A noncanonical splicing variant c.875-5 T > G in von Willebrand factor causes in-frame exon skipping and type 2A von Willebrand disease.

INTRODUCTION: A novel variant involving noncanonical splicing acceptor site (c.875-5 T > G) in propeptide coding region of von Willebrand factor (VWF) was identified in a patient with type 2A von Willebrand disease (VWD), who co-inherited with a null variant (p.Tyr271*) and presented characteristic discrepancy of plasma level of VWF antigen and activity, and a selective reduction of both intermediate-molecular-weight (IMWMs) and high-molecular-weight VWF multimers (HMWMs). MATERIALS AND METHODS: VWF mRNA transcripts obtained from peripheral leukocytes and platelets of the patients were investigated to analyze the consequence of c.875-5 T > G on splicing. The impact of the variant on expression and multimer assembly was further analyzed by in vitro expression studies in AtT-20 cells. The intracellular processing of VWF mutant and the Weibel-Palade bodies (WPBs) formation was evaluated by immunofluorescence staining and electron microscopy. RESULTS: The mRNA transcript analysis revealed that c.875-5 T > G variant led to exon 8 skipping and an in-frame deletion of 41 amino acids in the D1 domain of VWF (p.Ser292_Glu333delinsLys), yielding a truncated propeptide. Consistent with the patient's laboratory manifestations, the AtT-20 cells transfected with mutant secreted less VWF, with the VWF antigen level in conditioned medium 47 % of wild-type. A slight retention in the endoplasmic reticulum was observed for the mutant. Almost complete loss of IMWMs and HMWMs in the medium and impaired WPBs formation in the cell, indicating truncated VWF propeptide lost its chaperon-like function for VWF multimerization and tubular storage. CONCLUSIONS: The VWF splicing site variant (c.875-5 T > G) causes propeptide truncation, severely compromising VWF multimer assembly and tubular storage.

Type 2M/2A von Willebrand disease: a shared phenotype between type 2M and 2A.

ABSTRACT: Four variants have been continuously subjected to debate and received different von Willebrand disease (VWD) classifications: p.R1315L, p.R1315C, p.R1374H, and p.R1374C. We chose to comprehensively investigate these variants with full set of VWD tests, protein-modeling predictions and applying structural biology. Patients with p.R1315L, p.R1315C, p.R1374H, and p.R1374C were included. A group with type 2A and 2M was included to better understand similarities and differences. Patients were investigated for phenotypic assays and underlying disease mechanisms. We applied deep protein modeling predictions and structural biology to elucidate the causative effects of variants. Forty-three patients with these variants and 70 with 2A (n = 35) or 2M (n = 35) were studied. Patients with p.R1315L, p.R1374H, or p.R1374C showed a common phenotype between 2M and 2A using von Willebrand factor (VWF):GPIbR/VWF:Ag and VWF:CB/VWF:Ag ratios and VWF multimeric profile, whereas p.R1315C represented a type 2M phenotype. There was an overall reduced VWF synthesis or secretion in 2M and cases with p.R1315L, p.R1374H, and p.R1374C, but not in 2A. Reduced VWF survival was observed in most 2A (77%), 2M (80%), and all 40 cases with p.R1315L, p.R1374H, and p.R1374C. These were the only variants that fall at the interface between the A1-A2 domains. p.R1315L/C mutants induce more compactness and internal mobility, whereas p.R1374H/C display a more extended overall geometry. We propose a new classification of type 2M/2A for p.R1315L, p.R1374H, and p.R1374C because they share a common phenotype with 2M and 2A. Our structural analysis shows the unique location of these variants on the A1-A2 domains and their distinctive effect on VWF.

Von Willebrand Factor (VWF) multiplex activity assay differentiation of type 1 von Willebrand Disease (VWD) and variant VWD.

INTRODUCTION: VWD diagnosis is challenging requiring multiple VWF activity tests using many individual assays. We have developed an ELISA-based VWF Multiplex Activity Assay (VWF-MAA) to address this concern; however, the ability of the VWF-MAA to discriminate between type 1 VWD, variant VWD, and normal subjects has not been evaluated. AIM: To evaluate the VWF-MAA and its ability to differentiate between type 1 VWD, variant VWD and normal subjects in individuals undergoing an initial laboratory evaluation for bleeding. METHODS: A total of 177 plasma samples from the Zimmerman Program: Comparative Effectiveness in the Diagnosis of VWD were evaluated from 11 centres across the US and Canada. The VWF-MAA was compared to Versiti Blood Research Institute (VBRI) and Local Center (LC) assigned VWD diagnosis. RESULTS: Overall, 129/177 (72.9%) were correctly assigned as normal (non-VWD), type 1, or variant VWD compared to the VBRI assigned diagnosis. VWF-MAA assigned non-VWD accurately in 29/57 (50.9%) samples, and type 1 VWD accurately in 93/110 (84.6%) samples. Considering LC diagnosis where there was agreement with VWF-MAA and not VBRI diagnosis, type 1 VWD was accurate in 105/110 (95.5%) samples. Bland-Altman analysis demonstrated good correlation between laboratory methods. VWD, types 2A, 2B, 1C VWD were also assigned by the VWF-MAA. CONCLUSIONS: We demonstrate that the VWF-MAA has utility in differentiating type 1 VWD, variant VWD and normal subjects in individuals undergoing an initial laboratory evaluation for bleeding.

Periprocedural management of type 2N von Willebrand disease with efanesoctocog alfa.

Type 2 Normandy von Willebrand disease (type 2N VWD) is a rare qualitative defect in von Willebrand factor (VWF) that results in impaired factor VIII (FVIII) binding and consequently reduced FVIII levels. Current perioperative strategies require VWF concentrates to attain durable hemostatic FVIII levels. This case highlights the successful perioperative management of a 78-year-old female with type 2N VWD and coronary artery disease utilizing efanesoctocog alfa, a novel long-acting recombinant FVIII product approved for hemophilia A. By decoupling the FVIII-VWF interaction, efanesoctocog alfa achieves prolonged FVIII circulation independent of VWF. A single administration targeting 90% FVIII levels yielded sustained FVIII elevation without achieving supraphysiologic VWF levels, thus mitigating potential cardiovascular risks. This is the first report of efanesoctocog alfa use in type 2N VWD. Further clinical studies are necessary to corroborate its efficacy and safety for this indication.

Laboratory Testing for von Willebrand Factor: Factor VIII Binding for the Diagnosis or Exclusion of Type 2N von Willebrand Disease: An Update.

von Willebrand factor (VWF) is a large adhesive plasma protein that expresses several functional activities. One of these activities is to bind coagulation factor VIII (FVIII) and to protect it from degradation. Deficiency of, and/or defects in, VWF can give rise to a bleeding disorder called von Willebrand disease (VWD). The defect in VWF that affects its ability to bind to and protect FVIII is captured within type 2N VWD. In these patients, FVIII is produced normally; however, plasma FVIII quickly degrades as it is not bound to and protected by VWF. These patients phenotypically resemble those with hemophilia A, where instead, FVIII is produced in lower amount. Both hemophilia A and 2N VWD patients therefore present with reduced levels of plasma FVIII relative to VWF level. However, therapy differs, since patients with hemophilia A are given FVIII replacement products, or FVIII mimicking products; instead, patients with 2N VWD require VWF replacement therapy, since FVIII replacement will only be effective for a short term, given this replacement product will quickly degrade in the absence of functional VWF. Thus, 2N VWD needs to be differentiated from hemophilia A. This can be achieved by genetic testing or by use of a VWF:FVIII binding assay. The current chapter provides a protocol for the performance of a commercial VWF:FVIII binding assay.

von Willebrand factor neutralizing and non-neutralizing alloantibodies in 213 subjects with type 3 von Willebrand disease enrolled in 3WINTERS-IPS.

BACKGROUND: Type 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions. OBJECTIVE: The objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS. METHODS: An indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay. RESULTS: In total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14). CONCLUSIONS: Anti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used.

Challenges in the management of women with type 2B von Willebrand disease during pregnancy and the postpartum period: evidence from literature and data from an international registry and physicians' survey-communication from the Scientific and Standardization Committees of the International Society on Thrombosis and Haemostasis.

BACKGROUND: Management of women with type 2B von Willebrand disease (VWD) during pregnancy is challenging because of dysfunctional von Willebrand factor (VWF) and the complexity resulting from discrepant VWF/factor VIII (VWF/FVIII) levels, impaired platelet-dependent VWF activity, progressive thrombocytopenia, and risks associated with the use of desmopressin. There is a lack of high-quality evidence to support clinical decision making. OBJECTIVES: In this study, we examined the current diagnostic and management approaches and outcomes in women with VWD during pregnancy. METHODS: Data were collected via 3 avenues: literature review, an international registry, and an international survey on physicians' practices for the management of pregnancy in women with VWD. The registry and survey were supported by the International Society on Thrombosis and Haemostasis. RESULTS: Data on clinical and laboratory features, management and bleeding complications, and pregnancy outcomes of a total of 55 pregnancies from 49 women across the globe (literature: 35, registry: 20) and data reported by 112 physicians were analyzed. We describe the largest dataset on pregnancies in women with type 2B VWD available to date. The data highlight the following key issues: a) bleeding complications remain a concern in these patients, b) the target safe VWF level and the ideal monitoring approach are unknown, c) there is a wide range of hemostatic management practices in the type and timing of treatment, and d) physicians have diverse views on the mode of delivery and use of neuraxial anesthesia. CONCLUSION: We conclude that an international consensus and guidance are critically required for better care and improved outcomes in this patient cohort.

A nanobody against the VWF A3 domain detects ADAMTS13-induced proteolysis in congenital and acquired VWD.

von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A-group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high-molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.

Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain.

Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms. In this study, we produced recombinant glycosylated AIM-A1 fragments bearing type 2B VWD mutations and examined how each mutation affects the A1 domain's thermodynamic stability, conformational dynamics, and biomechanical regulation of the AIM. We found that the A1 domain with mutations associated with severe bleeding occupy a higher affinity state correlating with enhanced flexibility in the secondary GPIbα-binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wild-type (WT) but shares regions of solvent accessibility with both WT and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared with all variants. Lastly, examination of the mechanical stability of each variant revealed variable AIM unfolding. Together, these studies illustrate that the heterogeneity among type 2B VWD mutations is evident in AIM-A1 fragments.

Von Willebrand Factor Multimer Analysis and Classification: A Comprehensive Review and Updates.

Von Willebrand factor (VWF) is a multimeric glycoprotein with essential roles in primary hemostasis. Patients with von Willebrand disease (VWD), due to quantitative and/or qualitative defects of VWF usually experience mucocutaneous bleeding. Based on the laboratory results of VWF antigen, various VWF activities, factor VIII activity, and VWF multimer patterns, VWD can be categorized as type 1, 2, and 3 VWD. VWF multimer analysis by either manual or semi-automated electrophoresis and immunoblotting is a critical part of the laboratory testing to differentiate type 1, type 2 VWD, and subtypes of type 1 or 2 VWD. The multimer distribution patterns can also help to understand the underlying molecular mechanism of VWF synthesis, multimerization, and clearance defects in VWD. This review will cover VWF synthesis, multimerization, secretion, VWF multimer analysis, and VWF multimer interpretation of various types and subtypes of VWD.

Origin and timing of de novo variants implicated in type 2 von Willebrand disease.

Very few studies have shown the real origin and timing of de novo variants (DNV) implicated in von Willebrand disease (VWD). We investigated four families with type 2 VWD. First, we conducted linkage analysis using single nucleotide variant genotyping to recognize the possible provenance of DNV. Second, we performed amplification refractory mutation system-quantitative polymerase chain reaction to confirm the real origin of variant (~0% mutant cells) or presence of a genetic mosaic variant (0%-50% mutant cells) in three embryonic germ layer-derived tissues and sperm cells. Then, three possible timings of DNV were categorized based on the relative likelihood of occurrence according to the number of cell divisions during embryogenesis. Two each with type 2B VWD (proband 1 p.Arg1308Cys, proband 4 p.Arg1306Trp) and type 2A VWD (proband 2 p.Leu1276Arg, proband 3 p.Ser1506Leu) were identified. Variant origins were identified for families 1, 2 and 3 and confirmed to originate from the mother, father and father, respectively. However, the father of family 4 was confirmed to have isolated germline mosaicism with 2.2% mutant sperm cells. Further investigation confirmed the paternal grandfather to be the origin of variant. Thus, we proposed that DNV originating from the two fathers most likely occurred at the single sperm cell, the one originating from the mother occurred at the zygote during the first few cellular divisions; alternatively, in family 4, the DNV most likely occurred at the early postzygotic development in the father. Our findings are essential for understanding genetic pathogenesis and providing accurate genetic counselling.

External Quality Assessment Data for Investigation of von Willebrand Disease: Focus on Relative Utility of Contemporary Functional von Willebrand Factor Assays. The United Kingdom National External Quality Assessment Scheme (UK NEQAS) Experience.

Von Willebrand disease (VWD) is one of the most common hereditary bleeding disorders. Effective management of patients and their families depends on accurate diagnosis and subtype classification, and quality assurance including participation in proficiency testing programs is essential to ensure the accuracy of the panel of assays required to achieve this diagnosis. We report here findings from recent external quality assessment (EQA) exercises, as well as from a questionnaire about diagnostic practices employed by centers in the United Kingdom National Quality Assessment Scheme (UK NEQAS) performing von Willebrand factor (VWF) assays. Plasma samples from donors with VWD, "normal" donors, the International Society for Thrombosis and Haemostasis Scientific Subcommittee (ISTH SSC) plasma standard, and whole blood samples were sent to participants in the UK NEQAS BC program for VWF investigation. Calibration of lot#5 of the ISTH SSC plasma standard was shown to give improved comparability between the recovered value from an EQA exercise and the assigned potency for VWF activity assays. Diagnostic accuracy and precision amongst UK NEQAS participants was good, with an average 99% of centers reporting the correct interpretation for normal, type 1 and type 2 VWD samples, 100% diagnostic accuracy for centers performing FVIII binding assays, and good agreement amongst centers performing multimeric analysis. Genetic analysis of the VWF gene by specialist centers demonstrated errors in the genotyping process in one center, but also demonstrated failings in the interpretation of results in other centers. Despite evidence of good laboratory accuracy and precision in their assays, a questionnaire identified marked variation in diagnostic criteria employed, underlining the importance of guidelines to support the diagnosis of VWD.

Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange.

von Willebrand factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excessive bleeding of type 2A von Willebrand disease (VWD). Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report electron cryomicroscopy (cryo-EM) structures of VWF tubules before and after intermolecular disulfide bond formation. The structures provide evidence that VWF tubulates through a charge-neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states before and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VWD-causing mutations.