Elevated Von Willebrand Factor Levels Research Articles

Dynamic Assessment of Plasma von Willebrand Factor and ADAMTS13 Predicts Mortality in Hospitalized Patients with SARS-CoV-2 Infection.

Plasma levels of von Willebrand factor (VWF) are significantly elevated in patients with coronavirus disease 2019 (COVID-19). However, dynamic changes and prognostic value of this biomarker in hospitalized patients with COVID-19 have not been determined. A total of 124 patients infected with SARS-CoV-2 were prospectively recruited for the study. Serial blood samples were obtained at the time of admission (D1), 3-4 days following standard-care treatments (D2), and 1-2 days prior to discharge or any time collected prior to death (D3). Plasma VWF antigen, ADAMTS13 antigen, and ADAMTS13 proteolytic activity, as well as the ratio of VWF/ADAMTS13 were determined, followed by various statistical analyses. On admission, plasma levels of VWF in COVID-19 patients were significantly elevated compared with those in the healthy controls, but no statistical significance was detected among patients with different disease severity. Plasma ADAMTS13 activity but not its antigen levels were significantly lower in patients with severe or critical COVID-19 compared with that in other patient groups. Interestingly, the ratios of plasma VWF antigen to ADAMTS13 antigen were significantly higher in patients with severe or critical COVID-19 than in those with mild to moderate disease. More importantly, plasma levels of VWF and the ratios of VWF/ADAMTS13 were persistently elevated in patients with COVID-19 throughout hospitalization. Kaplan-Meier and Cox proportional hazard regression analyses demonstrated that an increased plasma level of VWF or ratio of VWF/ADAMTS13 at D2 and D3 was associated with an increased mortality rate. Persistent endotheliopathy, marked by the elevated levels of plasma VWF or VWF/ADAMTS13 ratio, is present in all hospitalized patients following SARS-CoV-2 infection, which is strongly associated with mortality.

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.

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.

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.

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.

Elevated von Willebrand factor levels during heavy menstrual bleeding episodes limit the diagnostic utility for von Willebrand disease

BackgroundHeavy menstrual bleeding (HMB) is often the first bleeding symptom for female individuals with inherited bleeding disorders. Guidelines recommend performing the hemostatic evaluation at HMB presentation. Von Willebrand factor (VWF) levels increase with stress, making it unclear if VWF studies during acute bleeding are beneficial in diagnosing von Willebrand disease (VWD). ObjectivesTo determine the utility of testing for VWD during acute HMB. Patients/MethodsThis retrospective cohort study evaluated VWF levels of individuals presenting to the emergency department (ED) with HMB from January 1, 2017, to December 31, 2018, after prospective implementation of a clinical practice guideline recommending hemostatic evaluation in the ED. We compared VWF and factor VIII (FVIII) levels between acute presentation and follow‐up visit after bleeding resolution. We compared the diagnostic accuracy of initial and follow‐up labs. ResultsDuring the study period, 221 individuals were seen in the ED for acute HMB, and 39 had VWD testing at both time points. Median FVIII and VWF levels were higher during acute bleeding than at follow‐up. The difference in VWF levels between visits was negligible when initial FVIII value was normal. Overall incidence of VWD was 7.5%; 69% of those with VWD had low VWF levels during acute HMB. ConclusionVWD testing during acute HMB detects the majority of individuals with VWD but also leads to elevated levels of VWF, potentially limiting at the accuracy of diagnostic labs during acute bleeding episodes. Delayed testing until resolution of anemia and active bleeding may provide more accurate diagnostic evaluation for VWD.

VWF/ADAMTS13 Imbalance, But Not Global Coagulation or Fibrinolysis, Is Associated With Outcome and Bleeding in Acute Liver Failure.

Background and AimsRecent studies of acute liver failure (ALF) in man and animals have suggested that rebalanced hemostasis occurs, with distinct hypercoagulable features clinically evidenced by a low risk of bleeding. Rodent models have shown a link between intrahepatic microthrombus formation and progression of ALF. We sought to confirm these earlier findings in a large series of patients with well‐characterized ALF from the Acute Liver Failure Study Group.Approach and ResultsCitrated plasma samples taken on admission from 676 patients with ALF or acute liver injury (international normalized ratio ≥2.0 without hepatic encephalopathy) were used to determine levels of von Willebrand factor (VWF), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity, thrombomodulin‐modified thrombin generation, and clot lysis time (CLT) and compared with the levels in 40 healthy controls. Patients had 3‐fold increased VWF levels, 4‐fold decreased ADAMTS13 activity, similar thrombin generating capacity, and 2.4‐fold increased CLT, compared with controls. Increasing disease severity was associated with progressively more elevated VWF levels as well as hypofibrinolysis. Patients who died or underwent liver transplantation within 21 days of admission had higher VWF levels, lower ADAMTS13 activity, but similar thrombin generation and a similar proportion of patients with severe hypofibrinolysis, when compared with transplant‐free survivors. Likewise, patients with bleeding complications had higher VWF levels and lower ADAMTS13 activity compared to those without bleeding. Thrombin generation and CLT did not differ significantly between bleeding and nonbleeding patients.ConclusionsRebalanced hemostatic status was confirmed in a large cohort of patients with acute liver injury/ALF, demonstrating that VWF/ADAMTS13 imbalance is associated with poor outcome and bleeding. The association between VWF/ADAMTS13 imbalance and bleeding suggests that bleeding in ALF relates more to systemic inflammation than a primary coagulopathy.

Characterization of ADAMTS13 and von Willebrand factor levels in septic and non-septic ICU patients.

Sepsis is a life-threatening disease characterized by excessive host response to infection that can lead to activation of the coagulation system. Von Willebrand Factor (VWF) and ADAMTS13 are important regulators of hemostasis and their dysregulation during sepsis progression is not well understood. Herein we characterize ADAMTS13 and VWF in septic and non-septic patients. ADAMTS13 activity, ADAMTS13 antigen, VWF antigen, myeloperoxidase, and protein C, were measured in plasma collected from 40 septic patients (20 non-survivors and 20 survivors) and 40 non-septic patients on the first and last day of their ICU stay. ADAMTS13 activity and ADAMTS13 antigen were reduced, whereas VWF antigen was elevated among septic patients compared to non-septic patients and healthy controls. Non-septic patients also exhibited elevated VWF antigen and reduced ADAMTS13 activity, but to a lesser extent than septic patients. Non-survivor septic patients exhibited the lowest levels of ADAMTS13 activity. ADAMTS13 activity:antigen ratio was similar across all patient cohorts suggesting that the specific activity of ADAMTS13 remains unchanged. Therefore, reduced ADAMTS13 function in circulation is likely due to a reduction in circulating levels. We suggest that massive release of VWF in response to inflammation consumes limited circulating ADAMTS13, resulting in the imbalance observed between VWF and ADAMTS13 among septic and to a lesser extent in non-septic ICU patients. Changes to ADAMTS13 did not correlate with myeloperoxidase or protein C levels. Reduced ADAMTS13 activity and antigen, and elevated VWF antigen observed among all patient cohorts on admission remained unchanged in survivors at ICU discharge. Prolonged reduction in ADAMTS13 activity and antigen in septic patients coincides with elevated levels of VWF. The persistent abnormalities in ADAMTS13 and VWF in sepsis patients discharged from the ICU may contribute to a sustained prothrombotic state.

Assessment Of Factors Of Vascular Wall Damage In Depressive Disorders In Patients With Myocardial Infarction

The study of pathogenetic predictors of the development of anxiety-depressive disorders in myocardial infarction will make it possible to develop ways of their correction, thereby reducing the frequency of complications of the postinfarction period. Clinical studies were based on the examination of 58 patients with MI (mean age 59.2 ± 4.7 years) who were admitted to the cardiological hospital for treatment, and the observation data for them at the stage of rehabilitation. On the basis of the presence of anxiety-depressive symptoms, the patients were divided into 2 groups. The first control group consisted of 14 patients with MI without depressive disorders. The second group included 44 age-matched patients who underwent MI with symptoms of anxiety and depression without comorbid cardiovascular pathology. The diagnosis of myocardial infarction was based on the results of clinical examination, ECG changes, laboratory parameters, and echocardiographic data. In order to assess the mental status of the subjects, subjective methods were used: the hospital anxiety and depression scale (HADS) for patients in a somatic hospital and recommended for use in patients with post-infarction period. Markers of endothelial dysfunction in blood plasma were determined by enzyme immunoassay using appropriate test systems. Since fibrinogen is one of the key factors in the blood coagulation process, disorders of which with a tendency to thrombotic changes are one of the central links in the pathogenesis of MI, the level of oxidized fibrinogen with parameters of the functional state of endothelial cells was studied. In the early stages after myocardial infarction, the level of oxidized fibrinogen was 1.7 times higher in the study group compared to the control, although, in general, the level of fibrinogen in the study group was within normal values. In the subsequent periods of the study, the level of oxidized fibrinogen was in high values and, on average, exceeded the control values by 1.64 times. Since one of the key roles in the development of dysfunction and endothelial destruction is assigned to the factors of oxidative stress, a correlation analysis of the relationship between the oxidative modification of fibrinogen and the parameters of endothelial function was carried out. A direct correlation was shown between the level of oxidized fibrinogen and the level of Endothelin-1 (r = 0.78, p &lt;0.01), and a direct correlation with the level of von Willebrand factor (r = 0.365, p &lt;0.01). Linear regression analysis confirmed the associations of oxidized fibrinogen with the indicated parameters of endothelial dysfunction. Based on the results obtained, it can be emphasized that with MI, in patients with developed DS, along with increased oxidative changes in lipids and plasma proteins, there is also a significant oxidative modification of fibrinogen, which does not depend on the concentration of fibrinogen. Oxidized fibrinogen potentiates potentially prothrombogenic changes in the vascular-platelet link of hemostasis, in particular, the acceleration of leukocyte-platelet aggregation. The revealed signs of thrombotic and hypercoagulant hemostasis disorders in patients with MI with depressive disorders, such as signs of endothelial dysfunction, elevated von Willebrand factor levels, are associated with oxidative changes in plasma fibrinogen in patients with MI with the development of DS, have a high diagnostic value.

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.

Von Willebrand Factor Is a Critical Mediator of Deep Vein Thrombosis in a Mouse Model of Diet-Induced Obesity.

Obesity is characterized by chronic low-grade inflammation and consequentially a hypercoagulable state, associating with an increased incidence of venous thromboembolism. Increased VWF (von Willebrand factor) plasma concentration and procoagulant function are independent risk factors for venous thromboembolism and are elevated in obese patients. Here, we explore the pathobiological role of VWF in obesity-associated venous thrombosis using murine models. Approach and Results: We first showed that diet-induced obese mice have increased VWF plasma levels and FVIII (factor VIII) activity compared with littermate controls. Elevated VWF levels appeared to be because of both increased synthesis and impaired clearance. Diet-induced obesity-associated venous thrombosis was assessed using the inferior vena cava-stenosis model of deep vein thrombosis. Diet-induced obese mice developed larger venous thrombi that were rich in VWF, erythrocytes, and leukocytes. Administering a polyclonal anti-VWF antibody or an anti-VWF A1 domain nanobody was protective against obesity-mediated thrombogenicity. Delayed administration (3 hours post-inferior vena cava stenosis) similarly reduced thrombus weight in diet-induced obese mice. This study demonstrates the critical role of VWF in the complex, thrombo-inflammatory state of obesity. It adds to the growing rationale for targeting VWF-specific interactions in thrombotic disease.

Coagulopathy and Thrombosis as a Result of Severe COVID-19 Infection: A Microvascular Focus.

Coronavirus disease of 2019 (COVID-19) is the clinical manifestation of the respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While primarily recognized as a respiratory disease, it is clear that COVID-19 is systemic illness impacting multiple organ systems. One defining clinical feature of COVID-19 has been the high incidence of thrombotic events. The underlying processes and risk factors for the occurrence of thrombotic events in COVID-19 remain inadequately understood. While severe bacterial, viral, or fungal infections are well recognized to activate the coagulation system, COVID-19-associated coagulopathy is likely to have unique mechanistic features. Inflammatory-driven processes are likely primary drivers of coagulopathy in COVID-19, but the exact mechanisms linking inflammation to dysregulated hemostasis and thrombosis are yet to be delineated. Cumulative findings of microvascular thrombosis has raised question if the endothelium and microvasculature should be a point of investigative focus. von Willebrand factor (VWF) and its protease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), play important role in the maintenance of microvascular hemostasis. In inflammatory conditions, imbalanced VWF-ADAMTS-13 characterized by elevated VWF levels and inhibited and/or reduced activity of ADAMTS-13 has been reported. Also, an imbalance between ADAMTS-13 activity and VWF antigen is associated with organ dysfunction and death in patients with systemic inflammation. A thorough understanding of VWF-ADAMTS-13 interactions during early and advanced phases of COVID-19 could help better define the pathophysiology, guide thromboprophylaxis and treatment, and improve clinical prognosis.

Effective hemostasis in children with Von Willebrand factor defects undergoing adenotonsillar procedures

Introduction Children with low von Willebrand factor (VWF) activity or type 1 von Willebrand disease (VWD) have increased risk of bleeding after adenotonsillar procedures and the optimal perioperative management to minimize bleeding is unknown. Aim To report the effectiveness and safety of an institutional protocol in minimizing postoperative bleeding in children with type 1 VWD or low VWF activity. Methods We conducted a retrospective chart review in children with type 1 VWD or low VWF activity treated via an institutional protocol that utilizes repeated doses of Desmopressin acetate (DDAVP, 1-deamino 8-D arginine- vasopressin) or VWF concentrate, brief hospitalization for observation and extended use of oral epsilon aminocaproic acid (EACA). Results From 2010 to 2017, 13 children underwent an adenotonsillar procedure and were treated with this protocol. Although 7.6% had minor immediate bleeding and 23% had minor delayed bleeding, no patients experienced major bleeding or required transfusion, additional surgery or other measures not specified by the protocol. Mild hyponatremia was observed in 80% of patients who received DDAVP. Conclusion Our institutional protocol specifying repeated dosing of DDAVP or VWF concentrate to sustain elevated VWF levels during periods of highest bleeding risk and extended use of EACA is effective at preventing major bleeding episodes after adenotonsillar procedures. However, this analysis raised safety concerns that prompted changes in the institutional protocol and highlight the need for further prospective studies to determine the optimal strategy for safely reducing bleeding complications in these patients.

Interaction networks of Weibel-Palade body regulators syntaxin-3 and syntaxin binding protein 5 in endothelial cells

The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. SignificanceThe hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health.

Common VWF sequence variants associated with higher VWF and FVIII are less frequent in subjects diagnosed with type 1 VWD

Unlabelled Box BackgroundGenetic variation in the VWF gene is associated with von Willebrand factor (VWF) and factor VIII (FVIII) levels in healthy individuals. ObjectivesWe hypothesized that VWF sequence variants associated with higher VWF or FVIII could impact the diagnosis of type 1 von Willebrand disease (VWD). MethodsWe examined VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF propeptide (VWFpp), and FVIII levels along with VWF gene sequencing in 256 healthy control and 97 type 1 VWD subjects as part of a cross‐sectional study. ResultsWe found several VWF sequence variants (VWF c.2880G>A and VWF c.2365A>G(;)c.2385T>C, found in linkage disequilibrium) associated with higher VWF and FVIII levels in healthy controls (P<.001 for both variants). In addition, these variants were significantly more common in controls than in subjects diagnosed with type 1 VWD and VWF:Ag <30 (P<.005). The decreased variant frequencies in type 1 VWD was not seen in other VWD types. VWF:Ag, VWF:RCo, and FVIII were not statistically different in type 1 VWD subjects who had these VWF variants compared to type 1 VWD patients without them. There was no difference in ABO blood group, VWF propeptide levels (excluding subjects with known VWF clearance defects), or bleeding score using the ISTH bleeding assessment tool. ConclusionsThese data suggest that certain VWF sequence variants associated with elevated FVIII and VWF levels may protect against reduced VWF levels. These findings were independent of other pathogenic sequence variants in VWF, suggesting a possible independent effect of c.2880G>A and c.2365A>G(;)c.2385T>C on VWF levels.

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.

Regulation of VWF expression, and secretion in health and disease.

Von Willebrand factor (VWF) is a large multidomain, multimeric glycoprotein that plays an essential role in regulating the balance between blood clotting and bleeding. Aberrant VWF regulation can lead to a spectrum of diseases extending from bleeding disorders [Von Willebrand disease (VWD)] to aberrant thrombotic thrombocytopenic purpura (TTP). Understanding the biology of VWF expression and secretion is essential for developing novel targeted therapies for VWF-related hemostasis disorders. A number of recent elegant in-vitro and in-vivo studies will be highlighted, including the discovery of intronic splicing in the VWF gene, microRNA-regulated VWF gene expression, and syntaxin binding protein and autophagy mediated VWF secretion. Compared with the already established critical role of VWF in VWD and TTP pathophysiology, additional clinical studies have clarified and reinforced the association of elevated plasma levels of VWF with an increased risk of stroke, myocardial infarction, venous thrombosis, and diabetic thrombotic complications. Moreover, experimental mouse models of ischemic stroke and myocardial infarction have further supported VWF as a potential therapeutic target. VWF biosynthesis, maturation, and secretion is a complex process, which mandates tight regulation. Significant progress has been made in our understandings of VWF expression and secretion and its association with thrombotic diseases, contributing to the development of novel targeting VWF drugs for prevention and treatment of deficient and enhanced hemostasis.

Plasma ADAMTS13, von Willebrand Factor (VWF), and VWF Propeptide Profiles in Patients With Connective Tissue Diseases and Antiphospholipid Syndrome.

Thrombotic thrombocytopenic purpura (TTP) frequently develops in patients with connective tissue diseases (CTDs). ADAMTS13 and von Willebrand factor (VWF) are closely related to the onset of TTP. We investigated the roles of ADAMTS13 and VWF in thrombotic events of patients with CTD. ADAMTS13 activity and VWF and VWF propeptide (VWFpp) levels in CTD, primary antiphospholipid antibody syndrome (pAPS), and controls were measured to examine their relationship with thrombosis. ADAMTS13 activity levels were significantly low in the patients with CTD but not in the patients with pAPS. No significant difference in the ADAMTS13 activity levels among the various CTD subgroups was found. The levels of VWF and VWFpp were significantly elevated in the patients with pAPS and CTD compared with that of control groups. Eleven patients with CTD developed TTP, and their ADAMTS13 activity levels were significantly lower than patients having CTD without TTP. However, the ADAMTS13 activity levels showed no difference between the patients having CTD with and without thrombotic events. The VWF antigen levels were significantly high in the patients having CTD with TTP. There were no significant differences in the VWF levels of the patients having CTD with TTP and thrombosis. The VWFpp levels were significantly high in the patients having CTD with TTP and thrombosis. The VWF and VWFpp levels were significantly high in the patients with pAPS. Decreased ADAMTS13 activity and elevated VWF and VWFpp levels were observed in patients with CTD. These abnormalities in patients with CTD may represent the increased risk of thrombosis in CTD.

Does deficiency of von Willebrand factor protect against cardiovascular disease? Analysis of a national discharge register

von Willebrand factor (VWF) plays a critical role in platelet adhesion and aggregation after vascular injury and at sites of high shear rate. Elevated VWF levels are associated with an increased risk of ischemic cardiovascular events; however, it is unclear whether VWF deficiency is protective against atherosclerosis. We aimed to compare the prevalence of cardiovascular disease (CVD) among patients with and without von Willebrand disease (VWD). A cross-sectional analysis was performed on discharge data for adults from the Nationwide Inpatient Sample (NIS) between the years 2009 and 2011. CVD was defined as ischemic heart disease, myocardial infarction, ischemic cerebrovascular disease, or peripheral vascular disease. For prevalence calculations and statistical analyses, we used discharge-level weights provided by the NIS to reflect national estimates. CVD was compared across groups by use of the Rao-Scott chi-square test. Multivariable logistic regression was used to estimate the likelihood of CVD in VWD patients after adjustment for age, gender, and CVD-related risk factors. The prevalence of CVD in VWD patients was less than the prevalence of CVD in non-VWD patients (15.0% versus 26.0%). VWD was associated with a decreased likelihood of CVD after adjustment for age, gender, and CVD-related risk factors (odds ratio0.85; 95% confidence interval0.79-0.92). These findings indicate that the risk of CVD is decreased among VWD patients, and that VWF deficiency may be protective against CVD.

Pregnancy Does Not Affect The Thrombotic Thrombocytopenic Purpura (TTP) phenotype Of ADAMTS13-Deficient Mice

Thrombotic thrombocytopenic purpura (TTP) is a life threatening disease, characterized by sudden onset of the pentad of fever, hemolytic anemia, thrombocytopenia, renal dysfunction and neurological findings. Previous studies in ADAMTS13-deficient mice demonstrated a dependence on both genetic and environmental factors for the expression of the disease. Though homozygous Adamts13-/- mice on a C57BL6/J (B6) background are indistinguishable from wild-type controls, introduction of the CAST/EiJ genetic background results in a spontaneous TTP-like syndrome in a subset of mice.The aim of this study was to evaluate the effect of pregnancy on TTP expression in this mouse model of TTP.Adamts13+/- mice were backcrossed for 20 generations to C57BL/6J to generate N20 B6 Adamts13+/- progeny. These mice were backcrossed for 2 generations into the CAST/EiJ mouse strain to generate N2F1 Adamts13-/- female mice that were tested and compared to N20F1 B6 Adamts13-/- female mice.Two groups of 20 Adamts13-/- female mice from each strain were tested. Mice from one group were mated with wild type males, and mice from the second group were housed separately from male mice. Pregnant Adamts13-/- mice were compared to control non-pregnant Adamts13-/- female mice. Mice were monitored for a full profile of blood parameters and peripheral blood smears weekly for 10 weeks. Data was collected for mortality, pregnancy outcome and litter size. Von Willebrand factor (VWF) levels were also measured.All Adamts13-/- mice in the B6 non-pregnant group appeared healthy and exhibited normal blood count parameters, without microangiopathic changes on peripheral blood smears. None of Adamts13-/- mice in the B6 pregnant group developed TTP. Mean platelet count in this group was 844,000±127,000 cells/µl compared to 666,000±56,000 cells/µl in the B6 non-pregnant group (p<0.01)In contrast to the B6 results, Adamts13-/- CAST/EiJ non-pregnant mice exhibited lower platelet counts 369,000±89,000 cells/µl with spontaneous TTP-like disease in 54% of mice, as expected from previous studies.Pregnancy did not worsen TTP expression in Adamts13-/- CAST/EiJ mice. Only 45% of mice in the CAST/EiJ pregnant group developed low platelet counts with a higher mean platelet count of 511,000±160,000 cells/µl, compared to non-pregnant group(p<0.01).The physiological rise in VWF levels of pregnancy had no effect on TTP susceptibility in this model. VWF antigen levels were 167%, 203%, 197% and 245% in B6 non-pregnant, B6 pregnant, CAST/EiJ non-pregnant and pregnant respectively, compared to wild-type B6 levels. Four mice died during follow-up, 2 in the B6 pregnant and 2 in the CAST/EiJ non-pregnant groups.In summary, TTP expression was unique to Adamts13-/- CAST/EiJ mice. Pregnancy and elevated VWF levels had no deleterious effect on TTP susceptibility in this mouse model. Disclosures:No relevant conflicts of interest to declare.