Increases In Plasma Von Willebrand Factor Research Articles

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.

Management of elective procedures in low von Willebrand factor patients in the LoVIC study

BackgroundMost individuals with mild to moderate reductions in plasma von Willebrand factor (VWF) levels do not demonstrate increased bleeding. However, some patients with plasma VWF levels of 30–50 IU/dl do have a significant bleeding phenotype. Management of these “low VWF” patients, who may have significant bleeding scores >10, around times of elective procedures continues to pose a common clinical challenge because of a lack of evidence. ObjectiveTo investigate the safety and efficacy of different periprocedural management options for adult patients with low VWF. MethodsTreatment and outcomes were retrospectively reviewed for 160 invasive procedures performed in 60 patients with well characterized low VWF enrolled in the previously described Low Von Willebrand factor Ireland Cohort study. ResultsWe demonstrate that 1‐desamino‐8‐D‐arginine vasopressin is efficacious in preventing bleeding for both minor or major elective procedures in adult low VWF patients, even in those with significant bleeding histories. In addition, tranexamic acid alone is effective for low VWF patients undergoing nondental minor procedures. Importantly, age‐related increases in plasma VWF:antigen levels above 50 IU/dl were not necessarily associated with complete correction of bleeding phenotype. Procedure‐related bleeding complications were increased in low VWF patients who did not receive any hemostatic cover before their procedure. ConclusionElective procedures in adult patients with low VWF should be managed in liaison with a comprehensive care tertiary referral center so that personalized treatment plans may be implemented before all minor or major elective procedures.

Correcting dominant‐negative von Willebrand disease

Correcting dominant‐negative von Willebrand disease

The common VWF single nucleotide variants c.2365A>G and c.2385T>C modify VWF biosynthesis and clearance

AbstractPlasma levels of von Willebrand factor (VWF) vary considerably in the general population and this variation has been linked to several genetic and environmental factors. Genetic factors include 2 common single nucleotide variants (SNVs) located in VWF, rs1063856 (c.2365A>G) and rs1063857 (c.2385T>C), although to date the mechanistic basis for their association with VWF level is unknown. Using genotypic/phenotypic information from a European healthy control population, in vitro analyses of recombinant VWF expressing both SNVs, and in vivo murine models, this study determined the precise nature of their association with VWF level and investigated the mechanism(s) involved. Possession of either SNV corresponded with a significant increase in plasma VWF in healthy controls (P < .0001). In vitro expression confirmed this observation and highlighted an independent effect for each SNV (P < .0001 and P < .01, respectively), despite close proximity and strong linkage disequilibrium between them both. The influence of c.2365A>G on VWF levels was also confirmed in vivo. This increase in VWF protein corresponded to an increase in VWF messenger RNA (mRNA) resulting, in part, from prolonged mRNA half-life. In addition, coinheritance of both SNVs was associated with a lower VWF propeptide-to-VWF antigen ratio in healthy controls (P < .05) and a longer VWF half-life in VWF knockout mice (P < .0001). Both SNVs therefore directly increase VWF plasma levels through a combined influence on VWF biosynthesis and clearance, and may have an impact on disease phenotype in both hemostatic and thrombotic disorders.

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.

An Arg760Cys mutation in the consensus sequence of the von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant

AbstractWe describe a von Willebrand disease (VWD) variant characterized by the persistence of von Willebrand factor (VWF) propeptide as a result of a C>T transition at nucleotide 2527 in exon 17 of the VWF gene. This mutation, which was present in the proband and his father, predicts the substitution of Cys for Arg at position 760 of pre–pro-VWF, 4 residues before the propeptide cleavage site belonging to a consensus sequence for substrate recognition by the processing enzyme paired dibasic amino acid–cleaving enzyme (PACE)/furin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) documented the presence of both processed and unprocessed VWF in the patient's plasma, with unprocessed VWF relatively less represented. The patient's hemostatic phenotype was characterized by a mild decrease in plasma factor VIII (FVIII) and VWF, a decrease in plasma VWF multimers, and a mild reduction in the FVIII binding capacity of VWF. The FVIII binding defect was more pronounced in the proband than in the father because he also inherited the type 2N Arg91Gln mutation from his mother. The persistence of VWF propeptide did not impair VWF synthesis because platelet VWF content was normal, nor did it compromise VWF storage in endothelial cells, because of the normal post–1-deamino-8-D-arginine vasopressin (DDAVP) increase in plasma VWF. Coexpression of wild-type and Arg760Cys VWF into a Furin-producing BHK cell line resulted in decreased VWF secretion and a defect in the FVIII binding capacity of VWF, together with the persistence of VWF propeptide. These findings confirm that a normal consensus sequence for VWF propeptide cleavage and efficient cleavage are required in vivo for normal FVIII binding capacity of VWF.