Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD

Abstract von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)–based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα235Y;239V) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.

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Fourteen Percent of Healthy African Americans Participating In the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB VWD) Are Heterozygous for the VWF Gene Mutation H817Q Associated with Type 2N Von Willebrand Disease

Blood ◽

10.1182/blood.v116.21.239.239 ◽

2010 ◽

Vol 116 (21) ◽

pp. 239-239

Author(s):

Kenneth D Friedman ◽

Daniel B. Bellissimo ◽

Pamela A. Christopherson ◽

Veronica H Flood ◽

Joan Cox Gill ◽

...

Keyword(s):

African American ◽

African Americans ◽

Racial Differences ◽

Healthy Subjects ◽

Von Willebrand Disease ◽

Compound Heterozygous ◽

Healthy Controls ◽

Clinical Biology ◽

A1 Domain ◽

Von Willebrand

Abstract Abstract 239 Von Willebrand disease (VWD) is a common hereditary bleeding disorder caused by reduced concentration or abnormal structure/function of von Willebrand Factor (VWF). Most published studies of normal VWF have been carried out in European or North American subjects without regard to racial differences. In the process of studying healthy controls in the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD), we identified a common polymorphism (D1472H) in the VWF A1-domain in African Americans that affects the measurement of VWF function by ristocetin cofactor (VWF:RCo) but does not appear associated with increased bleeding risk. We therefore explored whether other polymorphisms or mutations were identified more frequently in African Americans. VWF sequencing was performed on 191 healthy controls including 66 that were self-identified as African American. European Bleeding Score was obtained and normal in all healthy subjects. Among the African Americans, 9 individuals were heterozygous for the reported type 2N H817Q mutation and one was homozygous. Factor VIII binding to VWF (VWF:F8B) was determined with a standard FVIII binding assay using the subject's plasma VWF and recombinant FVIII. The VWF:F8B was significantly reduced in H817Q heterozygotes when compared to 10 healthy study subjects without the H817Q mutation (65 ± 11 versus 108 ± 11, p=0.003). The VWF:F8B was further reduced to 37 using the plasma VWF from the homozygous H817Q subject. However, the observed VWF:F8B in these individuals with H817Q are still considerably higher that that observed in patients enrolled in ZPMCB-VWD that are either homozygous or compound heterozygous with the common R854Q type 2N VWD (VWF:F8B < 13). Of the 116 self-identified Caucasian healthy subjects, none had the H817Q mutation, but 3 were heterozygous for the R854Q mutation; their mean plasma VWF:F8B was reduced to 51. While the homozygous R854Q patients had reduced plasma FVIII levels (mean FVIII=24 IU/dL), none of the sequenced healthy control subjects had plasma FVIII levels below 53 IU/dL, Some have advocated FVIII/VWF:Ag ratios as a screen for type 2N VWD. The subject with homozygous H817Q had only a mildly reduced FVIII/VWF:Ag ratio (0.59), while the heterozygous H817Q were not reduced (mean=0.90), thereby demonstrating that the VWF:F8B assay has greater sensitivity for type 2N VWF binding defects than the FVIII/VWF:Ag ratio. Since the previously reported A1-domain D1472H polymorphism was common in African Americans, we explored the prevalence of this polymorphism in the healthy subjects with the H817Q mutation. All H817Q heterozygous subjects were either homozygous (4) or heterozygous (5) for the D1472H polymorphism. The one individual who was H817Q homozygous was also D1472H homozygous, suggesting that there may be an extended haplotype present in African Americans. In summary, an H817Q type 2N mutation is commonly found in healthy African American subjects with an allele frequency of 0.083, predicting that approximately 7 in 1,000 African Americans would be homozygous for the H817Q type 2N mutation. Our data, and the rarity of diagnosis of type 2N VWD in African Americans suggests that while mutation H817Q may interfere with the interaction of FVIII with VWF, this mutation appears to confer little or no clinical symptoms. Disclosures: No relevant conflicts of interest to declare.

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Force-induced on-rate switching and modulation by mutations in gain-of-function von Willebrand diseases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1501689112 ◽

2015 ◽

Vol 112 (15) ◽

pp. 4648-4653 ◽

Cited By ~ 21

Author(s):

Jongseong Kim ◽

Nathan E. Hudson ◽

Timothy A. Springer

Keyword(s):

Single Molecule ◽

Von Willebrand Disease ◽

Bulk Phase ◽

Ligand Complex ◽

Gain Of Function ◽

Phase Measurements ◽

Conformational Regulation ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

Mutations in the ultralong vascular protein von Willebrand factor (VWF) cause the common human bleeding disorder, von Willebrand disease (VWD). The A1 domain in VWF binds to glycoprotein Ibα (GPIbα) on platelets, in a reaction triggered, in part, by alterations in flow during bleeding. Gain-of-function mutations in A1 and GPIbα in VWD suggest conformational regulation. We report that force application switches A1 and/or GPIbα to a second state with faster on-rate, providing a mechanism for activating VWF binding to platelets. Switching occurs near 10 pN, a force that also induces a state of the receptor−ligand complex with slower off-rate. Force greatly increases the effects of VWD mutations, explaining pathophysiology. Conversion of single molecule kon (s−1) to bulk phase kon (s−1M−1) and the kon and koff values extrapolated to zero force for the low-force pathways show remarkably good agreement with bulk-phase measurements.

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Identification of the Binding Site for an Alloantibody to von Willebrand Factor which Inhibits Binding to Glycoprotein Ib within the Amino-terminal Region Flanking the A1 Domain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614572 ◽

1999 ◽

Vol 81 (05) ◽

pp. 793-798 ◽

Cited By ~ 6

Author(s):

Masaru Shibata ◽

Yoshihiro Fujimura ◽

Yukihiro Takahashi ◽

Hiroaki Nakai ◽

Yoshihiko Sakurai ◽

...

Keyword(s):

Platelet Aggregation ◽

Von Willebrand Factor ◽

Inhibitory Effect ◽

Von Willebrand Disease ◽

Tryptic Fragment ◽

Amino Terminal ◽

A1 Domain ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

SummaryAn alloantibody to von Willebrand factor (vWF) which developed in a Japanese boy with type 3 von Willebrand disease has been characterized. The antibody was non-precipitating IgG and the main subclasses were IgG2 and IgG4. The antibody inhibited completely ristocetin-induced platelet aggregation (RIPA) and high shear stress-induced platelet aggregation (SIPA). Its predominant inhibitory role was focused, therefore, on the interaction between vWF and platelet gycoprotein Ib. The antibody reacted with a 52/48 kDa tryptic fragment of vWF (residues 449-728). No reaction was seen, however, with either a 39/34 kDa dispase fragment (480-718) or a recombinant vWF fragment (residues 465-728). These findings suggested that the essential epitope resided in the amino-terminal flanking region of the A1 domain. We synthesized overlapping peptides corresponding to the region containing D3/A1 boundary. A peptide, residues 458-472, bound to the antibody and dose-dependently blocked the antibody binding to the 52/48 kDa fragment. The same peptide neutralized the inhibitory effect of the alloanti-body on SIPA. The data are consistent with the presence of an epitope within residues 458-472 which reacted with the 52/48 kDa fragment.Furthermore, the specific component of the antibody, directed against residues 458-472, blocked vWF binding to GPIb in absence of exogenous agonist. Our results suggest that the region flanking the A1 domain plays an important role in regulating vWF binding to GPIb.

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Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614241 ◽

1998 ◽

Vol 79 (01) ◽

pp. 211-216 ◽

Cited By ~ 13

Author(s):

Lysiane Hilbert ◽

Claudine Mazurier ◽

Christophe de Romeuf

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Missense Mutations ◽

Inhibit Platelet Aggregation ◽

Wild Type ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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Evidence for the Misfolding of the A1 Domain within Multimeric von Willebrand Factor in Type 2 von Willebrand Disease

Journal of Molecular Biology ◽

10.1016/j.jmb.2019.09.022 ◽

2020 ◽

Vol 432 (2) ◽

pp. 305-323 ◽

Cited By ~ 1

Author(s):

Alexander Tischer ◽

Maria A. Brehm ◽

Venkata R. Machha ◽

Laurie Moon-Tasson ◽

Linda M. Benson ◽

...

Keyword(s):

Von Willebrand Factor ◽

Von Willebrand Disease ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

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Toward Personalized Treatment for Patients with Low von Willebrand Factor and Quantitative von Willebrand Disease

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0041-1722864 ◽

2021 ◽

Vol 47 (02) ◽

pp. 192-200

Author(s):

James S. O'Donnell

Keyword(s):

Von Willebrand Factor ◽

Bleeding Risk ◽

Von Willebrand Disease ◽

Personalized Treatment ◽

Treatment Plans ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

AbstractThe biological mechanisms involved in the pathogenesis of type 2 and type 3 von Willebrand disease (VWD) have been studied extensively. In contrast, although accounting for the majority of VWD cases, the pathobiology underlying partial quantitative VWD has remained somewhat elusive. However, important insights have been attained following several recent cohort studies that have investigated mechanisms in patients with type 1 VWD and low von Willebrand factor (VWF), respectively. These studies have demonstrated that reduced plasma VWF levels may result from either (1) decreased VWF biosynthesis and/or secretion in endothelial cells and (2) pathological increased VWF clearance. In addition, it has become clear that some patients with only mild to moderate reductions in plasma VWF levels in the 30 to 50 IU/dL range may have significant bleeding phenotypes. Importantly in these low VWF patients, bleeding risk fails to correlate with plasma VWF levels and inheritance is typically independent of the VWF gene. Although plasma VWF levels may increase to > 50 IU/dL with progressive aging or pregnancy in these subjects, emerging data suggest that this apparent normalization in VWF levels does not necessarily equate to a complete correction in bleeding phenotype in patients with partial quantitative VWD. In this review, these recent advances in our understanding of quantitative VWD pathogenesis are discussed. Furthermore, the translational implications of these emerging findings are considered, particularly with respect to designing personalized treatment plans for VWD patients undergoing elective procedures.

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Phage Display Broadly Identifies Inhibitor-reactive Regions in von Willebrand Factor

10.1101/2021.03.08.434464 ◽

2021 ◽

Author(s):

Andrew Yee ◽

Manhong Dai ◽

Stacy E. Croteau ◽

Jordan A. Shavit ◽

Steven W. Pipe ◽

...

Keyword(s):

Phage Display ◽

Von Willebrand Factor ◽

Gene Deletion ◽

Von Willebrand Disease ◽

Response To Treatment ◽

Phage Library ◽

Incomplete Removal ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

SummaryBackgroundCorrection of von Willebrand factor (VWF) deficiency with replacement products containing VWF can lead to the development of anti-VWF alloantibodies (i.e., VWF inhibitors) in patients with severe von Willebrand disease (VWD).ObjectiveLocate inhibitor-reactive regions within VWF using phage display.MethodsWe screened a phage library displaying random, overlapping fragments covering the full length VWF protein sequence for binding to a commercial anti-VWF antibody or to immunoglobulins from three type 3 VWD patients who developed VWF inhibitors in response to treatment with plasma-derived VWF. Immunoreactive phage clones were identified and quantified by next generation DNA sequencing (NGS).ResultsNGS markedly increased the number of phage analyzed for locating immunoreactive regions within VWF following a single round of selection and identified regions not recognized in previous reports using standard phage display methods. Extending this approach to characterize VWF inhibitors from three type 3 VWD patients (including two siblings homozygous for the same VWF gene deletion) revealed patterns of immunoreactivity distinct from the commercial antibody and between unrelated patients, though with notable areas of overlap. Alloantibody reactivity against the VWF propeptide is consistent with incomplete removal of the propeptide from plasma-derived VWF replacement products.ConclusionThese results demonstrate the utility of phage display and NGS to characterize diverse anti-VWF antibody reactivities.

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Rare Occurrence of Inhibitors in Von Willebrand Disease: A Case Report

Frontiers in Medicine ◽

10.3389/fmed.2021.807664 ◽

2022 ◽

Vol 8 ◽

Author(s):

Bipin P. Kulkarni ◽

Kirti Ghargi ◽

Chandrakala Shanmukhaiah ◽

Shrimati D. Shetty

Keyword(s):

Correct Diagnosis ◽

Severe Form ◽

Von Willebrand Disease ◽

Rare Occurrence ◽

Standard Assay ◽

Type 3 ◽

Von Willebrand ◽

High Degree ◽

Willebrand Factor ◽

Inhibitor Titer

Introduction: Type 3 Von Willebrand Disease (VWD) is the least common but the most severe form of a disease, with a prevalence of about 0. 5 to 1 per million in Western countries. The prevalence of type 3 VWD in the developing countries, with a high degree of consanguinity, is about 6 per million. Moreover, due to underdiagnosis of the milder cases, the prevalence of type 3 VWD is about 50% of the cases. Rarely, some patients develop the Von Willebrand Factor (VWF) inhibitors, which may subsequently develop severe anaphylactic reactions on further exposure to the VWF containing factor replacement therapy. The prevalence of inhibitor development in patients with type 3 VWD has been shown to be in the range of 5.8 to 9.5%. In the absence of a gold standard assay for the quantitation of VWF inhibitors, a correct diagnosis and management of these patients are often challenging.Objectives: The objective of this study is to standardize the Bethesda assay for the VWF inhibitors and to estimate the VWD inhibitor titer in two cases of congenital type 3 VWD, which developed the VWF inhibitors.Results and Conclusions: We could successfully standardize the Bethesda assay for the quantitation of VWF inhibitors in two patients with congenital type 3 VWD with inhibitors.

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Collagen Binding Provides a Sensitive Screen for Variant von Willebrand Disease

Clinical Chemistry ◽

10.1373/clinchem.2012.199000 ◽

2013 ◽

Vol 59 (4) ◽

pp. 684-691 ◽

Cited By ~ 35

Author(s):

Veronica H Flood ◽

Joan Cox Gill ◽

Kenneth D Friedman ◽

Pamela A Christopherson ◽

Paula M Jacobi ◽

...

Keyword(s):

Gel Electrophoresis ◽

Reference Intervals ◽

Von Willebrand Disease ◽

Type Iii Collagen ◽

Healthy Controls ◽

Collagen Binding ◽

Vessel Injury ◽

Clinical Biology ◽

Von Willebrand

BACKGROUND von Willebrand factor (VWF) is a multimeric protein that binds platelets and collagen, facilitating hemostasis at sites of vessel injury. Measurement of VWF multimer distribution is critical for diagnosis of variant von Willebrand disease (VWD), particularly types 2A and 2B, but the typical measurement by gel electrophoresis is technically difficult and time-consuming. A comparison of VWF collagen binding (VWF:CB) and VWF multimer distribution was performed to evaluate the utility of VWF:CB as a diagnostic test. METHODS Participants were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. VWF:CB was analyzed with type III collagen and multimer distribution by agarose gel electrophoresis. The study population included 146 healthy controls, 351 individuals with type 1 VWD, and 77 with type 2 VWD. Differences between individuals with multimer group results within (controls) and outside the reference intervals were assessed with Mann–Whitney tests. RESULTS The mean VWF:CB/VWF antigen ratio was 1.10 for individuals with multimer distribution within the reference intervals and 0.51 for those with multimer distribution outside the reference intervals (P < 0.001). Sensitivity of VWF:CB for multimer abnormalities was 100% for healthy controls, 99% for patients with type 1, and 100% for patients with type 2A and type 2B VWD using a VWF:CB/VWF antigen cutoff ratio of 0.6, and decreased to 99% for all patients with a ratio of 0.7. With the exception of individuals with novel or unclassified mutations, the VWF:CB was able to correctly categorize participants with variant VWD. CONCLUSIONS These findings suggest that VWF:CB may substitute for multimer distribution in initial VWD testing, although further studies are needed to validate the clinical utility of VWF:CB.

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Clinical cases of using coagulation factor VIII with von Willebrand factor in parturient women with type III von Willebrand disease

Тромбоз, гемостаз и реология ◽

10.25555/thr.2020.3.0938 ◽

2020 ◽

Author(s):

И.В. Куртов ◽

Е.С. Фатенкова ◽

Н.А. Юдина ◽

А.М. Осадчук ◽

И.Л. Давыдкин

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Coagulation Factor ◽

Von Willebrand Disease ◽

Coagulation Factor Viii ◽

Vitro Fertilization ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

Болезнь Виллебранда (БВ) может представлять определенные трудности у рожениц с данной патологией. Приведены 2 клинических примера использования у женщин с БВ фактора VIII свертывания крови с фактором Виллебранда, показана эффективность и безопасность их применения. У одной пациентки было также показано использование фактора свертывания крови VIII с фактором Виллебранда во время экстракорпорального оплодотворения. Von Willebrand disease presents a certain hemostatic problem among parturients. This article shows the effectiveness and safety of using coagulation factor VIII with von Willebrand factor for the prevention of bleeding in childbirth in 2 patients with type 3 von Willebrand disease. In one patient, the use of coagulation factor VIII with von Willebrand factor during in vitro fertilization was also shown.

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