Treatment of severe von Willebrand disease with a high-purity von Willebrand factor concentrate (Wilfactin®): a prospective study of 50 patients

BACKGROUND AND OBJECTIVES: A plasma-derived von Willebrand factor (VWF) concentrate with low factor VIII (FVIII) content was specifically developed to treat von Willebrand disease (VWD). Efficacy and safety were investigated by merging the results of two comparable protocols conducted prospectively in 5 European and 12 French centers. METHODS AND RESULTS: Fifty patients with clinically severe VWD (72% had VWF ristocetin cofactor activity less than 10 IU dL(-1) and 46% had FVIII < 20 IU dL(-1)) were treated with the concentrate as the only therapy, except for clinical situations requiring a priming dose of FVIII to rapidly correct an intrinsic coagulation defect. A total of 139 spontaneous bleeding episodes were treated; only 53 (38%) needed a concomitant FVIII dose. Outcome was excellent or good in 89% of the episodes. Forty-four patients underwent 108 surgical or invasive procedures. Outcome was excellent or good in 95 scheduled procedures (only VWF was infused) and 13 emergency procedures (a priming FVIII dose was co-administered with the first VWF infusion). There were no thrombotic complications and none of the 18 patients with type 3 VWD developed anti-VWF or anti-FVIII antibodies. CONCLUSIONS: This concentrate safely and effectively provides hemostasis in patients with clinically severe VWD.     

Increased clearance of von Willebrand factor antigen post‐DDAVP in Type 1 von Willebrand disease: is it a potential pathogenic process?

Summary.  The mechanism of von Willebrand factor (VWF) clearance is not fully understood. The factors that affect VWF clearance, and the normal in vivo mechanism of clearance, may be relevant to the pathogenesis of Type 1 von Willebrand disease (VWD), in which there is a partial deficiency of VWF. In order to investigate the clearance of VWF in Type 1 VWD, the current study assessed the half-life of VWF antigen ( t _½ VWF:Ag) in Type 1 VWD patients and individuals with mild hemophilia A following the administration of 1-deamino-8- d -arginine vasopressin (DDAVP; desmopressin). To date 20 individuals have been assessed, 13 with Type 1 VWD and seven with mild hemophilia A. The median t _½ VWF:Ag in the Type 1 VWD and mild hemophilia A groups were 4.6 h and 9.5 h, respectively. The difference between the t _½ VWF:Ag for the two groups was significant, P  < 0.02. Analysis of the data showed a correlation between the t _½ VWF:Ag and the baseline VWF:Ag level prior to administration of DDAVP: lower baseline VWF:Ag levels were associated with a shorter t _½ VWF:Ag. These data suggest that increased clearance of VWF may be the pathogenic mechanism in some cases of Type 1 VWD.     

Shear stress is required for the endocytic uptake of the factor VIII‐von Willebrand factor complex by macrophages

Summary. Background: Low‐density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. Objectives: It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte‐derived macrophages. Methods: A confocal microscopy study was employed to study the uptake by monocyte‐derived macrophages of a functional green fluorescent FVIII‐GFP derivative in the presence and absence of VWF. Results: The results revealed that FVIII‐GFP is internalized by macrophages. We found that FVIII‐GFP co‐localizes with LDL receptor‐related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII‐GFP. However, FVIII‐GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII‐VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII‐GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII‐GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII‐GFP and VWF was inhibited. Conclusion: The results show that shear stress is required for macrophages to internalize both constituents of the FVIII‐VWF complex.     

Developments in the diagnostic procedures for von Willebrand disease

Von Willebrand disease (VWD) is the most common inherited bleeding disorder but its diagnosis can be challenging due to the heterogeneity of the disease. VWD is mainly associated with mild mucocutaneous bleeding, although there are more severe phenotypes with bleeding from the gastrointestinal tract or even the joints. Also, surgical interventions and trauma may lead to critical bleeding events. These bleeding episodes are all related to quantitative or qualitative defects of von Willebrand factor (VWF), a multimeric glycoprotein produced by endothelial cells and megakaryocytes, which mediates platelet adhesion and aggregation and binds factor VIII (FVIII) in the circulation. This review describes the diagnostic procedures required for correct diagnosis. Accurate diagnosis and classification is required for proper treatment and counseling. Assessment of bleeding starts with the medical history. After a positive bleeding or family history, subsequent laboratory investigations will start with a panel of standard screening tests for hemostatic defects. Patients suspected of having VWD will be tested for plasma VWF antigen levels, the ability of VWF to bind platelets and FVIII activity. When VWD is confirmed, a set of subtyping tests can classify the patients as VWD types 1, 2 (A, B, M or N) or 3. The performance of some additional assays and analyses, such as VWF propeptide measurement or genetic analysis, may help in identifying the pathological mechanism behind certain defects or can guide in the choice of treatment.     

Correction of a dominant‐negative von Willebrand factor multimerization defect by small interfering RNA‐mediated allele‐specific inhibition of mutant von Willebrand factor

Essentials

• Substitution therapy for von Willebrand (VW) disease leaves mutant VW factor (VWF) unhindered.
• Presence of mutant VWF may negatively affect phenotypes despite treatment.
• Inhibition of VWF by allele‐specific siRNAs targeting single‐nucleotide polymorphisms is effective.
• Allele‐specific inhibition of VWF p.Cys2773Ser improves multimerization.

Summary

Background

Treatment of the bleeding disorder von Willebrand disease (VWD) focuses on increasing von Willebrand factor (VWF) levels by administration of desmopressin or VWF‐containing concentrates. Both therapies leave the production of mutant VWF unhindered, which may have additional consequences, such as thrombocytopenia in patients with VWD type 2B, competition between mutant and normal VWF for platelet receptors, and the potential development of intestinal angiodysplasia. Most cases of VWD are caused by dominant‐negative mutations in VWF, and we hypothesize that diminishing expression of mutant VWF positively affects VWD phenotypes.

Objectives

To investigate allele‐specific inhibition of VWF by applying small interfering RNAs (siRNAs) targeting common single‐nucleotide polymorphisms (SNPs) in VWF. This approach allows allele‐specific knockdown irrespective of the mutations causing VWD.

Methods

Four SNPs with a high predicted heterozygosity within VWF were selected, and siRNAs were designed against both alleles of the four SNPs. siRNA efficiency, allele specificity and siRNA‐mediated phenotypic improvements were determined in VWF‐expressing HEK293 cells.

Results

Twelve siRNAs were able to efficiently inhibit single VWF alleles in HEK293 cells that stably produce VWF. Transient cotransfections of these siRNAs with two VWF alleles resulted in a clear preference for the targeted allele over the untargeted allele for 11 siRNAs. We also demonstrated siRNA‐mediated phenotypic improvement of the VWF multimerization pattern of the VWD type 2A mutation VWF p.Cys2773Ser.

Conclusions

Allele‐specific siRNAs are able to distinguish VWF alleles on the basis of one nucleotide variation, and are able to improve a severe multimerization defect caused by VWF p.Cys2773Ser. This holds promise for the therapeutic application of allele‐specific siRNAs in dominant‐negative VWD.

     

Reduced von Willebrand factor survival in von Willebrand disease: pathophysiologic and clinical relevance

Summary.  Von Willebrand disease (VWD) is characterized by a wide heterogeneity of clinical and laboratory phenotypes. The complexity of the phenotype is further increased by a highly variable removal rate of von Willebrand factor (VWF) released by desmopressin, which is independent of post-infusion peak level. After the initial demonstration that a reduced VWF survival is present in patients with R1205H mutation (VWD Vicenza), several other mutations, mostly occurring in the VWF D3 domain, have been shown to be associated with accelerated removal of released VWF. Normal subjects with O blood group show reduced survival after desmopressin, underlining the role of different VWF glycosylation present in ABO blood group. Recent evidence suggests that liver and spleen macrophages are responsible for VWF clearance through uptake and endocellular degradation, but it is still not known why some VWF mutants are more prone to increased clearance.     

Measurement of von Willebrand factor activity: relative effects of ABO blood type and race

Summary.  Tests based on three different principles are reported to measure the activity of von Willebrand factor (VWF): ristocetin cofactor (VWF:RCo), collagen binding (VWF:CB), and the so-called 'activity ELISA' (VWF:MoAb). We measured these and other diagnostic parameters in a population of 123 randomly selected female study controls, age 18–45 years. Type O subjects had significantly lower levels than non-O subjects in each test. Race differences were seen in all tests except VWF:RCo, with Caucasians having significantly lower levels than African-Americans. ABO differences accounted for 19% of the total variance in VWF:Ag ( P  < 0.0001) and race for 7% ( P  < 0.0001), for a total of 26%. Both effects were mediated through VWF:Ag and were independent. VWF:Ag level was the primary determinant of VWF function, accounting for approximately 60% of the variance in VWF:RCo and VWF:CB and 54% of the variance in factor VIII. The ratio VWF:RCo/VWF:Ag differed significantly by race within blood group. The median ratios were 0.97 for type O Caucasians vs. 0.79 for type O African-Americans and 0.94 for non-O Caucasians vs. 0.76 for non-O African-Americans. The ratio VWF:CB/VWF:Ag did not vary. This suggests racial differences in the interaction of VWF with GP1b but not with subendothelium. Alternatively, VWF:RCo may be regulated to maintain a relatively constant plasma level in the presence of excessive VWF:Ag. This heterogeneity within the normal population is partially responsible for the difficulty in defining diagnostic limits for von Willebrand disease.     

Preclinical evaluation of a semi‐automated and rapid commercial electrophoresis assay for von Willebrand factor multimers

Background

The von Willebrand factor (VWF) multimer test is required to correctly subtype qualitative type 2 von Willebrand disease (VWD). The current VWF multimer assays are difficult, nonstandardized, and time‐consuming. The purpose of this study was to evaluate the clinical utility of the commercial VWF multimer kit by Sebia (Lisses, France), an electrophoresis technique yielding same‐day results.

Methods

Ten healthy volunteer plasma samples, in‐house reference plasma (IRP) and commercial normal plasma (CNP) samples, 10 plasma samples from patients with a known VWD type, 1 hemophilia A plasma sample, and 7 external quality assurance (EQA) samples were analyzed using the commercial VWF multimer kit. Additional coagulation testing included measurements of VWF antigen (VWF:Ag), VWF activity (VWF:Ac), and FVIII activity (FVIII:C).

Results

The CNP results revealed a relative loss of the highest molecular weight multimers; therefore, IRP was preferred as the reference sample. The interpretations of 10 patients with a known VWD type could be successfully reproduced and agreed with previous VWF multimer results. In all EQA surveys, the multimer results and final VWD diagnosis agreed with expert opinion.

Conclusions

The VWF multimer assay by Sebia is easy to perform and can be successfully implemented in any clinical laboratory for second‐stage evaluation of VWD. The resolution power of multimer distribution is adequate to correctly classify VWD types 1, 2A, 2B, and 3.

     

Evaluation of a microfluidic flow assay to screen for von Willebrand disease and low von Willebrand factor levels

Essentials

• von Willebrand factor (VWF) function is shear stress dependent.
• Platelet accumulation in a microfluidic assay correlates with VWF levels.
• The microfluidic assay discriminates type 1 von Willebrand disease from healthy controls.
• The microfluidic flow assay detects responses to therapeutic intervention (DDAVP).

Summary

Background

von Willebrand disease (VWD) is a mucocutaneous bleeding disorder with a reported prevalence of 1 in 10 000. von Willebrand factor (VWF) function and platelet adhesion are regulated by hemodynamic forces that are not integrated into most current clinical assays.

Objective

We evaluated whether a custom microfluidic flow assay (MFA) can screen for deficiencies in VWF in patients presenting with mucocutaneous bleeding.

Methods

Whole blood from individuals with mucocutaneous bleeding was assayed in a custom MFA.

Results

Thirty‐two patients with type 1 VWD (10/32) or reported mucocutaneous bleeding were enrolled. The platelet adhesion velocity (r = 0.5978 for 750 s^?1 and 0.6895 for 1500 s^?1) and the maximum platelet surface area coverage (r = 0.5719 for 750 s^?1 and 0.6633 for 1500 s^?1) in the MFA correlated with VWF levels. Furthermore, the platelet adhesion velocity at 750 s^?1 (type 1 VWD, mean 0.0009761, 95% confidence interval [CI] 0.0003404–0.001612; control, mean 0.003587, 95% CI 0.002455–0.004719) and at 1500 s^?1 (type 1 VWD, mean 0.0003585, 95% CI 0.00003914–0.0006778; control, mean 0.003132, 95% CI 0.001565–0.004699) differentiated type 1 VWD from controls. Maximum platelet surface area coverage at 750 s^?1 (type 1 VWD, mean 0.1831, 95% CI 0.03816–0.3281; control, mean 0.6755, 95% CI 0.471–0.88) and at 1500 s^?1 (type 1 VWD, mean 0.07873, 95% CI 0.01689–0.1406; control, mean 0.6432, 95% CI 0.3607–0.9257) also differentiated type 1 VWD from controls. We also observed an improvement in platelet accumulation after 1‐desamino‐8‐d ‐arginine vasopressin (DDAVP) treatment at 1500 s^?1 (pre‐DDAVP, mean 0.4784, 95% CI 0.1777–0.7791; post‐DDAVP, mean 0.8444, 95% CI 0.7162–0.9726).

Conclusions

These data suggest that this approach can be used as a screening tool for VWD.

     

Accelerated clearance alone explains ultra‐large multimers in von Willebrand disease Vicenza

See also Goodeve AC. Vicenza deciphered: modeling the von Willebrand disease enigma: commentary on accelerated clearance alone explains ultralarge multimers in VWD Vicenza. This issue, pp 1271–2. Summary. Background: von Willebrand disease (VWD) Vicenza is characterized by low plasma von Willebrand factor (VWF) levels, the presence of ultra‐large (UL) VWF multimers and less prominent satellite bands on multimer gels, and the heterozygous amino acid substitution R1205H in the VWF gene. The pathogenesis of VWD Vicenza has been elusive. Accelerated clearance is implicated as a cause of low VWF level. Objectives: We addressed the question, whether the presence of ultra‐large multimers is a cause, or a result of accelerated VWF clearance, or whether it is an unrelated phenomenon. Patients/methods: We studied the detailed phenotype of three Hungarian patients with VWD Vicenza, expressed the mutant VWF‐R1205H in 293T cells and developed a mathematical model to simulate VWF synthesis and catabolism. Results: We found that the half‐life of VWF after DDAVP was approximately one‐tenth of that after the administration of Haemate P, a source of exogenous wild‐type (WT) VWF (0.81 ± 0.2 vs. 7.25 ± 2.38 h). An analysis of recombinant mutant VWF‐R1205H showed that the biosynthesis and multimer structure of WT and mutant VWF were indistinguishable. A mathematical model of the complex interplay of VWF synthesis, clearance and cleavage showed that decreasing VWF half‐life to one‐tenth of normal reproduced all features of VWD Vicenza including low VWF level, ultra‐large multimers and a decrease of satellite band intensity. Conclusion: We conclude that accelerated clearance alone may explain all features of VWD Vicenza.     

Different bleeding risk in type 2A and 2M von Willebrand disease: a 2‐year prospective study in 107 patients

Summary. Background: Type 2A and 2M von Willebrand disease (VWD2A and VWD2M) are characterized by the presence of a dysfunctional von Willebrand factor (VWF) and a variable bleeding tendency. So far, a head‐to‐head comparison of the clinical history and bleeding risk between VWD2A and VWD2M has never been provided in a prospective manner. Aim of the study: We assessed the bleeding incidence rate and clinical characteristics in two cohorts of 17 families (46 patients) with VWD2A and 15 families (61 patients) with VWD2M prospectively followed‐up for 24 months. VWF gene mutations were characterized in all of them. Results: Mean bleeding score (BS) and VWF antigen at enrollment were significantly higher in VWD2A patients (P = 0.007). No correlation between VWF activity or factor VIII levels and the severity of BS was observed. The incidence rate of spontaneous bleeding requiring treatment was 107/100 patient‐years (95% CI, 88.3–131) in VWD2A compared with 40/100 patient‐years (95% CI, 30–53) in VWD2M (P < 0.001). The risk of bleeding was significantly higher in patients with BS ≥ 10 at enrollment compared with those with BS 0–2. Furthermore, 54 episodes of gastrointestinal bleeding occurred in 17/46 (36.9%) VWD2A patients and seven in 2/61 (3.3%) VWD2M patients (P < 0.0001). Conclusion: Bleeding tendency in VWD2A is greater than that of VWD2M, is not explained by factor VIII or VWF levels and is mainly due to an increased incidence of gastrointestinal bleeding.     

von Willebrand factor variant p.Arg924Gln marks an allele associated with reduced von Willebrand factor and factor VIII levels

Summary. Background: von Willebrand factor (VWF) variant c.2771G>A; p.R924Q has been described as a benign polymorphism or a possible marker for a null allele and been associated with mild bleeding phenotypes. It was identified in several patients in recent type 1 von Willebrand disease (VWD) studies. Objectives: To determine whether the p.R924Q allele contributes to reduced VWF levels and type 1 VWD. Methods: One thousand one hundred and fifteen healthy controls and 148 index cases from the MCMDM‐1VWD study were genotyped for c.2771G>A; VWF and FVIII levels were analyzed in ABO blood group stratified individuals and the p.R924Q variant was expressed in 293 EBNA cells. Results: c.2771G>A was present in six index cases, five of whom had a second VWF variant which probably contributed to the phenotype. A common core haplotype identified in families, which included the rare G allele of c.5843‐8C>G, was present in the majority of 35 c.2771G>A heterozygous controls. c.2771G>A contributed about 10% variance in VWF and FVIII levels in controls and 35% variance when co‐inherited with blood group O. Recombinant p.R924Q VWF had no effect on in vitro expression and heterozygous family members had normal VWF‐FVIII binding and normal clearance of VWF and FVIII. Conclusions: The allele bearing c.2771A leads to reductions in VWF and FVIII levels particularly in combination with blood group O. Its inheritance alone may be insufficient for VWD diagnosis, but it appears to be associated with a further VWF level reduction in individuals with a second VWF mutation and it contributes to population variance in VWF and FVIII levels.     

Homozygous type 2N R854W von Willebrand factor is poorly secreted and causes a severe von Willebrand disease phenotype

Summary. Background: von Willebrand disease (VWD) type Normandy (VWD 2N) is caused by mutations at the factor (F)VIII‐binding site of von Willebrand factor (VWF), located in the D′and D3 domains on the N‐terminus of mature VWF. The R854Q mutation is the most frequent cause of this phenotype. Objectives: We report the characterization of a homozygous VWD 2N mutation, R854W, detected in a patient with a severe VWD phenotype. Methods: The plasma VWF phenotype was studied, transient expression of recombinant mutant full‐length VWF in 293 EBNA cells was performed, and the results were compared with those obtained with wild‐type (WT) VWF. Furthermore, expression was also examined in HEK293 cells, which form Weibel–Palade body‐like granules when transfected with WT VWF. Results: The multimer analysis of plasma VWF showed the lack of the typical triplet structure, with the presence of the central band only, and a relative decrease in the high molecular mass multimers. Homozygous expression of recombinant R854W VWF resulted in normal amounts of cellular VWF, but with a severe reduction in secretion into the medium. Severe reductions in FVIII binding to R854W VWF, glycoprotein Ib binding activity and collagen binding of secreted W854 VWF was observed, and reproduced the phenotypic parameters of plasma VWF. In HEK293 cells, homozygous R854W VWF failed to form Weibel–Palade body‐like granules. Conclusions: Our results demonstrate that a homozygous R854W mutation in the D′ domain of VWF induces impaired secretion and activity of the protein, thereby explaining the severe phenotype of the patient.