scholarly journals Novel cysteine substitution p.(Cys1084Tyr) causes variable expressivity of qualitative and quantitative VWF defects

2022 ◽  
Author(s):  
Orla Rawley ◽  
Laura L. Swystun ◽  
Christine Brown ◽  
Kate Nesbitt ◽  
Margaret L Rand ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Expression System ◽  
Von Willebrand Disease ◽  
Phenotypic Data ◽  
Variable Expressivity ◽  
Structural Conformation ◽  
Hemostatic Activity ◽  
Normal Hemostasis ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand factor (VWF) is an extremely cysteine-rich multimeric protein that is essential for maintaining normal hemostasis. The cysteine residues of VWF monomers form intra- and inter-molecular disulfide bonds that regulate its structural conformation, multimer distribution and ultimately its hemostatic activity. In this study we investigated and characterized the molecular and pathogenic mechanisms through which a novel cysteine variant p.(Cys1084Tyr) causes an unusual, mixed phenotype form of von Willebrand disease (VWD). Phenotypic data including bleeding scores, laboratory values, VWF multimer distribution and desmopressin response kinetics were investigated in 5 members (2 parents and 3 daughters) of a consanguineous family. VWF synthesis and secretion were also assessed in a heterologous expression system and in a transient transgenic mouse model. Heterozygosity for p.(Cys1084Tyr) was associated with variable expressivity of qualitative VWF defects. Heterozygous individuals had reduced VWF:GPIbM (<0.40IU/mL) and VWF:CB (<0.35IU/mL) as well as relative reductions in high-molecular weight multimers, consistent with type 2A VWD. In addition to these qualitative defects, homozygous individuals also displayed reduced FVIII:C/VWF:Ag leading to very low FVIII levels (0.03-0.1IU/mL) as well as reduced VWF:Ag (<0.40IU/mL) and VWF:GPIbM (<0.30IU/ml). Accelerated VWF clearance and impaired VWF secretion contributed to the fully expressed homozygous phenotype with impaired secretion arising due to disordered disulfide connectivity.

Molecular Background of “Smeary” von Willebrand Factor Multimers.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2711-2711
Author(s):  
Reinhard Schneppenheim ◽  
Olivier Marggraf ◽  
Heike Eckert ◽  
Tobias Obser ◽  
Florian Oyen ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Von Willebrand Disease ◽  
Molecular Testing ◽  
Functional Deficits ◽  
Intermolecular Disulfide Bonds ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: Multimer analysis of von Willebrand factor (VWF) is a most important technique to classify patients with von Willebrand disease (VWD) type 2. Besides “classical” multimer patterns a “smeary” appearance of individual VWF oligomers is increasingly observed and has previously been regarded as a pre-analytical artifact. Objective: To phenotypically and genotypically assess the molecular background of “smeary” VWF multimers. Patients and methods: Samples of 8 VWD patients were analyzed in our reference lab (UB) for further classification and molecular testing. Multimer profiles were assessed by intermediate resolution gels. VWF:CB and VWF:GpIb binding were used as functional assays. VWF gene mutation analysis was performed in all index cases (IC). The causal relationship between genotype and phenotype was studied by analyzing recombinant mutants in comparison to wildtype VWF. Results: In all IC the phenotype correlated with particular mutations in the VWF D3 domain (G1172D), the A1 domain (R1315C, R1374S, R1374C, R1399C), the D4 domain (C2257R), the C1 domain (R2464C) and in the region close to the CK domain (C2671Y), respectively. The multimer patterns of recombinant mutant VWF was of a “smeary” appearance and closely resembled those of mutant plasma VWF. Mutations in the A1 domain additionally correlated with severe GpIb binding deficiency. Conclusions: Our data suggest a molecular cause of the “smeary” multimer structure rather than pre-analytical artifacts. Most of the mutations identified involved cysteine residues suggesting an influence on the VWF secondary structure which is determined by intra- and intermolecular disulfide bonds. This could explain the peculiar multimer appearance. The functional deficits, however, seem to depend on the location of the mutations with a significant impact on GpIb binding of mutants in the A1 domain.

Differential effects of the loss of intrachain- versus interchain-disulfide bonds in the cystine-knot domain of von Willebrand factor on the clinical phenotype of von Willebrand disease

2006 ◽  
Vol 96 (12) ◽  
pp. 717-724 ◽  
Author(s):  
Pernilla Tjernberg ◽  
Hans Vos ◽  
Caroline Spaargaren-van Riel ◽  
Brenda Luken ◽  
Jan Voorberg ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Von Willebrand Disease ◽  
Minor Effect ◽  
Cystine Knot ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Intrachain Disulfide Bond

SummaryVon Willebrand factor (VWF) contains a large number of cysteine residues, which all form disulfide bonds. Mutations of cysteines located in the cystine-knot (CK) domain of VWF have been identified in both qualitative type 2A (IID) and quantitative type 3 vonWillebrand disease (VWD).Our objective was to test the hypothesis that the difference in phenotype is related to whether the mutated cysteine residue is involved in either interchain- or intrachain-disulfide-bond formation. The effects of three cysteine mutations which are all located in the CK-domain of VWF, C2773S (type 2A(IID)), C2739Y (type 3), and C2754W (type 3), were studied by transient expression in 293T cells. Cotransfection of wild-type (wt) and C2773S VWF constructs reproduced the plasma phenotype of heterozygous type 2A(IID) patients, with normal to high levels of VWF antigen (VWF:Ag), absence of high-molecular-weight multimers, and the presence of intervening bands between the normal multimers.In contrast, single transfections of C2739Y or C2754W resulted in a quantitativeVWF defect with lowVWF:Ag levels, and co-transfections of wt and mutant constructs resulted in a 50% reduction of VWF:Ag and only a minor effect on VWF multimerization. We demonstrated N-terminal dimerization of VWF-C2773S and both Nand C-terminal dimerization of VWF-C2754W. Our data suggest that loss of a single disulfide bond in the CK-domain ofVWF leads toa recessive, quantitativeVWF deficiency if an intrachain-disulfide bond is involved, and to a dominant-negative, qualitative defect of VWF if an interchain-disulfide bond is involved.

Getting at the Variable Expressivity of Von Willebrand Disease

2001 ◽  
Vol 86 (07) ◽  
pp. 144-148 ◽  
Author(s):  
Gallia Levy ◽  
David Ginsburg
Keyword(s):  
General Population ◽  
Von Willebrand Factor ◽  
Genetic Factors ◽  
Von Willebrand Disease ◽  
Incomplete Penetrance ◽  
Large Component ◽  
Future Directions ◽  
Variable Expressivity ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by abnormalities of von Willebrand factor (VWF). VWF levels vary widely in the general population, and this variation is likely to be a major factor accounting for the incomplete penetrance and variable expressivity of VWD. In addition, variation in VWF level may play an important role in determining the risk of venous thrombosis. A large component of the variation in VWF level in the general population has been shown to be attributable to genetic factors. This review will focus on the current understanding of the genetic causes for variation in VWF level, and will highlight future directions for getting at the variable expressivity of von Willebrand disease.

scholarly journals Variable content of von Willebrand factor mutant monomer drives the phenotypic variability in a family with von Willebrand disease

Blood ◽  
2015 ◽  
Vol 126 (2) ◽  
pp. 262-269 ◽  
Author(s):  
Junmei Chen ◽  
Jesse D. Hinckley ◽  
Sandra Haberichter ◽  
Paula Jacobi ◽  
Robert Montgomery ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Phenotypic Variability ◽  
Von Willebrand Disease ◽  
Variable Expressivity ◽  
Key Points ◽  
Variable Content ◽  
Von Willebrand ◽  
Willebrand Factor

Key Points VWD is characterized by variable expressivity, even within families with the same VWF mutation. The content of mutant monomers in the final multimeric structure may explain the observed variability.

scholarly journals Porcine and Canine von Willebrand Factor and von Willebrand Disease: Hemostasis, Thrombosis, and Atherosclerosis Studies

Thrombosis ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Timothy C. Nichols ◽  
Dwight A. Bellinger ◽  
Elizabeth P. Merricks ◽  
Robin A. Raymer ◽  
Mark T. Kloos ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Potential Role ◽  
Von Willebrand Disease ◽  
Current Status ◽  
Bleeding Events ◽  
Coronary Artery Thrombosis ◽  
Normal Hemostasis ◽  
Von Willebrand ◽  
Willebrand Factor

Use of animal models of inherited and induced von Willebrand factor (VWF) deficiency continues to advance the knowledge of VWF-related diseases: von Willebrand disease (VWD), thrombotic thrombocytopenic purpura (TTP), and coronary artery thrombosis. First, in humans, pigs, and dogs, VWF is essential for normal hemostasis; without VWF bleeding events are severe and can be fatal. Second, the ADAMTS13 cleavage site is preserved in all three species suggesting all use this mechanism for normal VWF multimer processing and that all are susceptible to TTP when ADAMTS13 function is reduced. Third, while the role of VWF in atherogenesis is debated, arterial thrombosis complicating atherosclerosis appears to be VWF-dependent. The differences in the VWF gene and protein between humans, pigs, and dogs are relatively few but important to consider in the design of VWF-focused experiments. These homologies and differences are reviewed in detail and their implications for research projects are discussed. The current status of porcine and canine VWD are also reviewed as well as their potential role in future studies of VWF-related disorders of hemostasis and thrombosis.

Von Willebrand Disease

2018 ◽  
Author(s):  
Laura A. Downey ◽  
Nina A. Guzzetta
Keyword(s):  
Von Willebrand Factor ◽  
Treatment Plan ◽  
Correct Diagnosis ◽  
Postoperative Bleeding ◽  
Bleeding Disorder ◽  
Von Willebrand Disease ◽  
Normal Hemostasis ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand disease (vWD) is the most common bleeding disorder in humans. It is the result of an abnormality in the amount, structure, or function of von Willebrand factor (vWF), a glycoprotein important in maintaining normal hemostasis.. In children with vWD, the most frequent presentation is easy bruising and epistaxis. Other symptoms include hematomas, menorrhagia, and bleeding from minor wounds. Although intraarticular bleeding may occur, especially in certain subtypes, it is much more commonly seen with hemophilia. There are several subtypes of vWD based on the underlying defect in vWF, but, in general, they may be categorized as quantitative (types 1 and 3) or qualitative (all types 2). If vWD is suspected, consultation with a hematologist to establish the correct diagnosis and perioperative approach to hemostasis is essential. Avoidance of medications that interfere with coagulation, anticipation of intraoperative and postoperative bleeding, and an appropriate hemostatic treatment plan should be addressed.

scholarly journals The disulfide bond Cys2724-Cys2774 in the C-terminal cystine knot domain of von Willebrand factor is critical for its dimerization and secretion

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yuxin Zhang ◽  
Fengwu Chen ◽  
Aizhen Yang ◽  
Xiaoying Wang ◽  
Yue Han ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Von Willebrand Disease ◽  
Cystine Knot ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background Type 3 von Willebrand disease (VWD) exhibits severe hemorrhagic tendency with complicated pathogenesis. The C-terminal cystine knot (CTCK) domain plays an important role in the dimerization and secretion of von Willebrand factor (VWF). The CTCK domain has four intrachain disulfide bonds including Cys2724-Cys2774, Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806, and the single cysteine mutation in Cys2739-Cys2788, Cys2750-Cys2804 and Cys2754-Cys2806 result in type 3 VWD, demonstrating the crucial role of these three disulfide bonds in VWF biosynthesis, however, the role of the remaining disulfide bond Cys2724-Cys2774 remains unclear. Method and results In this study, by the next-generation sequencing we found a missense mutation a c.8171G>A (C2724Y) in the CTCK domain of VWF allele in a patient family with type 3 VWD. In vitro, VWF C2724Y protein was expressed normally in HEK-293T cells but did not form a dimer or secrete into cell culture medium, suggesting that C2724 is critical for the VWF dimerization, and thus for VWF multimerization and secretion. Conclusions Our findings provide the first genetic evidence for the important role of Cys2724-Cys2774 in VWF biosynthesis and secretion. Therefore, all of the four intrachain disulfide bonds in CTCK monomer contribute to VWF dimerization and secretion.

Assembly, Storage, and Secretion of Von Willebrand Factor.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-46-sci-46
Author(s):  
J. Evan Sadler
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Three Dimensional ◽  
Von Willebrand Disease ◽  
Inflammatory Stress ◽  
Von Willebrand ◽  
Willebrand Factor

Von Willebrand factor (VWF) plays a central role in hemostasis, and dysregulation of VWF can cause either bleeding or thrombosis. Endothelial cells assemble VWF multimers in two stages that occur in distinct intracellular compartments: proVWF subunits dimerize in the endoplasmic reticulum through “tail-to-tail” disulfide bonds between C-terminal CK domains, and the proVWF dimers form enormous multimers in the Golgi through “head-to-head” disulfide bonds between N-terminal D3 domains. The finished multimers are packaged into ordered tubules within storage organelles called Weibel-Palade bodies (WPB), and tubular packing is necessary for the secretion of VWF filaments that have normal hemostatic function. We have recreated VWF tubule assembly in vitro, starting with pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D’D3 domains. No other cellular proteins or components of WPB are needed. Assembly requires low pH and calcium ions, similar to conditions in the Golgi. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D’D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging. Secretion into the blood exposes VWF tubules to neutral pH conditions, releasing the constraints that maintain tubular packing and permitting the orderly unfurling of large VWF multimers. Some VWF multimers bind platelets and initiate the growth of platelet-rich thrombi. Under normal circumstances, these thrombi are limited in size by ADAMTS13, a metalloprotease that cleaves VWF multimers and releases the platelets. The absence of large VWF multimers causes bleeding that is typical of von Willebrand disease. Conversely, congenital or acquired deficiency of ADAMTS13 prevents the dissolution of VWF-platelet aggregates, which can cause the widespread microvascular thrombosis that characterizes thrombotic thrombocytopenic purpura (TTP). Interestingly, ADAMTS13 deficiency alone need not trigger TTP. Some patients persist for months or years without active disease but become ill whenever they suffer additional inflammatory stress associated with infection, surgery, or pregnancy. Thus, interactions between inflammatory mediators and endothelial cells can determine the course of VWF-dependent thrombosis. The mechanisms responsible for these phenomena remain poorly understood.

scholarly journals The Use of Phage Display and Yeast Based Expression System for the Development of a Von Willebrand Factor Propeptide Assay: Development of a Von Willebrand Factor Propeptide Assay

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
S. M. Meiring ◽  
B. D. P. Setlai ◽  
C. Theron ◽  
R. Bragg
Keyword(s):  
Phage Display ◽  
Von Willebrand Factor ◽  
Expression System ◽  
Plasma Concentrations ◽  
Cost Effective ◽  
Antibody Fragments ◽  
Von Willebrand Disease ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Von Willebrand Factor Propeptide

Background. The diagnosis of von Willebrand disease is complex due to the heterogeneity of the disease. About eighty percent of von Willebrand disease patients are diagnosed with a quantitative defect of von Willebrand factor (VWF) where fifty percent is due to an increased clearance of von Willebrand factor. These patients do not respond well to the treatment of choice, Desmopressin (DDAVP) due to decreased efficacy. The ratio between the VWF propeptide and the mature VWF antigen is used to diagnose these patients. Commercial VWF propeptide assays are too expensive for use in developing countries. In this study, we developed a cost-effective ELISA assay. Methods. We first displayed VWF propeptide on yeast. Antibody fragments were selected against the displayed VWF propeptide by using phage display technology. The antibodies were used to develop a cost-effective VWF propeptide assay and compared to a commercial VWF propeptide assay. Results. Two of these antibody fragments bound specific to the VWF propeptide and not to the yeast used for the expression of the propeptides. These purified antibody fragments were able to detect VWF propeptide in normal plasma. Conclusion. Our assay performed well when compared to a commercial kit. It also showed a higher binding affinity for VWF propeptide in plasma at especially lower plasma concentrations.

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

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
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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