Von Willebrand Disease Phenotype Research Articles

The Experimental Gene Therapy on Type 2A/Iic VWD Mouse Model with AAV Vector Delivering Only VWF Propeptide Expression cassette4

Type 2A/IIC von Willebrand Disease (VWD) is caused by mutations in the propeptide of von Willebrand Factor (VWF), and characterized by the impaired multimer assembly. The structural analysis revealed that VWF propeptide (D1D2 domain) serves as a pH-sensing template for VWF multimerization and tubular storage, which implies that D1D2 expression "in trans" has the potential to restore the multimerization defect of type 2A/IIC mutatns. The gene therapy of VWD is challenging due to the huge size of VWF gene, which cannot be packed to the virus vectors, particularly recombinant adeno-associated virus (rAAV). In the pilot studies, we had co-transfected AtT20 cell with plasmid encoding full length VWF, containing the Y87S mutation on propeptide region and causing type 2A VWD phenotype, and plasmid expressing only wild type propeptide of VWF. The co-transfection rescued the multimer formation in a concentration dependent manner (Figure 1A )and corrected the functional defects caused by the Y87S mutation (Figure 1B). Inspired by the results of in vitro studies, we thus have designed a novel gene therapy strategy for type 2A VWD utilizing the machinery of VWF multimer assembling. Instead of trying to squeeze the VWF gene into the AAV vector, we had prepared the AAV vector containing the expression cassette of propeptide region directed by endothelial cell specific promoter FLT-1. The mouse model representing natural occurring VWF Y87S mutation identified in type 2A VWD patient was established by criper9 gene editing technique. The AAV vectors was administered via tail vein injection at a dose of 5Ⅹ1011 genome copies to 6 weeks old male mice to deliver the propeptide of VWF gene and the plasma of the treated mice was collected retroorbital pretreatment and at 2- ,4- and 6-weeks post vector injection. The antigen level of VWF was determined by ELISA and the function activity of VWF was measured by collagen binding assay. The status of VWF multimerization was examined by agarose gel electrophoresis. Although the Y87S mutation led to significant reduce of high molecule weight multimer in mice, unlike observed in mutation harboring patient, the mice still had medium and low molecular weight multimer, maintaining ~30% of activity. The expression of propeptide by AAV delivered vector improved the VWF activity significantly to ~70% of wild type control (Figure 1C), and restored the formation of high molecular weight multimer (Figure 1D). The experimental gene therapy conducted on VWD mouse model showed that it would be efficient to express the propeptide alone to correct the type 2A/IIC VWD defect and the preliminary results provide a novel strategy to overcome technique limits on gene therapy of VWD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Von Willebrand factor propeptide variants lead to impaired storage and ER retention in patient‐derived endothelial colony‐forming cells

Backgroundvon Willebrand factor (VWF) is synthesized by vascular endothelial cells and megakaryocytes. The VWF propeptide is critical for multimerization and acts as an intra‐molecular chaperone for mature VWF in sorting to its storage organelles, Weibel‐Palade bodies (WPBs). In the Canadian Type 3 VWD study, almost half of the identified variants were in the VWF propeptide and these were associated with an increased bleeding phenotype. ObjectiveTo investigate VWF propeptide variants that cause quantitative von Willebrand disease (VWD) by utilizing patient‐derived endothelial colony‐forming cells (ECFCs). Patients/MethodsEndothelial colony‐forming cells were isolated from five Type 3 VWD patients from four families with the following variants: (1) homozygous p.Asp75_Gly178del (deletion of exons 4 and 5 deletion; Ex4‐5del), (2) homozygous p.Cys633Arg, (3) homozygous p.Arg273Trp, and (4) p.Pro293Glnfs*164 and p.Gln419* inherited in the compound heterozygous state. Additionally, ECFCs were isolated from six family members (two Type 1 VWD, four unaffected). ResultsEndothelial colony‐forming cells from the Type 3 patient with the compound heterozygous genotype exhibited a true null VWF cellular phenotype, with negligible VWF detected. In contrast, the other three propeptide variants presented a similar expression pattern in homozygous ECFCs where VWF was synthesized but not packaged in WPBs, and variant VWF had an increased association with the endoplasmic reticulum (ER) marker, protein disulfide‐isomerase (PDI), indicating an ER‐retention phenotype. The biosynthetic phenotype was similar but to a lesser degree in heterozygous ECFCs expressing the non‐null variants. ConclusionThis study further elucidates the importance of the VWF propeptide in the VWD phenotype using patient‐derived cells.

Laboratory characterization of obligate carriers of type 3 von Willebrand disease with a potential role for Platelet Function Analyzer (PFA-200).

Type 3 von Willebrand disease (VWD) is a rare autosomal recessive disorder characterized by undetectable von Willebrand Antigen (VWF:Ag). Carriers of type 3 VWD carry one null allele and have von Willebrand factor (VWF) at about 50% of normal. The aim of this study was to characterize type 3 VWD carriers and to study the role of Platelet Function Analyzer (PFA-200) in this cohort. This was a cross-sectional study where data were collected from carriers (parents/offspring) of type 3 VWD patients and evaluated with activated partial thromboplastin time, factor VIII, blood group, ristocetin cofactor assay (VWF:RCo), VWF:Ag, and closure time on PFA-200 with collagen/epinephrine (COL/EPI), and collagen/ADP (COL/ADP). One hundred carriers were included in the study of which 85 were included for PFA-200 analysis. The mean (SD) of VWF:Ag (IU/ml) and VWF:RCo (IU/ml) was 0.63 (0.24) and 0.61 (0.26), respectively. Among the 100 carriers, based on VWF levels (VWF:Ag and/or VWF:RCo) and bleeding history, there were 7 type 1 VWD, 10 type 2 VWD, 25 borderline VWF (0.30-0.50IU/ml and no bleeding), and 58 normal VWF (>0.50IU/ml). PFA-200 was prolonged in 71% of the carriers, all carriers with type 1 and type 2 VWD phenotype, 80% carriers with borderline VWF, and 59% with normal VWF. COL/EPI was more sensitive than COL/ADP and showed better correlation with VWF parameters than COL/ADP. Carriers of type 3 VWD can have a variable laboratory phenotype. PFA-200 showed good sensitivity among the carriers at VWF levels <0.50IU/ml.

IX international curse of continuing formation in haemophilia and other congenital coagulopathies. The role of the Laboratory in coagulation disorders. Diagnosis of von Willebrand disease.

Von Willebrand disease (VWD) is the most frequent inherited bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (VWF). This protein far from simplicity constitutes a very complex molecular model, remaining unravelled yet many aspects of it, even though the VWF gene (VWF) was cloned already in 1985 and the structure of VWF well defined. VWD diagnosis is difficult to achieve in a significant proportion of patients due to both disease heterogeneity and limitations in existing test processes. The cornerstone of diagnosis relies on interpretation of VWF test results, the presence of clinical manifestations of bleeding, especially mucocutaneous, and (in most cases) a positive family history. However, even with a significant bleeding history, a family history may not be positive due to factors of incomplete penetrance and variable expressivity that affect genetic changes. The laboratory diagnosis of VWD can be difficult, as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2 variants) VWD requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity. The latter is required for identifying and subtyping VWD, but the assay is poorly standardized. For that reason, novel VWF activity assays have been developed awaiting more extensive comparison data between different methodologies and requiring validation on larger patient series. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that measure other activities or the size distribution of VWF multimers. However, frequently, it may be difficult to correctly classify the VWD phenotype, and genetic analysis is through mutation identification may provide a tool to clarify the disorder.

Characterization of the Pathogenicity of Novel Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program for the Molecular and Clinical Biology Study

Von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Von Willebrand factor's (VWF) complex intracellular processing may result in variable VWF expression which requires exploring the underlying mechanism related to individual variants. The mechanisms underlying type 1 VWD are not fully understood, although reduced secretion and increased clearance have been implicated. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program in order to (1) define the underlying mechanism, (2) explore if mutations in a particular domain are mechanistically similar, and (3) correlate expression study data to patient phenotype.Homozygous and heterozygous expression in mammalian cells was used to study the effect of SV on VWF secretion, intracellular retention, function, and multimerization. Novel SV identified throughout the entire VWF protein were introduced into VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Variant VWF cDNA was transfected homozygously and cotransfected heterozygously 1:1 with wild type (WT) VWF pCIneo into Human Embryonic Kidney cells (HEK293T). Supernatants (sup) and cell lysates from 3 separate transfections were collected and analyzed by ELISA for VWF:Ag, VWF binding to platelet glycoprotein 1b (VWF:GPIbM), VWF binding to collagen type III (VWF:CB). Typically VWF:GP1bM/VWF:Ag > 0.7 indicates normal platelet binding and VWF:CB/VWF:Ag > 0.7 indicates normal multimer structure. VWF multimer structure is analyzed by SDS-agarose electrophoresis and western blotting with differential staining of the VWF secreted from variant and WT alleles.V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, and A2569E demonstrated secretion of VWF in sup comparable to that of WT-VWF both when transfected homozygously and hetereozygously, normal VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and normal multimer structure. Two of the stop codon SV W199X and E1660X were not expressed homozgyously (as expected) but demonstrated normal secretion, function, and multimerization when cotransfected 1:1 with WT. Homozygous expression of R960P and C2693F demonstrated reduced VWF secretion, VWF:GPIbM/VWF:Ag, VWF:CB/VWF:Ag and abnormal multimerization. Heterozygous transfection with WT improved VWF secretion, but not multimer defects,reflected by a reduced VWF:CB/VWF:Ag. Homozygous expression of C524Y, G994D, C996W, Q1353X, C2199Y, C2701Y, and C2754Y resulted in absent or greatly reduced secretion with increased intracellular retention. Heterozygous expression resulted in reduced VWF secretion (<50% of WT) for all 7 variants. Q1353X, C2199Y, C2701Y, and C2754Y had normal VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag, and multimer structure, but consisted primarily of secreted WT-VWF. G994D had VWF:CB/VWF:Ag ˂ 0.7 and reduced high molecular weight (HMW) multimers. Co-expression of C524Y and C996W with WT-VWF demonstrated nearly absent VWF secretion, indicating a strong dominant negative effect. The very low VWF:Ag levels precluded assessment of VWF function and multimerization.The VWF:CB/VWF:Ag ratio < 0.7 proved a reliable indicator of multimer structure. No clear domain-specific correlation of VWF secretion or multimer abnormality was observed. Co-expression results of Q1353X, C2199Y, C2701Y, C2754Y, C524Y, G994D, and C996W are consistent with the patient's phenotype, except that patients with C2701Y, and C524Y had slight defect in multimers. In contrast, the in-vitro G994D showed low VWF:CB/VWF:Ag and abnormal multimers but were normal in the patient. C2693F showed consistently abnormal multimer structure in both in vitro and in the patient while R960P had abnormal multimer structure in vitro only.In sum, 52.6% of the 19 SV theoretically are not causative of the VWD phenotype observed in the patients. These subjects had normal VWF propeptide/VWF:Ag suggesting reduced plasma survival is unlikely to be the causative mechanism. The VWD mechanism in these subjects remains undefined. Variants R960P, C2693F, and G994D (15.7%) behaved like Type 2 VWD SV with loss of HMW multimers. We identified 31.6% of the 19 VWF SV as likely to cause the type 1 VWD phenotype with reduced secretion. DisclosuresNo relevant conflicts of interest to declare.

ADAMTS‐13 and bleeding phenotype in von Willebrand disease

BackgroundThe bleeding phenotype of von Willebrand disease (VWD) varies highly between patients and can only partly be explained by von Willebrand factor (VWF) parameters. By cleaving large VWF multimers into smaller, less active multimers, ADAMTS‐13 is an important regulator of VWF activity. However, it is unknown what the role of ADAMTS‐13 is in individuals with VWD. ObjectivesWe therefore studied how ADAMTS‐13 activity is associated with the laboratory and bleeding phenotype in individuals with VWD. MethodsWe measured ADAMTS‐13 activity using the fluorescence resonance energy transfer substrate VWF 73 assay in 638 individuals with VWD in the nationwide cross‐sectional Willebrand in the Netherlands study and in 36 healthy controls. The bleeding phenotype was assessed using the Tosetto bleeding score. ResultsADAMTS‐13 activity was similar in individuals with VWD (109% ± 20.6%) and controls (110% ± 19.7%). ADAMTS‐13 activity was higher in individuals with VWD with type 3 than those with type 1 (mean difference, 11.8%; 95% confidence interval [CI], 2.9%‐20.8%) or type 2 (mean difference, 16.1%; 95% CI, 7.1%‐25.1%). ADAMTS‐13 activity was not associated with the Tosetto bleeding score (0.1 Tosetto bleeding score increase per 10% ADAMTS‐13 increase, 95% CI, −0.2 to 0.3). Furthermore, ADAMTS‐13 activity did not differ between individuals with and without a bleeding event during the year preceding blood sampling (mean difference, 1.4%; 95% CI, −2.1% to 4.9%). ConclusionADAMTS‐13 activity was highest in individuals with type 3 VWD, but it had only minor associations with VWF parameters. ADAMTS‐13 activity does not influence the bleeding phenotype in individuals with VWD.

Noncanonical type 2B von Willebrand disease associated with mutations in the VWF D′D3 and D4 domains

AbstractWe observed a 55-year-old Italian man who presented with mucosal and cutaneous bleeding. Results of his blood analysis showed low levels of von Willebrand factor (VWF) antigen and VWF activity (both VWF ristocetin cofactor and VWF collagen binding), mild thrombocytopenia, increased ristocetin-induced platelet aggregation, and a deficiency of high-molecular-weight multimers, all typical phenotypic hallmarks of type 2B von Willebrand disease (VWD). The analysis of the VWF gene sequence revealed heterozygous in cis mutations: (1) c.2771G>A and (2) c.6532G>T substitutions in the exons 21 and 37, respectively. The first mutation causes the substitution of an Arg residue with a Gln at position 924, in the D′D3 domain. The second mutation causes an Ala to Ser substitution at position 2178 in the D4 domain. The patient's daughter did not present the same fatherly mutations but showed only the heterozygous polymorphic c.3379C>T mutation in exon 25 of the VWF gene causing the p.P1127S substitution, inherited from her mother. The in vitro expression of the heterozygous in cis VWF mutant rVWFWT/rVWF924Q-2178S confirmed and recapitulated the ex vivo VWF findings. Molecular modeling showed that these in cis mutations stabilize a partially stretched and open conformation of the VWF monomer. Transmission electron microscopy and atomic force microscopy showed in the heterozygous recombinant form rVWFWT/rVWF924Q-2178S a stretched conformation, forming strings even under static conditions. Thus, the heterozygous in cis mutations 924Q/2178S promote conformational transitions in the VWF molecule, causing a type 2B–like VWD phenotype, despite the absence of typical mutations in the A1 domain of VWF.

Unraveling the Influence of Common von Willebrand factor variants on von Willebrand Disease Phenotype: An Exploratory Study on the Molecular and Clinical Profile of von Willebrand Disease in Spain Cohort.

The clinical diagnosis of von Willebrand disease (VWD), particularly type 1, can be complex because several genetic and environmental factors affect von Willebrand factor (VWF) plasma levels. An estimated 60% of the phenotypic variation is attributable to hereditary factors, with the ABO blood group locus being the most influential. However, recent studies provide strong evidence that nonsynonymous single nucleotide variants (SNVs) contribute to VWF and factor VIII phenotypic variability in healthy individuals. This study aims to investigate the role of common VWF SNVs on VWD phenotype by analyzing data from 219 unrelated patients included in the "Molecular and Clinical Profile of von Willebrand Disease in Spain project." To that end, generalized linear mixed-effects regression models were fitted, and additive and epistatic analyses, and haplotype studies were performed, considering five VWD-related measures (bleeding score, VWF:Ag, VWF:RCo, factor VIII:C, and VWF:CB). According to these analyses, homozygotes: for p.Thr789Ala(C) would be expected to show 39% higher VWF:Ag levels; p.Thr1381Ala(C), 27% lower VWF:Ag levels; and p.Gln852Arg(C), 52% lower VWF:RCo levels. Homozygotes for both p.Thr789Ala(C) and p.Gln852Arg(T) were predicted to show 185% higher VWF:CB activity, and carriers of two copies of the p.Thr1381Ala(T)/p.Gln852Arg(T) haplotype would present a 100% increase in VWF:RCo activity. These results indicate a substantial effect of common VWF variation on VWD phenotype. Although additional studies are needed to determine the true magnitude of the effects of SNVs on VWF, these findings provide new evidence regarding the contribution of common variants to VWD, which should be taken into account to enhance the accuracy of the diagnosis and classification of this condition. ClinicalTrials.gov identifier: NCT02869074.

Molecular Etiology and Laboratory Phenotypes of Recessive Von Willebrand Disease 2N Due to Mutations in the D’D3 Factor VIII-Binding Domain of the Von Willebrand Factor Gene: A Critical Appraisal of the Literature and Personal Experiences

The FVIII binding site on von Willebrand factor (VWF) is located in the D’ (766-864) and D3 (1054-1060) regions of the VWF gene. The cysteine residues in the D’ domain form disulfide bridges within the D’ trypsin-inhibitor-like (TIL’) and E’ regions,and these are of critically importance for the binding between TIL’E’ and FVIII. We analyzed the molecular etiology and laboratory phenotype of von Willebrand disease (VWD) 2N patients reported in the literature and added personal experiences from three European VWF VWD Research Centers.

Von Willebrand Disease: From In Vivo to In Vitro Disease Models.

Von Willebrand factor (VWF) plays an essential role in primary hemostasis and is exclusively synthesized and stored in endothelial cells and megakaryocytes. Upon vascular injury, VWF is released into the circulation where this multimeric protein is required for platelet adhesion. Defects of VWF lead to the most common inherited bleeding disorder von Willebrand disease (VWD). Three different types of VWD exist, presenting with varying degrees of bleeding tendencies.The pathophysiology of VWD can be investigated by examining the synthesis, storage and secretion in VWF producing cells. These cells can either be primary VWF producing cells or transfected heterologous cell models. For many years transfected heterologous cells have been used successfully to elucidate many aspects of VWF synthesis. However, those cells do not fully reflect the characteristics of primary cells. Obtaining primary endothelial cells or megakaryocytes with a VWD phenotype, requires invasive procedures, such as vessel collection or a bone marrow biopsy. A more recent and promising development is the isolation of endothelial colony forming cells (ECFCs) from peripheral blood as a true-to-nature cell model. Alternatively, various animal models are available but limiting, therefore, new approaches are needed to study VWD and other bleeding disorders. A potential versatile source of endothelial cells and megakaryocytes could be induced pluripotent stem cells (iPSCs).This review gives an overview of models that are available to study VWD and VWF and will discuss novel approaches that can be considered to improve the understanding of the structural and functional mechanisms underlying this disease.

Alteration in GPIIb/IIIa Binding of VWD-Associated von Willebrand Factor Variants with C-Terminal Missense Mutations.

The platelet receptor glycoprotein (GP) IIb/IIIa, formed by integrins αIIb and β3, plays an important role in platelet adhesion and aggregation. Its major binding site is the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. Upon platelet activation, inside-out signaling activates the complex permitting binding to RGD motif containing proteins, such as von Willebrand factor (VWF). VWF is a large multidomain plasma GP essential to primary hemostasis, which can directly interact with platelets because it exhibits binding sites for GPIbα and GPIIb/IIIa in its A1 and C4 domain, respectively. A vast variety of VWF variants have been identified in which domain-specific mutations affect distinct functions of VWF but reduced GPIIb/IIIa binding has barely been studied so far. Here, we strived to investigate the influence of C domain mutations, which have been identified in patients diagnosed with von Willebrand disease (VWD), on VWF-GPIIb/IIIa interaction. To determine binding to membrane-incorporated GPIIb/IIIa in the absence of GPIbα, we developed and validated a cell-based binding assay which uses HEK293 cells stably expressing a constitutively active form of the GPIIb/IIIa receptor complex on their plasma membrane. By employing this assay, we measured GPIIb/IIIa binding of 14 VWF C domain mutants identified in VWD patients. Mutants p.Cys2257Arg, p.Gly2441Cys, p.Cys2477Tyr, and p.Pro2722Ala exhibited significantly reduced binding. Summarizing, we have developed a useful research tool to specifically investigate GPIIb/IIIa interaction with its protein binding partners and identified four VWF variants that exhibit impaired GPIIb/IIIa binding. At least in the homozygous state, this defect could contribute to the VWD phenotype.

Bleeding Patterns in Type I VWD in Effect of VWF Levels: An Individual Participant Data Meta-Analysis of Three Cohorts

Type I von Willebrand disease (VWD) is the most frequent bleeding disorder, with a prevalence of 10-50 cases per 10,000 persons. VWD usually shows an autosomal dominant inheritance pattern, at least in families having VWF:Ag levels below 30 IU/dL. There is considerable uncertainty whether patients having only mildly reduced VWF levels (in the range 30-50 IU/dL) should be diagnosed as having VWD or if they should be considered as a separate clinical entity, broadly referred to as “reduced VWF patients.” We hypothesize that these patients may have a clinically distinct bleeding pattern, in terms of presenting symptoms at diagnosis, bleeding severity, and possibly even clinical.We pooled data from three cohorts of patients. The MCMDM-1 VWD Study is a multicenter survey on type 1 VWD that recruited 154 type 1 VWD families from nine European countries. The Kingston cohort recruited consecutive patients with type 1 VWD diagnosed in Kingston, Canada and includes patients screened because of bleeding symptoms or family history of VWD over a 10 year period. The Zimmerman Program for the Molecular and Clinical Biology of VWD is a multicenter study that enrolled VWD patients mostly from US clinical centres, primarily carrying a diagnosis of type 1 VWD (315 families) but also including type 2 and type 3 subjects. For the present study, only patients with a confirmed diagnosis of type 1 VWD were retained. From the three cohorts, demographic, laboratory and clinical data were abstracted, including severity of bleeding symptoms classified according to the MCMDM-1 study criteria. Only data from index cases and affected family members were retained for the present analysis. Bleeding symptoms receiving a score >=2 (and therefore requiring some type of medical intervention) were classified as “clinically relevant”. VWF:Ag and VWF:RCo were measured centrally by the reference core lab of each of the three studies, against the WHO International Standard. For this purpose of the present study, Type 1 VWD is defined as VWF:Ag levels (or VWF:RCo for the Zimmerman Program) below 30 IU/dL; affected family members and index cases with VWF:Ag levels equal or above 30 IU/dL were classified as “low-VWF” patients.Pooled data from the three studies resulted in 1411 patients. At multivariate analysis, blood group O females with lower bleeding scores were the characteristics associated with the “low-VWF” patients; interestingly, the number of clinically relevant bleeding symptoms was associated with the phenotype independently from the total bleeding score. For all considered bleeding symptoms, VWD patients had a higher prevalence of relevant bleeding, with the notable exception of menorrhagia. Particularly in index cases, menorrhagia but also post-surgical bleeding was increased in patients having the “low-VWF” phenotype, although only for menorrhagia the difference was statistically significant (p=0.022).Unsupervised cluster analysis of symptom distribution disclosed three subgroups of patients. The first two were composed by males (first group) or females (second group), both having few or none bleeding symptoms. The third identified by unsupervised cluster analysis group was composed of highly symptomatic patients, predominantly women. In these patients, mucous (“wet”) bleeding (epistaxis and menorrhagia) was common together with post-surgical or extraction bleeding. This pattern was not correlated with mean VWF:Ag level or “low-VWF” or Type 1 VWD phenotype. We conclude that “low-VWF” patients are more frequently blood group O, older females, with lower average bleeding scores and number of symptoms and possibly selected because of menorrhagia. A group of “severe bleeders” may be identified (n=270, 19%), having a similar distribution of “low VWF” and “VWD” phenotypes. DisclosuresTosetto:Stago, Novo-Nordisk, BMS: Speakers Bureau; Werfen: Other: Member of Advisory Board, Speakers Bureau. Eikenboom:CSL: Research Funding. James:CSL Behring: Research Funding; Shire: Research Funding; Bayer: Research Funding. Montgomery:BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

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

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.

Abnormal angiogenesis in blood outgrowth endothelial cells derived from von Willebrand disease patients.

: Bleeding associated with angiodysplasia is a common, often intractable complication in patients with von Willebrand disease (VWD). von Willebrand factor (VWF), the protein deficient or defective in VWD, is a negative regulator of angiogenesis, which may explain the pathologic blood vessel growth in VWD. This study explores the normal range of angiogenesis in blood outgrowth endothelial cells (BOECs) derived from healthy donors and compares this to angiogenesis in BOECs from VWD patients of all types and subtypes. BOECs were assessed for VWF and angiopoietin-2 (Ang-2) gene expression, secretion, and storage. To explore angiogenic potential, we characterized cellular proliferation, matrix protein adhesion, migration, and tubule formation. We found great angiogenic variability in VWD BOECs with respect to each of the angiogenesis parameters. However, type 1 and 3 VWD BOECs had higher Ang-2 secretion associated with impaired endothelial cell migration velocity and enhanced directionality. Type 2A and 2B BOECs were the most proliferative and multiple VWD BOECs had impaired tubule formation in Matrigel. This study highlights the angiogenic variability in BOECs derived from VWD patients. Abnormal cell proliferation, migration, and increased Ang-2 secretion are common features of VWD BOECs. Despite the many abnormalities of VWD BOECs, significant heterogeneity among individual VWD phenotypes precludes a simple description of relationship between VWD type and in vitro surrogates for angiodysplasia.

Expression of Sequence Variants Identified in Type 1 VWD Subjects in the Zimmerman Program Study Reveals Defects in VWF Secretion and Multimerization

von Willebrand Disease (VWD) is the most prevalent inherited bleeding disorder. Type 1 is the most common form of VWD and results in a partial quantitative deficiency of von Willebrand Factor (VWF). The mechanisms underlying type 1 VWD are still not very well understood although reduced VWF secretion and increased VWF clearance have been implicated in causing VWD. We aimed to characterize novel sequence variants (SV) identified in the VWF gene in type 1 VWD patients recruited through the Zimmerman Program for the Molecular and Clinical Biology of VWD in order to define the underlying mechanism and explore if SV in a particular domain are mechanistically similar.We utilized homozygous expression in human embryonic kidney cells (HEK-293T) to study the effect of VWF SV on VWF secretion, intracellular retention, multimerization, and function. Novel SV have been identified throughout the entire VWF protein. We introduced the following variants into a VWF-mycHis plasmid vector: V86M, W199X, C524Y, M947V, R960P, G994D, C996W, R1204W, Q1353X, E1660X, R1763Q, C2199Y, Q2256H, T2282I, P2524L, A2569E, C2693F, C2701Y, and C2754Y. Sequence variants were confirmed by Sanger sequencing. Variant VWF cDNA is transfected homozygously into HEK-293T cells. The supernatants and cell lysates from 3 independent transfections are collected and analyzed by ELISA for VWF:Ag and VWF binding to collagen type III (VWF:CB). VWF multimer structure is analyzed by SDS-agarose gel electrophoresis and western blotting. The genotype-phenotype patient data is correlated with the data from expression studies to explore a model to predict the impact of the SV on the VWD phenotype.Variants V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F demonstrated secretion comparable to that of wild type (WT)-VWF. In contrast, VWF variants R960P and C2701Y showed reduced VWF secretion (<50% of WT) with increased VWF in the cell lysate. VWF variants W199X, C524Y, G994D, C996W, Q1353X, E1660X, C2199Y, and C2754Y demonstrated a complete absence of secreted VWF. Not unexpectedly, homozygous expression of stop codon variants W199X, Q1353X, and E1660X demonstrated no VWF in the cell lysate. However, non-secreted VWF variants C524Y, G994D, C996W, C2199Y, and C2754Y showed intracellular retention with detectable VWF in the cell lysate. SV occurring at cysteine residues (C524Y, C996W, C2199Y, C2701Y, and C2754Y) all had reduced secretion and increased intracellular retention, consistent with altered conformation leading to increased intracellular chaperone interaction and proteasomal degradation. VWF binding to collagen is dependent on the presence of high molecular weight multimers (HMWM). VWF:CB/VWF:Ag is used to predict multimer structure with VWF:CB/VWF:Ag < 0.7 indicative of loss of the HMWM. VWF variants V86M, M947V, R1763Q, Q2256H, P2524L, C2701Y had VWF:CB/VWF:Ag ≥ 0.7 consistent with normal multimer structure, while variants R960P, R1204W, T2282I, A2569E, and C2693F had VWF:CB/VWF:Ag < 0.7 indicating abnormal multimer structure.47.3% of the 19 VWF variants studied had normal VWF secretion, 10.5% had reduced secretion with increased intracellular retention, and 26.3% revealed absent secretion with intracellular retention. Variants with a premature stop codon did not synthesize VWF at all. Some SV had normal secretion and multimerization (V86M, M947V, R1204W, R1763Q, Q2256H, T2282I, P2524L, A2569E, and C2693F) implying that the VWD phenotype in these patients results from yet unidentified mechanisms and may not be associated with these SV. Reduced plasma survival is unlikely as these patients had normal VWFpp/VWF:Ag level consistent with normal VWF clearance. Among the VWF variants with normal or decreased secretion, 45.4% had reduced VWF:CB/VWF:Ag consistent with abnormal multimer structure. Heterozygous expression, as observed in the patient, is expected to normalize these multimerization defects. The decreased or absent secretion observed for 52.7% of the variants studied correlates with the patient phenotype, indicating reduced secretion is the mechanism underlying these patients' type 1 VWD phenotype. No domain-specific correlation of VWF secretion or multimer abnormality was observed.In summary, VWF expression studies confirmed the causative nature of many, but not all of the novel sequence variants identified in type 1 VWD subjects in the Zimmerman Program. DisclosuresNo relevant conflicts of interest to declare.

The Von Willebrand Factor A1–Collagen III Interaction Is Independent of Conformation and Type 2 Von Willebrand Disease Phenotype

The blood von Willebrand factor (VWF) mediates platelet adhesion to injured vessels by sequestering platelets from blood flow and depositing them to collagen and other exposed subendothelial matrix proteins. This process of capturing platelets to facilitate formation of platelet plugs occurs through transient interactions with platelet glycoprotein Ibα via the VWF A1 domain which also binds collagen. Using a conformationally diverse collection of natively folded and mutation-induced misfolded von Willebrand disease (VWD) variants, we test a recently proposed affinity up-regulation hypothesis which states that collagen binding changes the conformation of the A1 domain to a high-affinity GPIbα binding competent state. With surface plasmon resonance (SPR), we present this diversified collection to collagen and quantify the kinetics of association and dissociation to ascertain the conformational selectivity of collagen. With analytical rheology, we quantify real-time platelet pause times and translocation velocities across a Cu2+ HisTag-chelated and collagen-bound A1 single domain and A1A2A3 tridomain fragment of VWF under shear stress in an ex vivo shear flow microfluidic chamber. In contrast to expected hypothetical outcomes, collagen has limited conformational selectivity for binding A1. A1-collagen binding is independent of gain- or loss-of-function phenotype and under shear stress, platelet translocation pause times on collagen-bound A1A2A3 are either normal or shorter depending on whether A1 is concertedly bound with the A3 domain to collagen. With respect to A1, collagen has an inhibitory role that provides an explanation for the lack of thrombosis in patients with gain-of-function VWD.

Rapid discrimination of the phenotypic variants of von Willebrand disease

AbstractApproximately 20% to 25% of patients with von Willebrand disease (VWD) have a qualitative defect of the von Willebrand factor (VWF) protein activities. Variant VWD typically is classified as type 1C, 2A, 2B, 2M, or 2N depending on the VWF activity defect. Traditionally, diagnosis has relied on multiple clinical laboratory assays to assign VWD phenotype. We developed an enzyme-linked immunosorbent assay (ELISA) to measure the various activities of VWF on a single plate and evaluated 160 patient samples enrolled in the Zimmerman Program for the Molecular and Clinical Biology of von Willebrand Disease with type 2 VWD. Using linear discriminate analysis (LDA), this assay was able to identify type 1C, 2A, 2B, 2M, or 2N VWD with an overall accuracy of 92.5% in the patient study cohort. LDA jackknife analysis, a statistical resampling technique, identified variant VWD with an overall accuracy of 88.1%, which predicts the assay's performance in the general population. In addition, this assay demonstrated correlation with traditional clinical laboratory VWF assays. The VWF multiplex activity assay may be useful as a same-day screening assay when considering the diagnosis of variant VWD in an individual patient.

Assessing the clinical severity of type 1 von Willebrand disease patients with a microchip flow‐chamber system

The clinical phenotype of von Willebrand disease (VWD) is heterogeneous, and von Willebrand factor ristocetin cofactor activity (VWF:RCo) does not always reflect clinical severity, especially in VWD type 1. We have reported the potential of a microchip flow-chamber system (Total-Thrombus Formation Analysis System [T-TAS®]) for assessing physiologic hemostasis in VWD. Aim To evaluate the relationship between T-TAS, bleeding score (BS) and laboratory test results in type 1 VWD patients. Microchips coated with collagen (platelet chip [PL-chip]) or collagen/thromboplastin (AR-chip) were used to assess platelet thrombus formation (PTF) at high shear rates or fibrin-rich PTF at low shear rates, respectively, in whole blood from 50 patients. The times needed for the flow pressure to increase by 10 kPa and 30 kPa (T10 and T30 ) from baseline were calculated from flow pressure curves. BS was determined by the use of a standardized questionnaire. PL-T10 values correlated with BS (R(2) ~ 0.45) better than VWF:RCo (R(2) ~ 0.36), irrespective of the flow rate, whereas AR-T10 showed only a weak correlation with BS (R(2) ~ 0.18). Patients with PL-T10 > 10 min or AR-T10 > 30 min had lower VWF levels and higher BS than those with PL-T10 ≤ 10 min or AR-T10 ≤ 30 min, and the greatest differences were observed with PL-T10. Clinical severity appeared to correlate best with PL-T10 > 8 min. BS was significantly higher in patients with VWF:RCo of < 10 IU dL(-1) than in those with VWF:RCo of 10 IU dL(-1) to < 25 IU dL(-1) and 25-40 IU dL(-1). In patients with VWF:RCo of < 10 IU dL(-1) , BS was significantly higher in those with PL-T10 > 8 min than in those with PL-T10 ≤ 8 min. T-TAS could be a useful technique for discriminating and predicting BS in VWD type 1 patients.

Phenotypic Variability in Carriers of Von Willebrand Factor Truncating Sequence Variants in the Zimmerman Program

▪Von Willebrand disease (VWD) is caused by quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). VWD type 1 and 2 are autosomal dominant with variable expression while type 3 is autosomal recessive. The majority of patients have quantitative defects in VWF. The clinical phenotype is highly variable. This variability is evident in the normal population as the VWF antigen levels range from 50-240 IU/dl. In order to investigate variability in clinical expression of VWD, we examined type 1 or type 3 VWD index cases and their family members enrolled in the Zimmerman program where a VWF truncating variant wasfound. These variants included nonsense, splice site, deletions and duplications. All subjects had factor VIII (FVIII), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and multimer distribution analyzed in a central laboratory. VWF gene sequencing was performed for all index cases. Targeted sequencing was performed for family members to ascertain the presence or absence of sequence variations found in the index case. Bleeding symptoms were quantified using the ISTH bleeding assessment tool and reported as bleeding scores. TableNumber of Truncating Variants210Number of Subjects257515VWF:Ag (IU/dl): Avg ±SD2.0±2.545±2099±32VWF:Ag Range0-106-11657-182VWF:Ag 25th-75th Percentile0-3.032-5671-112ISTH Bleeding Score: Avg±SD17.6±8.14.4±4.32.1±2.5ISTH Bleeding Score: 25th-75th Percentile10.0-23.51-60-4.0The VWF:Ag level was significantly different between all three groups (P<0.0001). As expected, type 3 subjects with two truncating variants had the significantly lower VWF:Ag and higher bleeding scores. There was less variability in VWF:Ag and bleeding score compared to the subjects in the other two groups. In subjects with no truncating variants, the unaffected family members, the average VWF:Ag and bleeding score were similar to values observed in healthy controls. The subjects with one variant were the largest group and the most variable in VWD phenotype. The average VWF:Ag was 45±20, slightly below the cutoff of the normal range but the antigen level in this group ranged from 6-116. Thirty-five percent of subjects had VWF:Ag greater than 50 and in the normal range. Approximately 65% had VWF:Ag below 50; about one half were blood type O. 17% had VWF:Ag levels below 30. Although the bleeding score was only slightly higher than the subjects with no variants (P=0.038), the bleeding score ranged from 0-17. Even within a family, highly variable Ag levels were observed.Although numbers were small, when only families with type 3 VWD were considered, mean antigens were higher and mean bleeding scores lower for subjects heterozygous for a truncating allele compared to a non-truncating allele. For a series of subjects with identical truncating alleles, those heterozygous subjects from families with a diagnosis of type 3 VWD had lower bleeding scores than heterozygous subjects from families with only type 1 VWD, despite similar antigen levels, suggesting less concern for mild bleeding symptoms in families where some members have severe bleeding.In summary, carriers of a single VWF truncating allele have a variable phenotype. A wide range of Ag levels and bleeding scores were observed. In 35% of subjects, the Ag level is normal (>50) suggesting the lack of expression from one VWF allele can be compensated by the other VWF allele. The majority of subjects with a single VWF truncating allele had antigen levels typical for type 1 VWD. This suggests that additional environmental or genetic factors are required for symptomaticVWD. DisclosuresNo relevant conflicts of interest to declare.

Laboratory aspects of von Willebrand disease: test repertoire and options for activity assays and genetic analysis.

The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory diagnosis of VWD can be difficult as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2) VWD variants requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity, determining the capacity of VWF to interact with the platelet GPIb-receptor. Knowing the VWF:RCo activity is essential for identifying, subtyping and monitoring VWD, but the assay is poorly standardized and many protocols do not fulfil the clinical need in all situations. This has led to the development of novel activity assays, independent of ristocetin, with enhanced assay characteristics. Results from the first independent clinical evaluations are promising, showing that they are reliable and suitable for VWD diagnosis. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that explore other activities or the size distribution of VWF multimers. These methods are discussed herein. However, in a number of patients it may be difficult to correctly classify the VWD phenotype and genetic analysis may provide the best option to clarify the disorder, through mutation identification.