Upstream Mechanisms Causing Type 1C Von Willebrand Disease (VWD): Contribution Of Defective Von Willebrand Factor (VWF) Multimerization, Regulated Storage, and Secretion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3571-3571
Author(s):  
Sandra L. Haberichter ◽  
David A Jakab ◽  
Paula M. Jacobi
Keyword(s):  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Essentially Normal ◽  
Intracellular Processing ◽  
Clinical Biology ◽  
And Function ◽  
Von Willebrand ◽  
Index Cases

Abstract One mechanism causing type 1 VWD is the reduced survival of VWF in plasma (type 1C VWD), characterized by markedly decreased VWF:Ag and VWF half-life, essentially normal multimers, increased ratio of VWF propeptide (VWFpp) to VWF:Ag, robust response to DDAVP, and normal ratios of VWF:CB, FVIII, or VWF:RCo to VWF:Ag. We enrolled 502 index cases with a pre-existing diagnosis of type 1 VWD through the Zimmerman Program for the Molecular and Clinical Biology of VWD. We confirmed 262 of the index cases as type 1 VWD (VWF:Ag or VWF:RCo ≤ 40 IU/dL). Of these, 58 met the criteria for type 1C VWD with VWFpp/VWF:Ag ≥ 3 and VWF:Ag ≤ 30 IU/dL. Sequence variations were identified in the VWF D3, A1, A2, and D4 domains. Little is known regarding the mechanisms causing type 1C VWD, but it has been assumed that VWF undergoes normal intracellular processing and secretion with rapid clearance upon release into plasma. We hypothesized that defective intracellular processing may contribute to the type 1C phenotype. We studied 10 type 1C variants including C1130Y, W1144G, R1205H, N1231S, R1315C, V1411E, R1527W, N2041S, Y2160C, and S2179F. Variants were expressed alone (homozygously) or with wild-type (WT) VWF (heterozygously) in HEK293T cells and VWF secretion, multimer structure, and binding to collagen (types III and VI), GPIb-alpha, and FVIII was analyzed. To assess regulated storage, variants were expressed homozygously in HEK293 cells where WT VWF forms elongated pseudo-Weibel-Palade bodies (pWPB). Five variants (C1130Y, R1315C, V1411E, N2041S, Y2160C) had severely decreased secretion and defective multimerization when homozygously expressed. These variants did not form pWPB, but appeared to co-localize with the endoplasmic reticulum, consistent with the severely impaired secretion. One variant, W1144G, had mildly reduced secretion, formed only dimeric VWF, and unexpectedly did not form pWPB. These multimer defective variants demonstrated decreased collagen binding and GPIb-alpha binding as would be predicted. The remaining variants (R1205H, N1231S, R1527W, S2179F) were normally secreted, multimerized, stored in pWPB, and had normal binding to FVIII, collagen, and GPIb-alpha. Interestingly, FVIII binding to homozygous VWF D3 variants C1130Y and W1144G was substantially reduced. This result is not entirely unexpected as the FVIII binding region in VWF has been mapped to the D’-D3 region. Co-expression with WT VWF essentially corrected defective secretion, although some variants still had moderately reduced secretion. Multimer structure appeared normal for all heterozygous variants, although staining which discriminates between variant and WT alleles revealed that for some variants, little variant VWF was actually expressed when transfected at a 1:1 ratio with WT. In sum, when variants were homozygously expressed, we observed a constellation of processing and functional defects. Only R1205H, N1231S, R1527W, and S2179F variants demonstrated normal processing and function. Heterozygous expression (consistent with patients) corrected most of the observed defects, although reduced secretion persisted for a subset of variants. We can conclude that while reduced plasma survival of VWF is a major determinant of the type 1C phenotype, additional upstream processing defects may contribute to the severity of the overall VWD phenotype. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Critical Importance Of VWF Propeptide (VWFpp) In The Diagnosis Of Type 1 Von Willebrand Disease (VWD)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 331-331 ◽  
Author(s):  
Sandra L. Haberichter ◽  
Pamela A. Christopherson ◽  
Veronica H. Flood ◽  
Joan Cox Gill ◽  
Daniel B. Bellissimo ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Promoter Polymorphisms ◽  
Normal Platelet ◽  
Bleeding Score ◽  
Clinical Biology ◽  
Type 3 ◽  
Severe Type ◽  
Index Cases

Abstract Type 1 VWD is the most common form of VWD and is characterized by quantitative deficiency of VWF. Mechanisms causing type 1 VWD include decreased VWF synthesis due to promoter polymorphisms, decreased VWF secretion with intracellular retention/degradation, and increased clearance of VWF from plasma (type 1C VWD). VWF and its propeptide (VWFpp) are released into plasma in equimolar amounts but have very different half-lives (8 – 12 hours and 2-3 hours, respectively). The assay of VWFpp can be used to assess VWF synthesis, secretion, and clearance. Reduced VWFpp level indicates reduced VWF synthesis or secretion. An increased VWFpp/VWF:Ag ratio is expected when VWF is cleared rapidly from plasma (type 1C VWD), while decreased VWF secretion/intracellular retention results in a normal ratio. We enrolled 502 type 1 VWD index cases with a pre-existing diagnosis of type 1 VWD through the Zimmerman Program for the Molecular and Clinical Biology of VWD. We confirmed 262 index cases as type 1 VWD (VWF:Ag or VWF:RCo ≤ 40 IU/dL). Of the confirmed type 1 VWD cases, 58 met the criteria for type 1C VWD with VWFpp/VWF:Ag ≥ 3 and VWF:Ag ≤ 30 IU/dL, and 12 met the criteria for “Type 1 – Severe” with VWF:Ag of 1 – 5 IU/dL and VWFpp/VWF:Ag < 3. Type 1C VWD comprised 22% of all type 1 VWD cases. The type 1C cohort included several individuals previously diagnosed as type 2A (11), type 2M (2), and “Unclassified” (3). The most significant reclassification involved 7 cases previously diagnosed as type 3 VWD – these patient had detectable VWF:Ag (2 – 6 IU/dL) and VWFpp (14 – 66 IU/dL), and elevated VWFpp/VWF:Ag (4.2 – 33.0). Although plasma VWF:Ag is very low in these patients, they might be expected to have normal platelet stores of VWF:Ag, unlike type 3 VWD patients. Type 3 VWD patients in our study had undetectable (<1 IU/dL) VWFpp and VWF:Ag. In fact, type 1C VWD comprised the majority of severe type 1 VWD cases. At very low VWF:Ag levels (2 – 10 IU/dL), 76% of index cases had a type 1C phenotype, while the remainder had normal VWFpp/VWF:Ag consistent with a decreased secretion phenotype. In the VWF:Ag of 11 – 20 IU/dL group, 38% were type 1C. At still higher VWF:Ag levels (21 – 30 IU/dL), only 7% had a type 1C phenotype and the majority had a decreased secretion phenotype. Additionally, 43% of the type 1C cohort had VWFpp levels below the normal range, indicating reduced synthesis/secretion is also involved in the mechanism causing type 1C VWD. Mutations associated with type 1C were located in the VWF D3, A1, and D4 domains while decreased secretion variants were located primarily in the D1, D2, and D3 domains. Somewhat surprisingly, no difference in bleeding score was identified between type 1C and other type 1 patients. We can conclude that a reduced VWF survival phenotype (type 1C) is very frequent in severe type 1 VWD patients with VWF:Ag < 20 IU/dL. Identification of type 1C VWD is important because the rapid clearance of VWF in these patients may preclude the use of desmopressin, which is commonly given in type 1 VWD, but may be insufficient to achieve hemostasis in type 1C patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Collagen Binding Provides a Sensitive Screen for Variant von Willebrand Disease

2013 ◽  
Vol 59 (4) ◽  
pp. 684-691 ◽  
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 &lt; 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.

Novel VWF Sequence Variations Identified in Normal Controls and Index Cases Enrolled in the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 709-709 ◽  
Author(s):  
Daniel B. Bellissimo ◽  
P.A. Christopherson ◽  
S.L. Haberichter ◽  
V.H. Flood ◽  
J.C. Gill ◽  
...  
Keyword(s):  
Normal Control ◽  
Normal Control Group ◽  
Control Group ◽  
Normal Individuals ◽  
Sequence Variations ◽  
Clinical Biology ◽  
Von Willebrand ◽  
Index Cases ◽  
Normal Controls

Abstract Von Willebrand disease (VWD) is caused by quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). The TS Zimmerman Program for the Molecular and Clinical Biology of VWD is a multinational Program Project established to further the study of VWD in the United States and to contrast these studies with the studies initiated previously in the EU and Canada. As one of the components of this study we sought further insight into the clinical expression and penetrance of established types of VWD by performing full gene DNA sequence analysis in VWD patients and normal controls. This report is an interim report of the first 50 index cases and 113 normal individuals recruited into this study. Twenty four of these index cases were found to have known mutations, four of which had a second new mutation, and 11 cases had 1 or 2 new mutations. In cases where mutations were identified, 46% of the identified mutations were new mutations that have not been reported in the Sheffield VWF Mutation Database. In 15 patients, no mutations were identified in the coding region, although analysis of the non-coding regions is still in progress. Five of the mutations were deletions, insertions, or nonsense mutations that have clear functional consequences. The other 12 mutations were missense mutations. Since VWF polymorphisms are not well characterized in all exons, we have also completed studies of the first 113 normal control individuals in our study. These are individuals without a bleeding history and in whom full VWF laboratory testing and VWF sequencing was also undertaken. Since some estimates in the EU and Canadian studies have determined the prevalence of VWF mutations varies by the severity of type 1 VWD patients, we wanted to determine the frequency of VWF variation in a normal population and determine if sequence variations correlate with VWF levels. There were three linked common polymorphims identified in normal African Americans that are discussed elsewhere and are not included in this present analysis. We found 19 new sequence variations in the normal control group of which three (2900G>A, 6554G>A, 7997C>T) were found individually in 4–6% of the normal control samples. In addition, in 12 normal control samples we identified 6 sequence variations that were previously reported as VWF mutations. Four were reported as type 1 mutations (2220 G>A, 3686T>G, 3692A>C, 6859C>T) and two as type 2N mutations (2451T>A, 2771G>A). The 2220G>A and 2451T>A mutations were seen in 6 normal controls (5%) and 5 of these 6 normal controls had both mutations. In another normal control, both 3686T>G and 3692A>C were identified. Although the reported prevalence of VWD is 1% or greater, the frequency of these mutations in our normal controls is higher than expected (as high as 5%). In our normal control group, the mean VWF:Ag concentration in the patients with polymorphisms/mutations did not differ from the normal control group as a whole and did not cluster on the lower end of the normal range. Thus, the data on our normal individuals suggest that VWF gene variation is considerable and that many mutations and polymorphisms remain to be identified. Differentiation of those that affect the diagnosis of VWD and/or hemorrhagic risk continues to be difficult.

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.

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.

Spectrum of Type 2 Von Willebrand Disease in the Zimmerman Program

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 472-472 ◽  
Author(s):  
Veronica H Flood ◽  
Pamela A Christopherson ◽  
Daniel B Bellissimo ◽  
Joan Cox Gill ◽  
Sandra L Haberichter ◽  
...  
Keyword(s):  
Sequence Variation ◽  
Von Willebrand Disease ◽  
Sequence Variations ◽  
Platelet Binding ◽  
The Mean ◽  
Bleeding Score ◽  
Von Willebrand ◽  
Index Cases

Abstract While von Willebrand disease (VWD) is the most common inherited bleeding disorder, most patients have quantitative defects in von Willebrand factor (VWF). The qualitative variants, collectively termed type 2 VWD, are less common, but also in general more severe than type 1 VWD. However, despite a common laboratory phenotype of decreased VWF:RCo/VWF:Ag ratio for types 2A, 2B, and 2M VWD, the clinical phenotype is highly variable. We examined index cases and affected family members enrolled in the Zimmerman Program with a phenotypic diagnosis of type 2 VWD. All subjects had factor VIII (FVIII), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and multimer distribution analyzed in a central laboratory. For calculation of mean VWF:RCo values, a level of 5 was assigned to subjects with VWF:RCo below the laboratory lower limit of detection of 10 IU/dL. A platelet binding assay was also performed using a gain of function GPIb containing 2 mutations that enable spontaneous binding to VWF in the absence of ristocetin (VWF:GPIbM). Full length 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 (BS). Mean FVIII, VWF:Ag, VWF:RCo, and BS are listed in the table below for each type 2 variant. For type 2A VWD, 113 subjects have been enrolled to date. All had an abnormal multimer distribution with loss of high molecular weight multimers. 6 type 2A subjects had a VWF:RCo/VWF:Ag ratio of ≤0.7. The lowest VWF:RCo levels were seen in the type 2A cohort with 60% <10. 98% of type 2A subjects had an identified sequence variation on full length sequencing. 25% had the p.R1597W sequence variation and an additional 4 subjects had p.R1597Q. The mean bleeding score for the subjects with sequence variations at 1597 was 10.6. 11% of subjects had p.R1374H, which correlated with a higher mean bleeding score of 12.4. Mean bleeding score for the remainder of the type 2A subjects was lower, at 6.6, suggesting that differences in VWF genetics may account for differences in phenotype, despite the common type 2A laboratory presentation of reduced VWF:RCo and loss of high molecular weight multimers. 44 type 2B subjects have been enrolled to date, all with abnormal multimer distribution and either documented abnormal VWF-platelet binding or a presence of a known type 2B sequence variation. Sequence variations were found in 100% of subjects. The most common sequence variations were p.V1316M (20%), p.R1306W (18%), p.R1341Q (11%), and p.H1268Y (9%). Mean VWF:RCo/VWF:Ag ratios ranged from 0.32-1.12, suggesting that a normal VWF:RCo/VWF:Ag ratio cannot completely exclude the possibility of type 2B VWD. Most (94%) had increased VWF:GPIbM. Subjects with p.V1316M and p.R1306W/Q sequence variations had lower VWF:RCo compared to subjects with p.R1341Q/W but mean bleeding scores did not differ. 59 type 2M subjects have been enrolled to date. Mean VWF:RCo/VWF:Ag ratio was 0.46 (range 0.14-0.7). Sequence variations were found in 93% of subjects. R1374C was found in 13 members from one family. While mean VWF levels were similar to the entire 2M group, a wide range in VWF:Ag and VWF:RCo/VWF:Ag ratio was observed, accompanied by a corresponding range in BS from 0-8. This suggests that other modifiers of phenotype may be present aside from the VWF sequence variation. 11 type 2N subjects have been enrolled to date, all with low VWF binding to FVIII. Sequence variations were found in 100% of this cohort. R854Q was present in 89% of subjects. Bleeding scores were highest for homozygous 2N sequence variations. Overall, the mean BS for type 1 VWD subjects was 6.3, the mean BS for type 2 VWD subjects was 7.5, and the mean BS for type 3 VWD subjects was 16.8. Types 2A and 2N had higher bleeding scores on average than type 2B, and type 2M subjects had on average the lowest bleeding scores. Although heterogeneity was seen across all the type 2 variants, both laboratory testing and genetic testing are useful in categorizing and phenotyping type 2 VWD. Table. FVIII (mean) VWF:Ag (mean) VWF:RCo (mean) BS (mean) Type 2A 47 34 12 8.7 Type 2B 45 36 23 7.1 Type 2M 62 54 21 5.4 Type 2N 30 69 76 8.3 Disclosures Montgomery: Immucor: Patents & Royalties.

Quantitative Measurement of VWF Binding to the Platelet Integrin αIIbβ3 - Abnormalities Identified in Individuals with VWD

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1107-1107
Author(s):  
Robert R. Montgomery ◽  
Veronica H Flood ◽  
Jieqing Zhu ◽  
Pamela A Christopherson ◽  
Peter J. Newman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Conflicts Of Interest ◽  
Functional Screening ◽  
Integrin Αiibβ3 ◽  
Binding Ratio ◽  
Clinical Biology ◽  
The One ◽  
Index Cases ◽  
Normal Controls

Abstract Most studies of VWF function have focused on VWF interactions with platelet GPIb, FVIII, and specific sites and types of collagen. Early studies carried out by Ruggeri and Montgomery demonstrated that in addition to binding GPIb on platelets, VWF also bound to the GPIIb-IIIa receptor (also known as integrin αIIbβ3), but only in the absence of fibrinogen. Ruggeri and other groups have demonstrated that under flow, VWF interaction with GPIIb-IIIa is also critical to VWF-platelet biology. A large systematic study of VWF binding to GPIIb-IIIa (αIIbβ3) has not been undertaken previously in a cohort of type 1 and 2 VWD subjects. Studies were carried out on 619 VWD index cases (both type 1 and type 2 VWD) and 84 normal controls enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) using a novel assay of αIIbβ3 binding of VWF. Zhu and coworkers expressed the soluble αIIbβ3 headpiece and previously characterized this in the open position when bound to ligand. This αIIbβ3 headpiece was used for our studies. An antigen capture ELISA was developed using the αIIbβ3 headpiece to capture VWF and to compare binding to the VWF:Ag as a ratio of VWF-αIIbβ3/VWF:Ag. ELISA plates were coated with the GPIIIa-specific monoclonal antibody (MAb), AP3 and used to capture the αIIbβ3 headpiece. Since fibrinogen blocks VWF binding to αIIbβ3, plasma samples were heated at 56°C to precipitate fibrinogen, and then centrifuged to remove the precipitate. The resulting heat defibrinated plasma was utilized in the assay. Bound VWF was quantified using biotin-labeled AVW15, a MAb to VWF. The reference was the ISTH SSC Plasma Standard. As specific positive and negative controls, we used wild-type recombinant VWF containing the normal RGDS sequence at position 2507-2510, and mutant VWF containing the RGDE sequence at the same position. As an additional control, the monoclonal antibody 7E3, known to block aggregation by binding to β3 was used to block αIIbβ3/VWF interactions. While binding of wt-VWF to αIIbβ3 was robust, no binding of the RGDE mutant was observed (<1% of wt). 7E3 completely inhibited binding of VWF to αIIbβ3. Interestingly, two other MAbs known to block the binding of Fg to αIIbβ3, AP2 and 10E5, did not inhibit VWF binding. The 84 normal controls had a mean VWF-αIIbβ3/VWF:Ag binding ratio of 0.87 with a 5% and 95% confidence limit of 0.53-1.39. 152 type 1 VWD (VWF <30IU/dL) samples had a modestly reduced binding ratio of 0.62 (0.36-0.85) with a significance of p<0.0001. The binding ratios of 55 type 2A and 41 2B VWD samples were markedly reduced with a ratio of 0.293 (0.12-0.67) and 0.40 (0.23-0.81) both with a significance of 0.0001. Since both of these latter VWF variants have abnormal VWF multimers, we tested recombinant D-pro VWF (no VWF propeptide present) and Y87S mutant VWF (absent VWF multimerization). No binding to αIIbβ3 was observed with VWF from either of these constructs. Among our ZPMCB-VWD index cases, we also had individuals with low VWF (VWF:Ag 30-50 IU/dL) that had an intermediate reduction between the normal controls and type 1 subjects. Type 2N VWD samples demonstrated no abnormal binding to αIIbβ3 and type 2M subjects had minimal differences. While VWF binding to αIIbβ3 has been recognized previously, it has not been systematically studied in subjects with VWD. We identified a modest decrease in αIIbβ3 binding of VWF in type 1 VWD and in clinical subjects with low VWF. Furthermore, type 2A and 2B subjects have a much more profound reduction in αIIbβ3 binding and suggest an importance of normal multimeric VWF to the functional binding of VWF to αIIbβ3. Mutating the RGDS sequence in VWF, or adding 7E3 to block αIIbβ3, abrogate this VWF binding. To date we have not identified any of our enrolled ZPMCB-VWD subjects with mutation of the RGDS sequence or in the C4 domain of VWF, although mutations in this region recently have been reported by Legendre et al. at the ISTH2015 meeting. Such mutations are not identified with current VWF functional screening assays, but specific assays of αIIbβ3-VWF interaction, such as the one described here, can be used and suggest there are specific potential mutations and variants with abnormal multimers that have abnormal αIIbβ3 binding. The qualitative and quantitative assessment of VWF function continues to be complicated. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document