Increased Plasma Von Willebrand Factor Research Articles

The aptamer BT200 blocks interaction of K1405-K1408 in the VWF-A1 domain with macrophage LRP1

AbstractRondaptivon pegol (previously BT200) is a pegylated RNA aptamer that binds to the A1 domain of von Willebrand factor (VWF). Recent clinical trials demonstrated that BT200 significantly increased plasma VWF–factor VIII levels by attenuating VWF clearance. The biological mechanism(s) through which BT200 attenuates in vivo clearance of VWF has not been defined. We hypothesized that BT200 interaction with the VWF-A1 domain may increase plasma VWF levels by attenuating macrophage-mediated clearance. We observed that full-length and VWF-A1A2A3 binding to macrophages and VWF-A1 domain binding to lipoprotein receptor–related protein 1 (LRP1) cluster II and cluster IV were concentration-dependently inhibited by BT200. Additionally, full-length VWF binding to LRP1 expressed on HEK293T (HEK-LRP1) cells was also inhibited by BT200. Importantly, BT200 interacts with the VWF-A1 domain in proximity to a conserved cluster of 4 lysine residues (K1405, K1406, K1407, and K1408). Alanine mutagenesis of this K1405-K1408 cluster (VWF-4A) significantly (P < .001) attenuated binding of VWF to both LRP1 clusters II and IV. Furthermore, in vivo clearance of VWF-4A was significantly (P < .001) reduced than that of wild-type VWF. BT200 did not significantly inhibit binding of VWF-4A to LRP1 cluster IV or HEK-LRP1 cells. Finally, BT200 interaction with the VWF-A1 domain also inhibited binding to macrophage galactose lectin and the SR-AI scavenger receptor. Collectively, our findings demonstrate that BT200 prolongs VWF half-life by attenuating macrophage-mediated clearance and specifically the interaction of K1405-K1408 in the VWF-A1 domain with macrophage LRP1. These data support the concept that targeted inhibition of VWF clearance pathways represents a novel therapeutic approach for von Willebrand disease and hemophilia A.

Plasma von Willebrand factor levels in patients with cancer: A meta‑analysis.

von Willebrand Factor (VWF) is well recognized for being dysregulated in various malignancies and has emerged as a potential biomarker for cancer detection. The present meta-analysis aimed to elucidate the association between plasma VWF and the incidence and metastasis of cancer. For this purpose, a comprehensive search was conducted across multiple databases from their inception until March 3, 2023. This culminated in the selection of 15 original studies on various types of cancer, including a collective sample of 1,403 individuals. The standardized mean difference (SMD) and 95% confidence intervals (CIs) were employed as statistical parameters to determine the association between plasma VWF and the incidence and metastasis of cancer. These were estimated using a random-effects model. The pooled data revealed that the plasma VWF levels of patients with cancer were significantly elevated compared with those of healthy controls (SMD, 0.98; 95% CI, 0.59-1.36), and a significant association was observed between plasma VWF levels and cancer metastasis (SMD, 0.69; 95% CI, 0.33-1.06). The symmetry of the Begg's funnel plots indicated that no significant bias was present in the analyses of VWF in cancer and its metastasis. In summary, the results of the present meta-analysis support the hypothesis that increased plasma VWF levels may serve as a biomarker for cancer and metastatic progression.

Diagnostic pitfalls and conundrums in type 1 von Willebrand disease

Most people with von Willebrand disease (VWD) have a partial quantitative deficiency of plasma von Willebrand factor (VWF) or type 1 VWD. In contrast to type 2 and type 3 VWD, laboratory assays will not always establish the diagnosis in type 1 VWD. This is because plasma VWF levels in type 1 VWD, especially those with levels closer to 50 IU/dL, overlap with the general population. Assessment is further complicated by increased plasma VWF levels in response to physiologic stressors or aging. Diagnosis of those with type 1 VWD with plasma VWF levels 30 to 50 IU/dL (previously referred to as "low VWF") requires expert assessment of bleeding phenotype as well as an understanding of the limitations of both bleeding assessment tools (BATs) and laboratory testing. Using the available evidence and highlighting research gaps, we discuss common dilemmas facing providers relating to assessment of adolescents, transition from pediatrics to adult care, and older individuals with type 1 VWD.

Von Willebrand disease and von Willebrand factor.

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

Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19

Thrombosis affecting the pulmonary and systemic vasculature is common during severe COVID-19 and causes adverse outcomes. Although thrombosis likely results from inflammatory activation of vascular cells, the mediators of thrombosis remain unconfirmed. In a cross-sectional cohort of 36 severe COVID-19 patients, we show that markedly increased plasma von Willebrand factor (VWF) levels were accompanied by a partial reduction in the VWF regulatory protease ADAMTS13. In all patients we find this VWF/ADAMTS13 imbalance to be associated with persistence of ultra-high-molecular-weight (UHMW) VWF multimers that are highly thrombogenic in some disease settings. Incubation of plasma samples from patients with severe COVID-19 with recombinant ADAMTS13 (rADAMTS13) substantially reduced the abnormally high VWF activity, reduced overall multimer size and depleted UHMW VWF multimers in a time and concentration dependent manner. Our data implicate disruption of normal VWF/ADAMTS13 homeostasis in the pathogenesis of severe COVID-19 and indicate that this can be reversed ex vivo by correction of low plasma ADAMTS13 levels. These findings suggest a potential therapeutic role for rADAMTS13 in helping restore haemostatic balance in COVID-19 patients.

Von Willebrand factor propeptide in severe coronavirus disease 2019 (COVID-19): evidence of acute and sustained endothelial cell activation.

Endothelial cell (EC) activation plays a key role in the pathogenesis of pulmonary microvascular occlusion, which is a hallmark of severe coronavirus disease 2019 (COVID-19). Consistent with EC activation, increased plasma von Willebrand factor antigen (VWF:Ag) levels have been reported in COVID-19. Importantly however, studies in other microangiopathies have shown that plasma VWF propeptide (VWFpp) is a more sensitive and specific measure of acute EC activation. In the present study, we further investigated the nature of EC activation in severe COVID-19. Markedly increased plasma VWF:Ag [median (interquatile range, IQR) 608·8(531-830)iu/dl] and pro-coagulant factor VIII (FVIII) levels [median (IQR) 261·9(170-315)iu/dl] were seen in patients with severe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Sequential testing showed that these elevated VWF-FVIII complex levels remained high for up to 3weeks. Similarly, plasma VWFpp levels were also markedly elevated [median (IQR) 324·6(267-524)iu/dl]. Interestingly however, the VWFpp/VWF:Ag ratio was reduced, demonstrating that decreased VWF clearance contributes to the elevated plasma VWF:Ag levels in severe COVID-19. Importantly, plasma VWFpp levels also correlated with clinical severity indices including the Sequential Organ Failure Assessment (SOFA) score, Sepsis-Induced Coagulopathy (SIC) score and the ratio of arterial oxygen partial pressure to fractional inspired oxygen (P/F ratio). Collectively, these findings support the hypothesis that sustained fulminant EC activation is occurring in severe COVID-19, and further suggest that VWFpp may have a role as a biomarker in this setting.

Hemostatic Abnormalities in Dementia: A Systematic Review and Meta-Analysis.

Alzheimer's disease (AD) is considered the most frequent cause of dementia. It is known that vascular risk factors play an important role in the development and progression of this condition. Alterations in vascular walls represent documented findings in patients with AD and other dementias affecting elderly people. The authors performed a systematic review and meta-analysis, aiming to synthesize observational studies that evaluated how the hemostatic system may contribute to cognitive decline in the elderly, using papers published until April 2018 and as indexed in Medline (PubMed), Scopus, Web of Science, ScienceDirect, Lilacs, Cinahl, PsycINFO, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews. Among 5,278 studies identified, 32 were included in the final synthesis, and these included 485 patients with mild cognitive impairment, 568 with vascular dementia (VD), 1,781 with AD, and 2,855 participants without dementia. AD patients had increased plasma von Willebrand factor (VWF) (standardized mean difference [SMD]: 2.53; 95% confidence interval [CI]: 0.10-4.95), D-dimer (SMD: 0.50; 95% CI: 0.35-0.66), plasminogen activator inhibitor-1 (SMD: 3.34; 95% CI: 1.01-5.67), thrombomodulin (SMD: 1.08; 95% CI: 0.53-1.62), and homocysteine levels (SMD: 0.65; 95% CI: 0.15-1.15). In contrast, the VD group showed increased fibrinogen levels (SMD: 0.77; 95% CI: 0.13-1.41), activated factor VII (SMD: 0.36; 95% CI: 0.05-0.67), factor VIII (SMD: 0.57; 95% CI: 0.22-0.91), VWF (SMD: 2.34; 95% CI: 0.38-4.29), D-dimer (SMD: 1.14; 95% CI: 0.51-1.78), and homocysteine (SMD: 2.17; 95% CI: 1.67-2.68). AD showed an elevation in some markers of endothelial dysfunction, whereas VD presented mostly an involvement of coagulation cascade components.

ADAMTS13 protects mice against renal ischemia-reperfusion injury by reducing inflammation and improving endothelial function.

Acute kidney injury (AKI) is a serious condition without efficient therapeutic options. Recent studies have indicated that recombinant human a disintegrin and metalloprotease with thrombospondin motifs 13 (rhADAMTS13) provides protection against inflammation. Therefore, we hypothesized that ADAMTS13 might protect against AKI by reducing inflammation. Bilateral renal ischemia-reperfusion injury (I/R) was used as AKI models in this study. Prophylactic infusion of rhADAMTS13 was employed to investigate potential mechanisms of renal protection. Renal function, inflammation, and microvascular endothelial function were assessed after 24 h of reperfusion. Our results showed that I/R mice increased plasma von Willebrand factor levels but decreased ADAMTS13 expression. Administration of rhADAMTS13 to I/R mice recovered renal function, histological injury, and apoptosis. Renal inflammation was reduced by rhADAMTS13, accompanied with the downregulation of p38/extracellular signal-regulated protein kinase phosphorylation and cyclooxygenase-2 expression. rhADAMTS13 restored vasodilation in afferent arterioles in I/R mice. Furthermore, rhADAMTS13 treatment enhanced phosphorylation of Akt at Ser473 and eNOS at Ser1177. Administration of the Akt pathway inhibitor wortmannin reduced the protective effect of rhADAMTS13. Our conclusions are that treatment with rhADAMTS13 ameliorates renal I/R injury by reducing inflammation, tubular cell apoptosis, and improving microvascular endothelial dysfunction. rhADAMTS13 could be a promising strategy to treat AKI in clinical settings.

Novel Insights into the Clinical Phenotype and Pathophysiology Underlying Low VWF Levels: The Low Von Willebrand Factor in Ireland Cohort (LoVIC) Study

▪Cohort studies have identified VWF gene mutations in ~65% of patients with type 1 Von Willebrand Disease (VWD). However, in patients with mild reductions in Von Willebrand Factor (VWF) levels, VWF mutations are less common and linkage studies suggest that the reduced plasma VWF levels are often independent of the VWFgene. Important clinical questions therefore remain unanswered regarding diagnosis and management of patients with Low VWF (levels 30-50 IU/dL). Evidence-based diagnostic criteria are needed for establishing the diagnosis of Low VWF, and the relationship between Low VWF levels and bleeding phenotype needs to be defined. Furthermore, the molecular mechanisms responsible for the reduced plasma VWF levels remain unclear. To address these questions, we established the Low VWF Ireland Cohort (LoVIC) study. In contrast to previous Type 1 VWD studies, this prospective longitudinal cohort study recruited only patients with Low VWF levels, (defined by bleeding history and lowest VWF levels 30-50 IU/dL on two occasions, three months apart) and recruited 140 adult Irish patients.Although Bleeding Assessment Tools (BATs) have been used in type 1 VWD, their utility has not been studied in patients with Low VWF. As determined using either the ISTH BAT or the Condensed MCMDM-1 VWD score, we observed significant bleeding histories in the majority of LoVIC patients. For example, among female patients (n=112), 77% had positive ISTH BAT scores (≥6) and 72% had positive MCMDM-1 scores (≥4). Importantly, bleeding tendency did not correlate with plasma VWF levels within the 30-50 IU/dL range. To further investigate this bleeding phenotype, hemostatic studies (including platelet aggregation and coagulation factor assays) were performed. Additional mild coagulation defects were identified in only 10 subjects. Furthermore, abnormal multimer patterns were identified in only 3 patients. To investigate the pathophysiology underlying Low VWF levels, plasma FVIII:C and VWF propeptide (VWF:pp) levels were defined. Interestingly, plasma FVIII:C/VWF:Ag ratios were significantly increased in LoVIC patients compared to normal controls (mean 1.3 versus 1.07; p<0.001). In contrast, increased plasma VWF:pp/VWF:Ag ratios > 3 were observed in only 6% of the total cohort. Taken together, these data demonstrate that the reduced plasma VWF levels in patients with Low VWF are predominantly attributable to decreased VWF synthesis and/or secretion rather than enhanced VWF clearance. To determine whether quantitative and/or qualitative abnormalities in platelet (plt)-VWF may influence bleeding phenotype in patients with Low VWF, we studied plt-VWF:Ag and VWF:CB in 50 consecutive LoVIC patients. In keeping with the hypothesis that reduced VWF synthesis plays a key role in the pathogenesis underlying Low VWF levels, plt-VWF:Ag and plt-VWF:CB levels were both significantly reduced in LoVIC patients compared to controls (mean plt-VWF:Ag 0.16 versus 0.21 IU/109/L, p<0.05; mean plt-VWF:CB 0.18 versus 0.34 IU/109/L, p<0.001).To further study the molecular mechanisms underlying Low VWF levels, plasma VWF:Ag and VWF:RCo responses following desmopressin (DDAVP) administration were examined. In 88% of patients, the post-DDAVP peak levels exceeded 100 IU/dL. Importantly, the response to DDAVP was also sustained with both VWF:Ag and VWF:RCo above 100 IU/dL after 4 hours in 72% subjects. These DDAVP responses demonstrate that Weibel Palade body stores of VWF are maintained in patients with Low VWF, and that the regulated pathway of VWF secretion is intact. Furthermore, the sustained plasma VWF response observed following DDAVP supports the hypothesis that enhanced VWF clearance does not play a major role in the pathogenesis of Low VWF levels.In conclusion, we demonstrate that Low VWF levels are associated with a significant bleeding phenotype. In addition, we further show that in the majority of patients with Low VWF levels, the bleeding phenotype cannot be explained by the presence of concomitant bleeding disorders. Finally, our novel data demonstrate that Low VWF levels are due in large part to reductions in VWF synthesis and/or constitutive secretion. Although enhanced VWF clearance may contribute to the pathophysiology in a minority of individuals with Low VWF, the absolute reduction in VWF plasma half-life is usually mild and not sufficient to significantly impact upon the duration of DDAVP-induced VWF response. DisclosuresLavin:Baxalta: Consultancy, Research Funding. O'Sullivan:Pzifer: Research Funding. O'Connell:Baxalta: Consultancy, Research Funding. James:Octapharma: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Biogen: Consultancy; Basalt: Consultancy. Di Paola:CSL BEhring: Consultancy; Biogen: Consultancy. O'Donnell:Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Leo Pharma: Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

A novel role for von Willebrand factor in the pathogenesis of experimental cerebral malaria

AbstractPlasmodium falciparum malaria infection is associated with an early marked increase in plasma von Willebrand factor (VWF) levels, together with a pathological accumulation of hyperreactive ultra-large VWF (UL-VWF) multimers. Given the established critical role of platelets in malaria pathogenesis, these increases in plasma VWF raise the intriguing possibility that VWF may play a direct role in modulating malaria pathogenesis. To address this hypothesis, we used an established murine model of experimental cerebral malaria (ECM), in which wild-type (WT) C57BL/6J mice were infected with Plasmodium berghei ANKA. In keeping with findings in children with P falciparum malaria, acute endothelial cell activation was an early and consistent feature in the murine model of cerebral malaria (CM), resulting in significantly increased plasma VWF levels. Despite the fact that murine plasma ADAMTS13 levels were not significantly reduced, pathological UL-VWF multimers were also observed in murine plasma following P berghei infection. To determine whether VWF plays a role in modulating the pathogenesis of CM in vivo, we further investigated P berghei infection in VWF−/− C57BL/6J mice. Clinical ECM progression was delayed, and overall survival was significantly prolonged in VWF−/− mice compared with WT controls. Despite this protection against ECM, no significant differences in platelet counts or blood parasitemia levels were observed between VWF−/− and WT mice. Interestingly, however, the degree of ECM-associated enhanced blood–brain barrier permeability was significantly attenuated in VWF−/− mice compared with WT controls. Given the significant morbidity and mortality associated with CM, these novel data may have direct translational significance.

ADAMTS13/VWF Axis Potentially Contributes to Diabetic Nephropathy By Regulating PAI-1 Levels

Background and objective: ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type I repeats-13) cleaves von Willebrand factor (VWF), a large multimeric protein that plays an important role in hemostasis and thrombosis. Severe deficiency or very low levels of ADAMTS13 in presence of external stimuli results in accumulation of thrombogenic ultra large VWF multimers (which are released from activated endothelium) known to trigger thrombotic microangiopathy. Activated endothelium/dysfunction is a prominent feature of diabetic nephropathy, and advanced diabetic glomerulopathy often exhibits thrombotic microangiopathy. Significantly reduced ADAMTS13 and increased plasma VWF levels have been found in diabetic patients with nephropathy. Although major site of ADAMTS13 synthesis is liver, ADAMTS13 is also expressed by podocytes in normal renal cortex. It remains unknown, however, whether VWF and ADAMTS13 imbalance plays a causal role in development of nephropathy in diabetic patients or rather is simply an associate marker of disease status, possibly secondary to endothelial function. We performed experiments in genetic models to determine whether ADAMTS13 and VWF axis contributes to diabetic nephropathy.Methods: Male, 8-10 weeks old wild-type (WT), Adamts13-/- and Vwf-/- mice were made diabetic by injecting multiple low doses of streptozotocin (60 mg/kg, i.p. for five consecutive days). Successful diabetes induction was tested after 2 weeks by measuring blood glucose. Mice having blood glucose levels above 300 mg/dL were included in the study. Controls were nondiabetic littermate mice treated with citrate buffer. The extent of renal injury was evaluated after 28 weeks of diabetes induction by measuring albuminuria and kidney to body weight ratio. Renal hypertrophy and extracellular matrix deposition was quantified by hematoxylin and immunostaining. PAI-1 mRNA and protein levels were measured by real time quantitative RT-PCR and ELISA.Results: Adamts13-/- diabetic mice exhibited significantly increased kidney to body weight ratio (P<0.05 vs. WT diabetic mice). Urine albuminuria, an index of renal injury was significantly elevated in Adamts13-/- diabetic mice (P<0.05 vs. WT diabetic mice). Increased renal injury in Adamts13-/- diabetic mice was concomitant with increased renal hypertrophy and extracellular matrix (ECM) deposition within glomeruli (P<0.05 vs. WT diabetic mice). Murine studies have shown that PAI-1 contributes to diabetic nephropathy by regulating TGF-beta and ECM deposition. A positive association exists between increased PAI-1 levels in glomeruli and microangiopathy in patients with diabetic nephropathy. We determined whether ADAMTS13 deficiency-induced microangiopathy in glomeruli increases PAI-1 levels. Adamts13-/- diabetic mice exhibited increased PAI-1 mRNA and protein levels (P<0.05 vs. WT diabetic mice). VWF remains the only known substrate of ADAMTS13 and increased plasma VWF levels have been associated with diabetic nephropathy. We determined the role of VWF in diabetic nephropathy. Vwf-/- diabetic mice exhibited significantly decreased kidney weight/body weight ratio, less urinary albuminuria, decreased kidney PAI-1 expression levels concomitant with improved kidney morphological changes (P<0.05 vs. WT diabetic mice).Conclusion: These findings provide experimental evidence for the first time that ADAMTS13/VWF axis potentially contributes to diabetic nephropathy, most likely by regulating PAI-1 levels. DisclosuresNo relevant conflicts of interest to declare.

Cocaine and Specific Cocaine Metabolites Induce von Willebrand Factor Release From Endothelial Cells in a Tissue-Specific Manner

Cocaine use is associated with arterial thrombosis, including myocardial infarction and stroke. Cocaine use results in increased plasma von Willebrand factor (VWF), accelerated atherosclerosis, and platelet-rich arterial thrombi, suggesting that cocaine activates the endothelium, promoting platelet-VWF interactions. Human umbilical vein endothelial cells, brain microvasculature endothelial cells, or coronary artery endothelial cells were treated with cocaine or metabolites benzoylecgonine, cocaethylene, norcocaine, or ecgonine methylester. Supernatant VWF concentration and multimer structure were measured, and platelet-VWF strings formed on the endothelial surface under flow were quantified. Cocaine, benzoylecgonine, and cocaethylene induced endothelial VWF release, with the 2 metabolites being more potent than the parent molecule. Brain microvasculature endothelial cells were more sensitive to cocaine and metabolites than were human umbilical vein endothelial cells or coronary artery endothelial cells. Coronary artery endothelial cells released VWF into the supernatant but did not form VWF-platelet strings. Intracellular cAMP concentration was not increased after treatment with cocaine or its metabolites. Both cocaine and metabolites benzoylecgonine and cocaethylene induced endothelial VWF secretion, possibly explaining thrombotic risk after cocaine ingestion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. These results suggest that clinical management of cocaine-induced ischemia may benefit from therapies aimed at disrupting the VWF-platelet interaction.

Neutrophil extracellular traps promote deep vein thrombosis in mice

Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be prothrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). To explore the source and role of extracellular chromatin in DVT. We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). We demonstrate that the levels of extracellular DNA increase in plasma after 6 h IVC stenosis, compared with sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs' structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development.

Polymicrobial sepsis and endotoxemia promote microvascular thrombosis via distinct mechanisms.

We reported recently that endotoxemia promotes microvascular thrombosis in cremaster venules of wild-type mice, but not in mice deficient in toll-like receptor 4 (TLR4) or von Willebrand factor (VWF). To determine whether the clinically relevant model of polymicrobial sepsis induced by cecal ligation/perforation (CLP) induces similar responses via the same mechanisms as endotoxemia. We used a light/dye-injury model of thrombosis in the cremaster microcirculation of wild-type mice and mice deficient in toll-like receptor-4 (C57BL/10ScNJ), toll-like receptor 2 (TLR2), or VWF. Mice underwent CLP or sham surgery, or an intraperitoneal injection of endotoxin (LPS) or saline. In the CLP model, we assessed the influence of fluid replacement on thrombotic responses. Both CLP and LPS enhanced thrombotic occlusion in wild-type mice. In contrast to LPS, CLP enhanced thrombosis in TLR4- and VWF-deficient strains. While TLR2-deficient mice did not demonstrate enhanced thrombosis following CLP, LPS enhanced thrombosis in these mice. LPS, but not CLP, increased plasma VWF antigen relative to controls. Septic mice, particularly those undergoing CLP, developed significant hemoconcentration. Intravenous fluid replacement with isotonic saline prevented the hemoconcentration and prothrombotic responses to CLP, though fluids did not prevent the prothrombotic response to LPS. Polymicrobial sepsis induced by CLP and endotoxemia promote microvascular thrombosis via distinct mechanisms; enhanced thrombosis induced by CLP requires TLR2 but not TLR4 or VWF. The salutary effects of intravenous fluid replacement on microvascular thrombosis in polymicrobial sepsis remain to be characterized.

Protein phosphatase 2B inhibition promotes the secretion of von Willebrand factor from endothelial cells.

Secretion of Weibel-Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the alpha and beta isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.

Severe Plasmodium falciparum malaria is associated with circulating ultra-large von Willebrand multimers and ADAMTS13 inhibition.

Plasmodium falciparum infection results in adhesion of infected erythrocytes to blood vessel endothelium, and acute endothelial cell activation, together with sequestration of platelets and leucocytes. We have previously shown that patients with severe infection or fulminant cerebral malaria have significantly increased circulatory levels of the adhesive glycoprotein von Willebrand factor (VWF) and its propeptide, both of which are indices of endothelial cell activation. In this prospective study of patients from Ghana with severe (n = 20) and cerebral (n = 13) P. falciparum malaria, we demonstrate that increased plasma VWF antigen (VWF∶Ag) level is associated with disproportionately increased VWF function. VWF collagen binding (VWF∶CB) was significantly increased in patients with cerebral malaria and severe malaria (medians 7.6 and 7.0 IU/ml versus 1.9 IU/ml; p<0.005). This increased VWF∶CB correlated with the presence of abnormal ultra-large VWF multimers in patient rather than control plasmas. Concomitant with the increase in VWF∶Ag and VWF∶CB was a significant persistent reduction in the activity of the VWF-specific cleaving protease ADAMTS13 (∼55% of normal; p<0.005). Mixing studies were performed using P. falciparum patient plasma and normal pooled plasma, in the presence or absence of exogenous recombinant ADAMTS13. These studies demonstrated that in malarial plasma, ADAMTS13 function was persistently inhibited in a time-dependent manner. Furthermore, this inhibitory effect was not associated with the presence of known inhibitors of ADAMTS13 enzymatic function (interleukin-6, free haemoglobin, factor VIII or thrombospondin-1). These novel findings suggest that severe P. falciparum infection is associated with acute endothelial cell activation, abnormal circulating ULVWF multimers, and a significant reduction in plasma ADAMTS13 function which is mediated at least in part by an unidentified inhibitor.

Increased Von Willebrand Factor Antigen Clearance in Type 1 Von Willebrand Disease: Investigation of Relationship with ABO Blood Group and Tyr1584Cys Polymorphism.

Type 1 von Willebrand disease (VWD) is a heterogeneous bleeding disorder in which genetic modifying factors, including ABO blood group, contribute towards the variability in von Willebrand factor (VWF) levels. Recent findings have reported an increased incidence of the Tyr1584Cys polymorphism in the VWF-A2 domain of patients with type 1 VWD. Presence of Cys1584 has been shown to cause increased intracellular retention of VWF, as well as lead to increased susceptibility of VWF to proteolysis by the metalloprotease ADAMTS13. An increased susceptibility to proteolysis by ADAMTS13 has also been demonstrated in the VWF of blood group O individuals. We have investigated the relationship between increased VWF antigen (VWF:Ag) clearance, ABO blood group and the presence of the Tyr1584Cys polymorphism in a group of patients with type 1 VWD. The VWF:Ag half-life (VWF:Ag t1/2) was prospectively evaluated in 45 patients with type 1 VWD, which included three families of two or more first-degree relatives. Median VWF:Ag level was 36.0iu/dl (range 4–50iu/dl); median VWF ristocetin cofactor activity (VWF:RCo) level was 35.0iu/dl (range 3–52iu/dl) and VWF:RCo/VWF:Ag ratio 0.97 (range 0.70–1.37). A normal multimeric pattern was demonstrated in all patients. 25 (55.5%) and 20 (44.5%) of the patients were of blood groups O and A respectively. The control group comprised eight patients with haemophilia A. VWF:Ag levels were measured over a 6 hour period following the administration of intravenous DDAVP. VWF:Ag t1/2 was calculated using the formula: C(t)=C0e−k.t, where C(t)=plasma [VWF:Ag] as a function of time; C0=[VWF:Ag] at time zero; e=base for natural logarithms; k=first order rate constant for the elimination phase (ß phase); t=time. The median value of the VWF:Ag t1/2 in the VWD group was 4.1 hours (95% C.I. 3.2–4.9h) and in the haemophilia A group 9.5 hours (95% C.I. 5.3–19h). This represents a significant difference in VWF:Ag t1/2 between the two groups (p<0.05). However, within the VWD patient group, there was no significant difference between the median VWF:Ag t1/2 values of patients of blood group O and those of blood group A (p>0.05). Within the three families, two affected family members of the same ABO blood group were studied and a concordant reduction in the VWF:Ag t1/2s was found in these subjects. To date, 24 of the VWD patients have been genotyped for the A/G polymorphism at nucleotide 24/1282 in the VWF gene, encoding a Tyr1584Cys polymorphism. The heterozygous presence of the G allele encoding Cys 1282 was demonstrated in one patient. The frequency of this polymorphism in normal and type 1 VWD individuals has been reported to be ~1% and 14% respectively. The median VWF:Ag t1/2 value of the 23 homozygous Tyr1584 patients was 3.4 hours (95% C.I. 3.2–4.8h), representative of the whole VWD group. The VWF:Ag t1/2 in the heterozygous patient was 4.8 hours. The finding of increased plasma VWF:Ag clearance as reflected by a reduction in VWF:Ag half-life in a significant number of patients with type 1 VWD, suggests that increased VWF:Ag clearance may be a contributory factor in the aetiology of type 1 VWD. However, this study suggests there is no relationship between increased VWF:Ag clearance and ABO blood group. Furthermore, the Tyr1584Cys polymorphism is not a major determinant of VWF:Ag clearance within this group of type 1 VWD patients.

An association of candidate gene haplotypes and bleeding severity in von Willebrand disease (VWD) type 1 pedigrees

Abstractvon Willebrand disease (VWD) type 1 is difficult to diagnose because of bleeding variability and low heritability of von Willebrand factor (VWF) levels. We compared a bleeding severity score and bleeding times to candidate gene haplotypes within pedigrees of 14 index cases, using a covariance components model for multivariate traits (Mendel: QTL Association). These pedigrees included 13 affected and 40 unaffected relatives, as defined by plasma ristocetin cofactor (VWF:RCo) levels. The bleeding severity score was derived from a detailed history. Donors were genotyped using a primer extension method, and 9 candidate genes were selected for analysis. VWF:RCo levels had the strongest influence on bleeding severity score and bleeding time. ITGA2 haplotype 2 (807C) and ITGA2B haplotype 1 (Ile843) were each associated with increased bleeding severity scores (P < .01 and P < .01, respectively). GP6 haplotype b (Pro219) was also associated with increased scores (P = .03) after adjustment for donor age. No association was observed with 6 other candidate genes, GP1BA, ITGB3, VWF, FGB, IL6, or TXA2R. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (–1793G; P = .02). These results establish that genetic differences in the adhesion receptor subunits α2, αIIb, and GPVI can influence the phenotype of VWD type 1.

Elevated plasma factor VIII in a mouse model of low-density lipoprotein receptor–related protein deficiency

AbstractIt has been established that low-density lipoprotein receptor-related protein (LRP) is involved in the cellular uptake and degradation of coagulation factor VIII (FVIII) in vitro. To address the physiologic role of LRP in regulating plasma FVIII in vivo, we used cre/loxP–mediated conditional LRP- deficient mice (MX1cre+LRPflox/flox). Upon inactivation of the LRP gene, MX1cre+LRPflox/flox mice had significantly higher plasma FVIII as compared with control LRPflox/floxmice (3.4 and 2.0 U/mL, respectively; P < .001). Elevated plasma FVIII levels in MX1cre+LRPflox/flox mice coincided with increased plasma von Willebrand factor (VWF) (2.0 and 1.6 U/mL for MX1cre+LRPflox/flox and control LRPflox/flox mice, respectively; P < .05). Elevation of plasma FVIII and VWF persisted for at least 6 weeks after inactivation of the LRP gene. Upon comparing plasma FVIII and VWF in individual mice, we observed an increase of the FVIII/VWF ratio in MX1cre+LRPflox/flox mice as compared with control LRPflox/flox mice. Administration of either a vasopressin analog or an endotoxin resulted in increased plasma VWF, but not FVIII. In clearance experiments, MX1cre+LRPflox/flox mice displayed a 1.5-fold prolongation of FVIII mean residence time. Adenovirus-mediated overexpression of the 39-kDa receptor–associated protein (RAP) in normal mice resulted in a 3.5-fold increase of plasma FVIII. These data confirm that the regulation of plasma FVIII in vivo involves a RAP-sensitive mechanism. Surprisingly, plasma FVIII in MX1cre+LRPflox/flox mice increased 2-fold after RAP gene transfer. We propose that RAP-sensitive determinants other than hepatic LRP contribute to the regulation of plasma FVIII in vivo.

Increased platelet activation in renal transplant patients

Background: Patients with functioning renal transplants are at risk of graft thrombosis in the postoperative period, and of fistula thrombosis and other thrombotic events thereafter. Investigation and therapeutic manipulation of haemostasis in these patients offers a means to counter this thrombotic tendency. Methods: Platelet aggregation in whole blood, plasma von Willebrand factor and plasma fibrinogen levels were measured in 32 stable renal transplant patients (creatinine <200 mu mol/litre, age of graft >4 months) and in 32 age, sex and smoking-habit matched normal controls. Results: In both patient and control groups, seven patients were smokers and the remaining 25 were non-smokers. There was no significant difference in age between patients and controls [patients, median: 39 (20-64) years; controls: 38 (24-60) years]. Spontaneous platelet aggregation was significantly higher in the patients at all time points studied [30s-6 min; at 4 min: patients median (interquartile range) 19.4 (11.3-27.3)%; controls, 8.0 (5.1-15.0)%, P < 0.0005]. ADP-induced aggregation was also increased at a concentration range of 0.1-3 mu M (at 1 mu M, 1 min, patients median (interquartile range) was 52.4 (30.5-70.0)%; controls was 16.5 (1.4-31.4)%, P < 0.0001). Transplant patients had significantly higher von Willebrand factor and fibrinogen levels compared with the controls (von Willebrand factor, patients median (range): 158 (13-269)%; controls: 85 (43-223)%, P < 0.00001; fibrinogen, patients: 3.29 (2.12-7.39) g/litre; controls: 2.81 (1.84-4.65) g/litre, P < 0.0002). Conclusion: Patients with stable renal transplants have in vitro evidence of enhanced platelet activation, and increased plasma von Willebrand factor and fibrinogen levels .