Willebrand factor (vWF), an endothelial activation marker, as prognostic marker in patients with chronc liver disease [cirrhosis and acute-on-chronic liver failure (ACLF)]. We searched published databases using predefined keywords to identify all studies up to June 2018, in which plasma vWF (antigen or activity assay) was used as prognostic marker predicting mortality in patients with chronic liver disease. Relevant extracted data from selected studies were narratively summarized. The individual study's area under ROC curve for plasma vWF as a predictor of mortality was pooled and meta-analyzed. Six studies (cirrhosis: 5; ACLF: 1) with an aggregate data of 765 patients (cirrhosis: 715 patients; ACLF: 50 patients) were included. Baseline plasma vWF-antigen was an independent predictor of medium-term mortality in patients with cirrhosis (summary area under the curve: 0.74; 95% confidence interval: 0.70-0.79) with an optimal cutoff of 318% (216-390%; median, range) over a period of 25.6 months (23.6-33 months). Plasma vWF also predicted short-term (over 7 days) mortality in patients with ACLF. Plasma vWF levels correlated with Child's score, model for end-stage liver disease (MELD) score and hepatic venous pressure gradient and performed as well as MELD score in predicting mortality in patients with cirrhosis and ACLF. Baseline plasma vWF level predicts mortality over a medium term (1-3 years) in cirrhosis and over a short term (1 week) in ACLF patients. The marked elevation of baseline plasma vWF levels in ACLF patients was associated with drastic truncation of survival when compared with cirrhosis patients.

Therapeutic monoclonal antibody interference in electrophoretic and immunofixation techniques

Consistent 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.This 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 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.We 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.These 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.

Lyso-SM-509 as highly sensitive biomarker for Niemann-Pick disease types A/B and C: Three years experience

Consistent 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.This 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 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.We 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.These 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.

Von Willebrand factor indicates bacterial translocation, inflammation, and procoagulant imbalance and predicts complications independently of portal hypertension severity.

Elevated plasma von Willebrand factor antigen (vWF) has been shown to indicate the presence of clinically significant portal hypertension, and thus, predicts the development of clinical events in patients with cirrhosis. To investigate the impact of bacterial translocation and inflammation on vWF, as well as the association between vWF and procoagulant imbalance. Moreover, we assessed whether vWF predicts complications of cirrhosis, independent of the severity of portal hypertension. Our study population comprised 225 patients with hepatic venous pressure gradient (HVPG)≥10mm Hg without active bacterial infections or hepatocellular carcinoma. vWF correlated with markers of bacterial translocation (lipopolysaccharide-binding protein [LBP; ρ=0.201; P=0.021]), inflammation (interleukin 6 [IL-6; ρ=0.426; P<0.001] and C-reactive protein [CRP; ρ=0.249; P<0.001]), and procoagulant imbalance (factor VIII/protein C ratio; ρ=0.507; P<0.001). Importantly, the associations between vWF and these parameters were independent of HVPG. Moreover, vWF (per 10%) independently predicted variceal bleeding (hazard ratio [HR]: 1.08 [95% confidence interval (95% CI): 1.01-1.16]; P=0.023), requirement of paracentesis (HR: 1.05 [95% CI: 1.01-1.1]; P=0.023) and bacterial infections (HR: 1.04 [95% CI: 1-1.09]; P=0.04) including spontaneous bacterial peritonitis (HR: 1.09 [95% CI: 0.999-1.18]; P=0.053) on a trend-wise level. After backward elimination, vWF (HR: 1.05 [95% CI: 1.02-1.08]; P=0.003) and CRP (per 10mg/L; HR: 1.53 [95% CI: 1.14-2.05]; P=0.005) remained in the final model for transplant-free mortality. Finally, the independent prognostic value of vWF/CRP groups for mortality was confirmed by competing risk analysis. Our results demonstrate that vWF is not only a marker of portal hypertension but also independently linked to bacterial translocation, inflammation and procoagulant imbalance, which might explain its HVPG-independent association with most clinical events. Prognostic groups based on vWF/CRP efficiently discriminate between patients with a poor 5-year survival and patients with a favourable prognosis.

Highly elevated plasma level of von Willebrand factor accelerates the formation of platelet thrombus under high shear stress in plasma with deficient ADAMTS13 activity

Upshaw-Schulman syndrome (USS) is a thrombo-hemorrhagic disease caused by congenital deficiency of ADAMTS13 due to ADAMTS13 gene mutations. USS is characterized by repeated episodes of thrombocytopenia and microangiopathic hemolytic anemia that respond dramatically to infusions of fresh frozen plasma. There are two phenotypic expressions of USS: one is the early-onset type and the other, the late-onset type, is asymptomatic during childhood with the first bout of thrombotic thrombocytopenic purpura (TTP) developing after adolescence or during adulthood. We found that gravida with the latter phenotype developed thrombocytopenia and hemolytic anemia during the second or third trimesters, often followed by thrombotic microangiopathies (TMAs). These phenomena suggest that elevated plasma von Willebrand Factor (VWF) might be crucial because plasma levels of VWF antigen usually increase by 200–500% during this period of gestation. Here, we performed platelet function assays using a mixture of anti-coagulated blood from normal volunteers, human VWF, anti-ADAMTS13 monoclonal antibody A10, and purified plasma-derived ADAMTS13 to investigate the effect of plasma VWF levels on platelet thrombus formation in the context of deficient ADAMTS13. In vitro studies showed that mural thrombus formation and platelet aggregation under high shear stress were markedly augmented by increasing the amounts of exogenously added VWF when ADAMTS13 activity was deficient, as may be the case in the in vivo circulation of gravida with USS. These results suggest that highly elevated plasma VWF might accelerate platelet thrombus formation not only in the circulation but also on the surface of vascular endothelial cells in the setting of ADAMTS13 deficiency in USS.

Von Willebrand factor deficiency reduces liver fibrosis in mice

Liver diseases are associated with complex changes in the hemostatic system and elevated levels of the platelet-adhesive protein Von Willebrand factor (VWF) are reported in patients with acute and chronic liver damage. Although elevated levels of VWF are associated with fibrosis in the general population, the role of VWF in acute and chronic liver injury has not been examined in depth in experimental settings. We tested the hypothesis that VWF deficiency inhibits experimental liver injury and fibrosis. Wild-type (WT) and VWF-deficient mice were challenged with carbon tetrachloride (CCl4) and the impact of VWF deficiency on acute liver injury and chronic liver fibrosis was determined. VWF deficiency did not significantly affect acute CCl4-induced hepatocellular necrosis in mice. Chronic CCl4 challenge, twice weekly for 6weeks, significantly increased hepatic stellate cell activation and collagen deposition in livers of WT mice. Interestingly, hepatic induction of several profibrogenic and stellate cell activation genes was attenuated in VWF-deficient mice. Moreover, birefringent sirius red staining (indicating type I and III collagens) and type I collagen immunofluorescence indicated a reduction in hepatic collagen deposition in CCl4-exposed VWF-deficient mice compared to CCl4-exposed WT mice. The results indicate that VWF deficiency attenuates chronic CCl4-induced liver fibrosis without affecting acute hepatocellular necrosis. The results are the first to demonstrate that VWF deficiency reduces the progression of liver fibrosis, suggesting a mechanistic role of elevated plasma VWF levels in cirrhosis.