Intracellular Retention, Enhanced Clearance, and Defective FVIII Binding Are Common Features of Von Willebrand Factor D'-D3 Domain Mutations in Patients with Von Willebrand Disease Type 1 From the European Mcmdm-1VWD Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 99-99 ◽  
Author(s):  
Reinhard Schneppenheim ◽  
Ulrich Budde ◽  
Javier Batlle ◽  
Giancarlo Castaman ◽  
Jeroen C. J. Eikenboom ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Recombinant Expression ◽  
Von Willebrand Disease ◽  
Phenotypic Characterization ◽  
Clinical Markers ◽  
In Trans ◽  
Von Willebrand ◽  
In Cis

Abstract Abstract 99 Background: Von Willebrand disease (VWD) type 1 is characterized by a partial reduction of structurally and functionally normal VWF with normal VWF multimers. As part of a large European study (Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD) patients previously diagnosed with VWD type 1 were studied systematically to assess the phenotypic and genotypic spectrum. Objective: To confirm the pathogenicity of VWF gene mutations and to elucidate the molecular mechanisms of VWD type 1. Patients and methods: VWD type 1 patients were recruited by twelve expert centers in nine European countries. VWF genotyping was performed in all index cases (IC). The eight mutations studied here are located in the VWF D'-D3 domain and corresponded to 57 patients from 19 families. They were reproduced by recombinant expression with subsequent phenotypic characterization, two of them in cis and one in trans with a second mutation. Results and Discussion: Intracellular VWF:Ag of all mutants was normal or near normal suggesting normal expression levels. However, seven mutations (p.M771I, p.I1094T, p.C1130R, p.C1130G, p.C1130F, p.W1144G and p.Y1146C) caused intracellular retention and impaired VWF secretion. In addition, we observed a major loss of high molecular weight multimers as in type 2A and a novel finding of a severe VWF:FVIII binding defect in most of the homozygously expressed mutants. Additional mutations either in cis or in trans had no modifying effect. The recombinant VWD type 1 Vicenza mutation p.R1205H with or without the allelic variant p.M740I seen in three Italian IC was secreted normally and had normal function leaving enhanced clearance of mutant VWF as the only pathomechanism. In conclusion, the majority of mutations in the D3 domain impair VWF multimerization, cause intracellular retention and correlate with defective FVIII binding. An elevated ratio of VWF propeptide to VWF:Ag suggests enhanced VWF clearance as an important pathomechanism of most mutations and particularly of p.R1205H. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Low VWF: insights into pathogenesis, diagnosis, and clinical management

2020 ◽  
Vol 4 (13) ◽  
pp. 3191-3199 ◽  
Author(s):  
James S. O’Donnell
Keyword(s):  
Clinical Management ◽  
Molecular Mechanisms ◽  
Optimal Choice ◽  
Von Willebrand Disease ◽  
Limited Information ◽  
Von Willebrand ◽  
The Relationship ◽  
Willebrand Factor ◽  
Choice Of Therapy

Abstract von Willebrand disease (VWD) constitutes the most common inherited human bleeding disorder. Partial quantitative von Willebrand factor (VWF) deficiency is responsible for the majority of VWD cases. International guidelines recommend that patients with mild to moderate reductions in plasma VWF antigen (VWF:Ag) levels (typically in the range of 30-50 IU/dL) should be diagnosed with low VWF. Over the past decade, a series of large cohort studies have provided significant insights into the biological mechanisms involved in type 1 VWD (plasma VWF:Ag levels <30 IU/dL). In striking contrast, however, the pathogenesis underpinning low VWF has remained poorly understood. Consequently, low VWF patients continue to present significant clinical challenges with respect to genetic counseling, diagnosis, and management. For example, there is limited information regarding the relationship between plasma VWF:Ag levels and bleeding phenotype in subjects with low VWF. In addition, it is not clear whether patients with low VWF need treatment. For those patients with low VWF in whom treatment is deemed necessary, the optimal choice of therapy remains unknown. However, a number of recent studies have provided important novel insights into these clinical conundrums and the molecular mechanisms responsible for the reduced levels observed in low VWF patients. These emerging clinical and scientific findings are considered in this review, with particular focus on pathogenesis, diagnosis, and clinical management of low VWF.

Genetic Background of von Willebrand Disease: History, Current State, and Future Perspectives

2019 ◽  
Vol 46 (04) ◽  
pp. 484-500 ◽  
Author(s):  
Jana Zolkova ◽  
Juraj Sokol ◽  
Tomas Simurda ◽  
Lubica Vadelova ◽  
Zuzana Snahnicanova ◽  
...  
Keyword(s):  
Genetic Background ◽  
Molecular Mechanisms ◽  
Diagnostic Algorithm ◽  
Coagulation Factors ◽  
Von Willebrand Disease ◽  
Molecular Testing ◽  
Gene Encoding ◽  
Gene Panels ◽  
Von Willebrand

AbstractSequencing of the gene encoding for von Willebrand factor (VWF) has brought new insight into the physiology of VWF as well as its pathophysiology in the context of von Willebrand disease (VWD). Molecular testing in VWD patients has shown high variability in the overall genetic background of this condition. Almost 600 mutations and many disease-causing mechanisms have been described in the 35 years since the VWF gene was identified. Genetic testing in VWD patients is now available in many centers as a part of the VWD diagnostic algorithm. Molecular mechanisms leading to types 2 and 3 VWD are well characterized; thus, information from genetic analysis in these VWD types may be beneficial for their correct classification. However, the molecular basis of type 1 VWD is still not fully elucidated and most likely represents a multifactorial disorder reflecting a combined impact of environmental and genetic factors within and outside of VWF. Regarding sequencing methods, the previous gold-standard Sanger sequencing is gradually being replaced with next-generation sequencing methods that are more cost- and time-effective. Instead of gene-by-gene approaches, gene panels of genes for coagulation factors and related proteins have recently become a center of attention in patients with inherited bleeding disorders, especially because a high proportion of VWD patients, mainly those with low VWF plasma levels (type 1), appear to be free of mutations in VWF. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) are accessible in a very limited number of laboratories. Results from these studies have presented several genes other than VWF or ABO possibly affecting VWF levels, and such findings will need further validation studies.

Reduced Von Willebrand Factor Secretion Is Associated with Loss of Weibel-Palade Formation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 541-541
Author(s):  
Giancarlo Castaman ◽  
Sofia Helene Giacomelli ◽  
Paula M. Jacobi ◽  
Tobias Obser ◽  
Reinhard Schneppenheim ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Negative Impact ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Wild Type ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 541 Background. Von Willebrand Disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes patients with quantitative plasma VWF deficiency with normal VWF structure and function. Aim of the study. We report three different novel type 1 VWF mutations (A1716P, C2190Y and R2663C) which although located in different VWF domains are associated with reduced secretion and lack of formation of Weibel-Palade body-like granules. Methods. Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding, and GpIb binding assessed in comparison to wild-type VWF. Furthermore, expression was also examined in HEK293 cells that form Weibel-Palade body (WPB)-like granules when transfected with wt VWF. Results. The multimer analysis of plasma VWF was compatible with type 1 VWD. The results of 3 different expression experiments showed a slightly reduced VWF synthesis and drastically impaired secretion into the medium with homozygous expression. In HEK293 cells, homozygous A1716P and C2190Y VWF variants failed to form WPB-like granules, while R2663C was capable of forming granules, but had fewer cells with granules and more with ER-localized VWF. Heterozygous expression of A1716P and C2160Y VWF variants had a negative impact on wild-type VWF and WPB-like granules were observed in transfected cells. Conclusions. Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations can be associated with inability to form endothelial Weibel-Palade-like granules and mutations in different VWF domains can affect the formation of these organelles. Disclosures: No relevant conflicts of interest to declare.

Molecular Analysis of Type 1 VWD Mutations: Effects of Mutant:Wild-Type Transfection Ratio and Protein Degradation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1210-1210
Author(s):  
Tara C White-Adams ◽  
Paula M Jacobi ◽  
Sandra L Haberichter ◽  
Jorge A Di Paola
Keyword(s):  
Conflicts Of Interest ◽  
Phenotypic Variability ◽  
Statistical Significance ◽  
Von Willebrand Disease ◽  
Incomplete Penetrance ◽  
Shear Stresses ◽  
Dependent Manner ◽  
Von Willebrand

Abstract Abstract 1210 Background: Von Willebrand disease (VWD), the most frequently diagnosed bleeding disorder, is characterized by variable expressivity and incomplete penetrance. Bleeding severity in type 1 VWD does not always correlate with plasma VWF levels, except in cases of severe deficiency. It is possible that the phenotypic variability observed in type 1 VWD is related to the final ratio of mutant vs. wild-type (WT) subunits in the mature VWF multimeric structure. The aim of this study was to determine the role of mutant:WT transfection ratio on von Willebrand factor (VWF) expression, secretion and degradation in VWD type 1 mutations. Methods: Type 1 VWD mutations with reported normal multimer distribution were chosen from the D'-D3 region of VWF. Mutations of cysteine residues were eliminated to avoid interference with inter- and intra-chain disulfide linkages. Mutations were generated by performing site-directed mutagenesis on full-length human VWF cDNA within the pcDNA3.1(-)A vector, which appends VWF with a Myc-His tag (denoted mH). The following mutations were generated: M771I, R782Q, R924W, I1094T and T1156M. Mutant VWF was co-transfected with WT VWF contained within the pCIneo vector (mutant mH:WT pCIneo ratios investigated were 1:3, 2:2, 3:1, 4:0). Recombinant (r)VWF expression was measured using ELISA and concentrations were determined by comparison to a standard curve generated with pooled normal plasma. Multimer composition was analyzed using SDS-agarose gel electrophoresis followed by Western blot. Statistical significance was determined using one-way ANOVA with post-hoc Tukey test. Results: Homozygous expression of R924W or I1094T had no effect on rVWF expression or secretion compared to WT, while M771I, R782Q and T1156M significantly increased intracellular protein retention. Co-expression of M771I or R782Q at varying ratios with WT was able to partially correct rVWF secretion, although intracellular retention remained significantly higher than WT at all ratios (n=3, * p<0.05, Figure 1). Co-expression with WT cDNA was also able to correct T1156M retention in a dose-dependent manner (n=3, Figure 1), as described previously [Lethagen, Thromb and Haemost, 2002]. Multimer analysis of co-transfection supernatants exhibited normal and full distribution of multimers, as expected for type 1 VWD mutations. Others have shown previously that heterodimers of WT and C1149R VWF, a type 1 VWD mutation, are degraded by the proteasome [Bodo et al, Blood, 2001], presumably via recognition of a folding defect within the mutant subunit. In order to determine the role of proteasomal degradation in the decreased secretion levels of our mutants, we performed experiments in the presence of the proteasome inhibitor MG-132. Treatment of co-transfected cells (mutant:WT 2:2) with 1 mM MG-132 for 16 hours prior to harvesting did not significantly affect secretion or overall expression of rVWF, suggesting that this pathway is not involved in the regulation of the expression of our mutants. Discussion: Our data demonstrate that M771I, T1156M and R782Q induce a significant increase in intracellular retention compared to WT protein, which could contribute to a quantitative deficiency in type 1 VWD, while R924W and I1094T do not appear to interfere with VWF production or secretion. Variable levels of intracellular retention have been observed in a previous study of VWF mutations identified in type 1 VWD patients [Eikenboom, et al, J Thromb Haemost, 2009]. While one interpretation of these results is that R924W or I1094T may not be causative mutations in type 1 VWD, other mechanisms including protein clearance and function remain to be explored. Although type 1 VWD mutations variably affect expression and secretion levels in vitro, studying platelet rolling on these mutants at a range of physiological shear stresses will provide valuable information regarding whether the degree of incorporation of mutant subunits into VWF multimers can affect supramolecular structure, and ultimately, hemostatic function. Disclosures: No relevant conflicts of interest to declare.

Phase II Biologic Effects Trial of Recombinant Interleukin-11 (rhIL-11, Neumega) in Moderate or Mild Hemophilia A or Von Willebrand Disease Unable to Use DDAVP,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3308-3308
Author(s):  
Margaret V. Ragni ◽  
Enrico M. Novelli ◽  
Anila Murshed ◽  
Elizabeth P. Merricks ◽  
Mark T. Kloos ◽  
...  
Keyword(s):  
Phase Ii ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Postoperative Bleeding ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Fluid Restriction ◽  
Biologic Effects ◽  
Von Willebrand

Abstract Abstract 3308 Background: DDAVP is the treatment of choice for individuals with type 1 von Willebrand disease (VWD), although 20% are unresponsive, and of the 80% who do respond, the VWF increase is transient, as endothelial stores are depleted after 3 days. Further, administration requires a 30- minute intravenous infusion in a medical facility. Plasma-derived concentrates may be used in these settings, but are more costly and have potential risk of transmissible infection. We recently demonstrated that recombinant human IL-11 (rhIL-11, Neumega®), a gp-130 signaling cytokine with hematopoietic and anti-inflammatory activity, increases VWF activity up to 2-fold when given daily by subcutaneous injection, with levels persisting each day it is given, and reduces menstrual and postoperative bleeding. The effects of rhIL-11 in individuals with VWD unresponsive or allergic to DDAVP, or hemophilia A, however, have not been evaluated. Methods: We conducted a phase II trial to evaluate the safety and biologic effects of rhIL-11 in VWD patients unresponsive or allergic to DDAVP (VWD-Un) or mild hemophilia A (HemA). rhIL-11 was given subcutaneously at 25 μg/kg daily for 4 days in the non-bleeding state, followed on day 4, 30 minutes after rhIL-11, by one dose of DDAVP intravenously, 0.3 μg/kg, if not contraindicated (pt. 2). Fluid restriction was recommended. Fluid status was assessed by height, weight, and exam. Pre- and post-dosing laboratory assays included the VWD profile, VWF multimers by SDS gel electrophoresis, and platelet VWF mRNA by qPCR. Results: The results of the first six subjects, including three with VWD (one type IIB and two type 1 VWD), VWF:RCo 0.10–0.20 U/ml, and three with mild hemophilia A, F.VIII 0.08–0.12 U/ml, are presented. All subjects were healthy, with no hypertension or cardiac disease, and all had normal physical exams and normal EKGs. By day 4, among VWD-Un subjects, there was a 1.2-fold increase in VWF:RCo (15±3% vs. 12±0%); a 1.6-fold increase in VWF:Ag (22±8% vs.14±6%); and a 1.3-fold increase in VIII:C (34±36% vs. 27±10%), as compared with pre-rhIL-11 levels (Figure). Following DDAVP (except pt. 2), there was an additional 2.0-fold, 1.7-fold, and 2.6-fold increase in VWF:RCo, VWF:Ag, and VIII:C, respectively. Among HemA subjects, by day 4, there was a 1.8-fold increase in VWF:RCo (160±25% vs. 88±12%); a 1.8-fold increase in VWF:Ag (182±28% vs.99±18%), p<0.01; and a 1.5-fold increase in VIII:C (21±8% vs. 14±5%), as compared with pre-rhIL-11 levels. Following DDAVP, there was an additional 1.5-fold (p<0.01), 1.7-fold, and 2.8-fold (p<0.05) increase in VWF:RCo, VWF:Ag, and VIII:C, respectively. The drug was well tolerated well with less than grade 1 mild conjunctival erythema, local erythema and tenderness at the injection site; in one subject transient hyponatremia, Na 129 meq/L, occurred after excess oral fluid intake for diabetic hyperglycemia, which resolved with fluid restriction. Discussion: These data suggest that rhIL-11 increases VWF and VIII levels modestly in VWD patients unresponsive/allergic to DDAVP, and in mild hemophilia A, suggesting the potential use in treatment of clinical bleeding in these disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bleeding scores: are they really useful?

Hematology ◽  
2012 ◽  
Vol 2012 (1) ◽  
pp. 152-156 ◽  
Author(s):  
Sarah H. O'Brien
Keyword(s):  
Laboratory Testing ◽  
Clinical Applications ◽  
Von Willebrand Disease ◽  
Assessment Tools ◽  
Bleeding Disorders ◽  
Clinical Markers ◽  
Precise Quantification ◽  
Bleeding Score ◽  
Von Willebrand

Abstract Given the commonality of bleeding symptoms in the general population and the diagnostic limitations of available laboratory testing for mild bleeding disorders, there has been increasing interest in a more precise quantification of bleeding symptoms. The Vicenza bleeding score (and its successor, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand disease [MCMDM-1 VWD]) and its pediatric counterpart, the Pediatric Bleeding Questionnaire, are validated research tools that have demonstrated their ability to discriminate between healthy subjects and those with VWD. These instruments collect data regarding both the presence and severity of a variety of bleeding symptoms and generate a bleeding score by summing the severity of all symptoms reported by a subject. More recent work demonstrates the promise of these tools as a diagnostic aid in the evaluation of patients with a suspected inherited mild bleeding disorder, as well as the development of a condensed score with increased clinical applicability. This review focuses on the development of these bleeding assessment tools, recent publications applying and refining these instruments, and current limitations of bleeding scores. Needed research studies and potential clinical applications of bleeding scores are also discussed. The ultimate goal would be for bleeding scores to be integrated with the results of standardized laboratory testing to allow for a universal diagnostic approach to patients with suspected bleeding disorders.

Quantitative Analysis of VWF Multimer Structure: Discrimination Between VWD Subtypes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1215-1215 ◽  
Author(s):  
Sandra L Haberichter ◽  
Paula M Jacobi ◽  
Veronica H Flood ◽  
Pamela A. Christopherson ◽  
Joan Cox Gill ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Quantitative Analysis ◽  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Similar Distribution ◽  
Image Analyzer ◽  
Healthy Controls ◽  
Essentially Normal ◽  
Von Willebrand

Abstract Abstract 1215 The diagnosis of von Willebrand disease (VWD) and discrimination between its subtypes includes analysis of VWF:Ag, VWF:RCo, and VWF multimer structure. VWF multimer analysis is qualitative, and therefore a subjective assessment open to interpretation. It is often difficult to assess subtle differences in multimer structure. To address these shortcomings we have developed a quantitative method for analysis of VWF multimers. We have analyzed multimer structure for VWD patients and healthy controls recruited through the Zimmerman Program for the Molecular and Clinical Biology of von Willebrand Disease (ZPMCB-VWD). The patient population includes type 1 and type 2 VWD with well-defined genotypes and phenotypes. Multimer analysis was performed using a 0.65% LiDS-agarose gel electrophoresis system and western blotting with chemilumiscent detection using the Fujifilm LAS-3000 luminescent image analyzer. Densitometry was performed and area-under-the-curve calculated using MultiGauge analysis software. We calculated the percentage of low molecular weight (LMW) multimers defined as bands 1 – 5, mid-molecular weight (MMW) multimers (bands 6 – 10) and high molecular weight (HMW) multimers (bands >10). For healthy controls, the distribution of multimer density (mean ± standard deviation) was 25.3 ± 2.7% HMW, 56.1 ± 4.9% MMW, and 18.6 ± 3.4% LMW. Type 1 VWD (including type 1C) patients had a similar distribution of multimers (22.5 ± 7.6% HMW, 48.5 ± 6.7% MMW, 29.0 ± 7.2 % LMW), although there was a slight shift in distribution to increased LMW. For some type 1C patients with mutations including C1130Y and W1144G, we observed a small loss of HMW multimers (14.2 ± 0.8% HMW, 51.1 ± 1.4% MMW, 34.7 ± 2.3% LMW), as has been previously reported in patients with a C1130F variation. In contrast, some patients with the type 1C “Vicenza” mutation, R1205H, demonstrated increased HMW multimers (32.6 ± 1.0% HMW, 42.2 ± 4.0% MMW, 25.2 ± 3.0% LMW) as previously reported. Although the multimers in the type 1 patients are essentially normal, quantitative analysis reveals subtle abnormalities in structure. In type 2B VWD patients with mutations including V1316M, R1306W, and R1341W, a loss of HMW and MMW multimers was observed (7.1 ± 3.2% HMW, 40.4 ± 8.3% MMW, and 52.5 ± 11.4% LMW). A greater loss of HMW and MMW multimers was observed in patients with type 2A VWD with mutations including Y1349C, R1597W, G1609R, I1628T, G1631D, and G1670S (3.5 ± 6.2% HMW, 19.7 ± 20.4% MMW, and 76.9 ± 26.3% LMW). The type 2A subjects consisted of two groups: those with a virtually complete loss of HMW and MMW (0.0 ± 0% HMW, 4.0 ± 1.0% MMW, and 96.0 ± 1.0% LMW), and those with loss of HMW and decreased MMW (8.7 ± 7.5% HMW, 41.0 ± 14.7% MMW, and 50.3 ± 20.9% LMW). The latter group had a similar multimer distribution to that of type 2B VWD subjects. While most type 2A patients with mutations associated with increased susceptibility to ADAMTS13 proteolysis had severe multimer abnormalities (>95% LMW), some had only moderate abnormalities. Our study demonstrates that quantitative analysis of VWF multimer patterns more clearly distinguishes patients with various subtypes of VWD than subjective analysis. Although one of the two groups of type 2A patients is similar to the type 2B group, the other group is clearly different and is associated with specific genotypes, perhaps eliminating the need for DNA sequence analysis to make a definitive diagnosis for this group. This technique provides an objective measure of VWF structure to better characterize subtle changes observed in the subtypes of VWD and may help to determine the nature of any additional clinical laboratory testing to reach a clear-cut diagnosis. Disclosures: No relevant conflicts of interest to declare.

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