Survival Of Von Willebrand Factor Research Articles

Type 2A and 2M von Willebrand Disease: Differences in Phenotypic Parameters According to the Affected Domain by Disease-Causing Variants and Assessment of Pathophysiological Mechanisms.

Type 2A and 2M von Willebrand disease (VWD) broadly show similar phenotypic parameters, but involve different pathophysiological mechanisms. This report presents the clinical and laboratory profiles of type 2A and type 2M patients genotypically diagnosed at one large center. Higher bleeding score values and a higher incidence of major bleeding episodes were observed in type 2A compared with type 2M, potentially reflective of the absence of large and intermediate von Willebrand factor (VWF) multimers in 2A. In type 2A, most of disease-causing variants (DCVs) appeared to be responsible for increased VWF clearance and DCV clustered in the VWF-A1 domain resulted in more severe clinical profiles. In type 2M, DCV in the VWF-A1 domain showed different laboratory patterns, related to either reduced synthesis or shortened VWF survival, and DCV in the VWF-A2 domain showed patterns related mainly to shortened survival. VWF-type 1 collagen binding/Ag (C1B/Ag) showed different patterns according to DCV location: in type 2A VWD, C1B/Ag was much lower when DCVs were located in the VWF-A2 domain. In type 2M with DCV in the VWF-A1domain, C1B/Ag was normal, but with DCV in the VWF-A2 domain, C1B/Ag was low. The higher frequency of major bleeding in VWD 2M patients with DCV in the VWF-A2 domain than that with DCV in the VWF-A1 domain could be a summative effect of abnormal C1B/Ag, on top of the reduced VWF-GPIb binding. In silico modeling suggests that DCV impairing the VWF-A2 domain somehow modulates collagen binding to the VWF-A3 domain. Concomitant normal FVIII:C/Ag and VWFpp/Ag, mainly in type 2M VWD, suggest that other nonidentified pathophysiological mechanisms, neither related to synthesis/retention nor survival of VWF, would be responsible for the presenting phenotype.

Von Willebrand disease type Vicenza: In search of a classification for the archetype of reduced von Willebrand factor survival.

Type Vicenza von Willebrand disease (VWD) features a von Willebrand factor (VWF) with a very short half‐life, and is classified as a form of type 1 VWD. To test the appropriateness of type Vicenza VWD classification, the main features of 17 patients from eight unrelated families were analysed. They had low VWF antigen levels and function (always below 20 U/dl); ristocetin‐induced platelet aggregation sometimes normal, sometimes reduced/absent (even in the same patient); normal platelet VWF levels; an increased VWF propeptide to VWF antigen ratio (8.74 ± 1.65 vs. normal 1.04 ± 0.28) and a reduced VWF half‐life. Plasma VWF multimer levels were homogeneously reduced, and unusually large VWF multimers were sometimes present. Recombinant p.R1205H VWF showed a normal synthesis, release, function, and multimer pattern, with no ultra‐large VWF multimers. The mathematical model by Galvanin et al. was used to explore the kinetic changes in VWF after DDAVP. It showed that the release, but especially the proteolysis (k proteol 1.0−3 ± 2.5−3 vs. normal 4.5−4 ± 6.4−4) and elimination (k el 1.0−2 ± 5.2−3 vs. normal 1.1−3 ± 6.8−4) of type Vicenza VWF were significantly higher than normal. The increased elimination is consistent with the short half‐life, while the increased proteolysis was unexpected. As a shorter survival of VWF is wholly responsible for the type Vicenza VWD phenotype (VWF synthesis, structure and function are normal), it might be better to classify it as a type 2 VWD (rather than type 1) to emphasise the greater interaction with clearance receptors as a new VWF functional defect.

Von Willebrand factor propeptide to antigen ratio identifies platelet activation and reduced von Willebrand factor survival phenotype in mice

Background Reduced plasma survival of von Willebrand factor (VWF) is characteristic of patients with type 1C von Willebrand disease (VWD). These subjects can be identified by an increased steady-state ratio of plasma VWF propeptide (VWFpp) to VWF antigen (VWF:Ag). A similar phenotype occurs in mice with the Mvwf1 allele. Objectives To (i) determine if the VWFpp/VWF:Ag ratio can be used to identify a 'type 1C' phenotype in mice, (ii) determine the most reliable method for murine blood sampling, and (iii) identify the source of VWF released during problematic blood collection. Methods 'Platelet-VWF' and 'endothelial-VWF' mice were generated by bone marrow transplantation between C57BL/6J and VWF-/- mice. Several blood sampling methods were used and murine VWFpp and VWF:Ag levels determined. Plasma and platelet VWF:Ag and VWFpp, VWF multimers and VWF half-life were examined in mouse strains with and without Mvwf1. Results A single retro-orbital bleed and vena cava collection were found to be the optimal methods of blood collection. Problematic collection resulted in release of VWF from platelets and endothelium. The VWFpp/VWF:Ag ratio identified strains of mice with reduced VWF survival. Conclusion Assay of murine VWFpp and VWF:Ag has utility in determining the acceptability of murine blood samples for coagulation testing and in identification of a reduced VWF survival phenotype in mice.

Von Willebrand factor arginine 1205 substitution results in accelerated macrophage‐dependent clearance in vivo

Enhanced von Willebrand factor (VWF) clearance is important in the etiology of type 1 and type 2 von Willebrand disease (VWD). More than 20 different VWF point mutations have already been reported in patients with enhanced clearance. These include the VWD-Vicenza variant, which is characterized by an Arg1205His substitution in the VWF D3 domain. Critically, however, the molecular mechanisms through which single amino acid substitutions in VWF result in enhanced clearance of this complex multimeric glycoprotein have not been defined. In this study, we have investigated the biological basis underlying the enhanced clearance of the VWF-R1205H variant. Using VWF(-/-) mice, in vivo clearance rates were determined for a series of full-length and truncated recombinant VWF variants. In addition, the role of macrophages in modulating enhanced VWD-Vicenza clearance was investigated using clodronate liposome administration. Our findings demonstrate that substitutions of R1205 with histidine, cysteine or serine all result in markedly reduced survival of full-length recombinant VWF. Importantly, D'A3 fragments containing these same R1205 substitutions also demonstrated significantly enhanced clearance. In contrast to the reduced in vivo survival observed with R1205H, clearance of R1204H was not enhanced. Recent studies have demonstrated that hepatic and splenic macrophages play key roles in regulating VWF clearance. Importantly, macrophage-depletion also served to markedly attenuate the enhanced clearance phenotypes associated with VWF-R1205H, VWF-R1205S and VWF-R1205C. Collectively, these novel findings demonstrate a specific and critical role for the R1205 residue in modulating macrophage-mediated clearance of VWF in vivo.

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

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.

Type 1 von Willebrand disease due to reduced von Willebrand factor synthesis and/or survival: observations from a case series

It may be difficult to diagnose type 1 von Willebrand disease (VWD) because of its heterogeneous and sometimes elusive nature. To evaluate the contribution of a shorter von Willebrand factor (VWF) survival in modulating VWD phenotype, the VWF half-life was assessed in 45 type 1 VWD patients using a 24-h 1-desamino-8-d-arginine vasopressin (DDAVP) test. A shorter VWF survival was observed in patients with C1130F mutations (T(1/2) elimination [T(1/2)el]=4.6+/-1.0h vs normal=15.8+/-2.3h, P<0.0001), in those with other missense mutations investigated (T(1/2)el=9.5+/-0.9h, P<0.02), and in patients not carrying VWF mutations (T(1/2)el=7.0+/-0.7h, P<0.001); the decrease mainly depended on a greater VWF clearance. VWF survival and clearance were normal in patients who carried nonsense mutations. The VWF-propeptide-to-VWF-antigen (VWF:Ag) ratio (VWFpp ratio) was higher in patients with a shorter VWF survival, and the values were inversely correlated with the VWF half-life (P<0.01). The response of VWF to DDAVP administration, which is useful to explore the synthesis and storage of VWF, was normal in patients with no mutations, whereas it decreased in patients with missense and nonsense mutations. Three scenarios, thus, are recognizable in type 1 VWD; one is associated mainly with a shorter survival of VWF, another is associated with its reduced synthesis and release, and a third is characterized by a combination of the two. The shorter VWF half-life found in patients with no VWF mutations suggests that mechanisms other than VWF might be involved in the pathogenesis of type 1 VWD.

An Assay for the Discrimination of Increased VWF Clearance in VWD.

Abstract 3478Poster Board III-415The quantitative deficiency in von Willebrand Factor (VWF) levels observed in Type 1 VWD can be caused by ineffective synthesis and storage or by a decrease in the half-life of the VWF in the circulation. To date, a number of different point mutations in VWF have been shown to cause a reduced VWF half-life. Clinically it is important to recognize this enhanced clearance phenotype because the increased clearance of VWF can reduce the efficacy of desmopressin treatment in these patients. The VWF propeptide is synthesized as part of a pro-VWF protein and is subsequently cleaved, stored and secreted in an equi-molar ratio with mature VWF. The level of VWF propeptide in the circulation can be used as a marker of VWF synthesis. In individuals with low VWF synthesis, the propeptide level is similarly decreased yielding a propeptide: VWF ratio near 1.0. In individuals with normal levels of VWF synthesis and decreased survival of VWF in circulation, an increased propeptide:VWF ratio is observed.GTI Diagnostics, Inc. (Waukesha, Wisconsin) has developed a fluorescent ELISA for the quantitative measurement of VWF levels and VWF propeptide levels in plasma and for the calculation of a propeptide:VWF ratio. All reagents necessary to run the assay are included in the kit as well as an analysis workbook for easy calculation of results. Assay incubation steps are only 15 minutes, therefore the assay can be completed in 90 minutes. In performance testing the VWF & Propeptide Assay showed excellent within-run, between-run and total imprecision. The limit of detection is 0.02 IU/dL for VWF and 0.02 U/dL for propeptide. The assay range varies depending on the calibrator stock included in the kit however the assay range is at least 1 – 273 IU/dL VWF or U/dL propeptide. No patient sample conditions tested were shown to interfere with the assay.Clinical studies were conducted to evaluate if the VWF & Propeptide Assay can be used to distinguish Type 1 VWD patients with mutations known to cause an increased VWF clearance phenotype (Type 1C) from those without these mutations. One hundred-fifteen Type 1 VWD patients diagnosed on the basis of VWF antigen level, ristocetin co-factor activity, and past bleeding history were tested and 24 Type 1 VWD patients with increased clearance of VWF (Type 1C) diagnosed on the basis of VWF antigen level, ristocetin co-factor activity, past bleeding history, and the presence of a point mutation previously shown to cause increased clearance of VWF. Patients with the following increased clearance mutations were included in the study: R1205H, S2179F, and W1144G (although the majority of the samples contained the R1205H mutation). Using the VWF & Propeptide Assay VWF levels, propeptide levels, and propeptide:VWF ratios were determined for each patient sample. The propeptide:VWF ratios were used to determine an appropriate diagnostic cutoff by receiver operating characteristics (ROC) curve analysis. From the ROC analysis, a propeptide:VWF ratio cutoff value of ≥3.3 provided optimal clinical sensitivity and specificity when distinguishing between Type 1 VWD and Type 1 VWD with increased VWF clearance (Type 1C). Using the ratio cutoff of ≥3.3 yielded 100.0% sensitivity, characterizing all known Type 1C patients correctly and yielded 97.4% specificity, where 3 Type 1 patients were characterized as Type 1C. Two of the 3 mischaracterized patients had Type O blood and the blood group of the third sample was unknown. Since it has been demonstrated that patients with a Type O blood generally have a lower VWF level and correspondingly a slightly elevated propeptide:VWF ratio, we suggest the use of the following grey zone. Propeptide:VWF ratios of 3.3 – 4.1 may be due to increased VWF clearance or the result of a Type 1 VWD patient with Type O blood. Ratios of ≥ 4.2 are indicative of Type 1C VWD. Disclosures:Stapleton:GTI Diagnostics: Employment. Ladvienka:GTI Diagnostics: Employment. Wuitschick:GTI Diagnostics: Employment.

Models for Prediction of Factor VIII Half-Life in Severe Haemophiliacs: Distinct Approaches for Blood Group O and Non-O Patients

BackgroundVon Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life.MethodologyStandardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15–44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated.Principal FindingsFVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5±2.6 h versus 14.3±3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%).ConclusionOur approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se.

Identification of type 1 von Willebrand disease patients with reduced von Willebrand factor survival by assay of the VWF propeptide in the European study: Molecular and Clinical Markers for the Diagnosis and Management of Type 1 VWD (MCMDM-1VWD)

AbstractThe decreased survival of von Willebrand factor (VWF) in plasma has been implicated as a mechanism in a subset of type 1 von Willebrand disease (VWD) patients. We have previously reported that the ratio of plasma levels of VWF and its propeptide (VWFpp) can be used to identify patients with reduced VWF survival. In this study, we report the assay of VWFpp and VWF:Ag in 19 individuals recruited from 6 European centers within the MCMDM-1VWD study. Eight individuals had a VWF:Ag level less than 30 IU/dL. Seven of these patients had a robust desmopressin response and significantly reduced VWF half-life that was predicted by a markedly increased steady-state plasma VWFpp/VWF:Ag ratio. VWF mutations previously associated with reduced VWF survival were identified in each of the 7 individuals. Thus, a substantially increased ratio of steady-state VWFpp/VWF:Ag predicted a reduced VWF half-life in patients with markedly decreased VWF:Ag levels. These data indicate that a reduced VWF survival is found in a subpopulation of patients with type 1 VWD. The systematic assay of both plasma VWF and the VWF propeptide in moderately severe type 1 VWD patients may identify patients with a reduced VWF survival phenotype.

Survival of von Willebrand factor released following DDAVP in a type 1 von Willebrand disease cohort: Influence of glycosylation, proteolysis and gene mutations

Reduced plasma survival of von Willebrand factor (VWF) may contribute towards the pathogenesis of type 1 von Willebrand disease (VWD). However, little is known about mechanism(s) of VWF clearance and factors that may affect it. The half-life of VWF-related parameters following the administration of DDAVP was measured in 26 patients with type 1 VWD and 10 haemophilia A controls. Binding of lectins Ricinus communis (RCA-I) and Erythina crystagalli (ECA) agglutinins to VWF and VWF susceptibility to ADAMTS-13-mediated proteolysis were investigated. Sequence analysis of targeted regions of the VWF gene was performed to inspect for mutations that have been associated with increased clearance. Post-DDAVP clearance of VWF was increased approximately three-fold in the type 1 VWD cohort overall. However this was not shown to consistently associate with steady-state VWF antigen (VWF:Ag) levels. Furthermore, increased VWF clearance was not consistently associated with increased ratios of VWF propeptide (VWFpp) to VWF:Ag indicating that a normal ratio does not necessarily reflect normal post-DDAVP survival in type 1 VWD patients. RCA-I and ECA binding to VWF were increased in type 1 VWD patients and, although inversely correlated with VWF levels, this was independent of VWF clearance. There was no association between VWF clearance and ADAMTS-13-mediated proteolysis. Three novel candidate mutations with an increased clearance phenotype were identified. The data are consistent with heterogeneity in pathogenic mechanisms in type 1 VWD and are consistent with type 1 VWD representing a complex genetic trait.

Clearance of von Willebrand factor

The life cycle of von Willebrand factor (VWF) comprises a number of distinct steps, ranging from the controlled expression of the VWF gene in endothelial cells and megakaryocytes to the removal of VWF from the circulation. The various aspects of VWF clearance have been the objects of intense research in the last few years, stimulated by observations that VWF clearance is a relatively common component of the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, improving the survival of VWF is now considered as a viable therapeutic strategy to prolong the half-life of factor VIII in order to optimise treatment of haemophilia A. The present review aims to provide an overview of recent findings with regard to the molecular basis of VWF clearance. A number of parameters have been identified that influence VWF clearance, including its glycosylation profile and a number of VWF missense mutations. In addition, in-vivo studies have been used to identify cells that contribute to the catabolism of VWF, providing a starting point for the identification of receptors that mediate the cellular uptake of VWF. Finally, we discuss recent data describing chemically modification of VWF as an approach to prolong the half-life of the VWF/FVIII complex.

The Prevalence of Reduced VWF Survival Phenotype (Type 1C VWD) in Type 1 Index Cases Recruited into the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB - VWD).

The decreased survival of von Willebrand factor (VWF) in plasma has recently been identified as a novel mechanism causing type 1 VWD. Patients with this phenotype are characterized by a substantially reduced plasma VWF:Ag (< 30 IU/dL), a robust response to DDAVP, and a reduced half-life of VWF:Ag after DDAVP administration. Although a normal distribution of plasma VWF multimers appears to be present, the multimer satellite bands are decreased in intensity. A few mutations have been identified in type 1C VWD patients including C1130F/G/R, W1144G, R1205H, and S2179F. Identification of type 1 VWD patients with this phenotype is clinically important because DDAVP may not be the treatment of choice in this subset of patients due to the reduced half-life of the VWF released into plasma. Although previous studies have facilitated the characterization of this phenotype, the prevalence of the reduced survival, type 1C VWD has not yet been determined. We have previously reported that assay of plasma VWF:Ag and VWF propeptide (VWFpp) can be used to identify type 1C VWD patients having reduced VWF survival. These individuals have a substantially increased ratio of steady-state plasma VWFpp/VWF:Ag in contrast to normal individuals where the ratio is equal to one. We subsequently established blood group-specific normal ranges for VWFpp/VWF:Ag by assay of 279 healthy blood donors. Here we report the assay of VWF:Ag and VWFpp in 192 normal controls and 61 type 1 VWD index cases recruited from eight US centers within the TS Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB - VWD) study. VWD type 1C individuals previously reported from our center were excluded from the current study. Using VWF:Ag and VWFpp levels determined for the 192 normal controls, we established a 2-standard deviation normal range for VWFpp/VWF:Ag ratio of 0.5 to 1.6, consistent with our previous studies. Of the 61 type 1 VWD index cases, three individuals were identified with a substantially increased VWFpp/VWF:Ag ratio (mean = 5.0). One individual was found to have the well-characterized R1205H VWF gene mutation that has been associated with reduced VWF survival. These individuals demonstrated laboratory parameters consistent with a reduced VWF survival phenotype including moderate to severely decreased plasma VWF:Ag (mean = 16.6 IU/dL), a diminished intensity of multimer satellite bands amongst an essentially normal distribution of plasma multimers, and an elevated bleeding score (mean = 4.7). Together these data indicate a reduced survival phenotype (type 1C) in 4.9% of type 1 VWD index cases in the ZPMCB - VWD study. Identification of type 1 VWD individuals with a reduced plasma VWF survival phenotype is clinically essential as these individuals may require alternative treatment strategies.

Clearance mechanisms of von Willebrand factor and factor VIII.

Clearance mechanisms of von Willebrand factor and factor VIII.

Assay of the von Willebrand factor (VWF) propeptide to identify patients with type 1 von Willebrand disease with decreased VWF survival

AbstractType 1 von Willebrand disease (VWD) is characterized by a partial quantitative deficiency of von Willebrand factor (VWF). Few VWF gene mutations have been identified that cause dominant type 1 VWD. The decreased survival of VWF in plasma has recently been identified as a novel mechanism for type 1 VWD. We report 4 families with moderately severe type 1 VWD characterized by low plasma VWF:Ag and FVIII:C levels, proportionately low VWF:RCo, and dominant inheritance. A decreased survival of VWF in affected individuals was identified with VWF half-lives of 1 to 3 hours, whereas the half-life of VWF propeptide (VWFpp) was normal. DNA sequencing revealed a single (heterozygous) VWF mutation in affected individuals, S2179F in 2 families, and W1144G in 2 families, neither of which has been previously reported. We show that the ratio of steady-state plasma VWFpp to VWF:Ag can be used to identify patients with a shortened VWF half-life. An increased ratio distinguished affected from unaffected individuals in all families. A significantly increased VWFpp/VWF:Ag ratio together with reduced VWF:Ag may indicate the presence of a true genetic defect and decreased VWF survival phenotype. This phenotype may require an altered clinical therapeutic approach, and we propose to refer to this phenotype as type-1C VWD.

Cysteine-mutations in von Willebrand factor associated with increased clearance

von Willebrand disease (VWD) is a bleeding disorder caused by the decrease of functional von Willebrand factor (VWF). Low levels of VWF can result from decreased synthesis, impaired secretion, increased clearance or combinations thereof. Several mutations lead to impaired synthesis or secretion of VWF, however, little is known about the survival of VWF in the circulation. To evaluate the effect of several VWF mutations on VWF clearance. The effect of three cysteine-mutations (C1130F, C1149R or C2671Y) on the in vivo survival of VWF was studied in patients carrying these mutations and in a VWF-deficient mice model. In patients carrying these mutations, we observed increased propeptide/mature VWF ratios and rapid disappearance of VWF from the circulation after desmopressin treatment. Detailed analysis of in vivo clearance of recombinant VWF in a VWF-deficient mice model revealed a fourfold increased clearance rate of the mutants. The mutations C1130F, C1149R and C2671Y are each associated with reduced survival of VWF in the circulation. Detailed analysis of the recombinant mutant VWF demonstrated that increased clearance was not due to increased proteolysis by ADAMTS-13. We did not identify functional or structural characteristics that the mutant proteins have in common and could be associated with the phenomenon of increased clearance. Cysteine-mutations in VWF may result in reduced in vivo survival. The observation that various mutations are associated with increased in vivo clearance may have major implications for the therapeutic strategies that rely on the rise of endogenous VWF after desmopressin administration.

An Experimental Model to Study the in Vivo Survival of von Willebrand Factor: BASIC ASPECTS AND APPLICATION TO THE R1205H MUTATION

To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t((1/2))alpha = 13 min) and a slow secondary phase (t((1/2))beta = 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of full-length VWF to truncated variants. Deletion of both the amino-terminal D'-D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D'-D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT = 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT = 4.5 h, p < 0.02). These results point to a role of the D'-D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT = 0.3 h; p = 0.004). Therefore, this mutation seems to abrogate the protective effect of the D'-D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide.

Biochemistry and genetics of von Willebrand factor.

Von Willebrand factor (VWF) is a blood glycoprotein that is required for normal hemostasis, and deficiency of VWF, or von Willebrand disease (VWD), is the most common inherited bleeding disorder. VWF mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet membrane glycoproteins and to constituents of exposed connective tissue. These activities appear to be regulated by allosteric mechanisms and possibly by hydrodynamic shear forces. VWF also is a carrier protein for blood clotting factor VIII, and this interaction is required for normal factor VIII survival in the circulation. VWF is assembled from identical approximately 250 kDa subunits into disulfide-linked multimers that may be > 20,000 kDa. Mutations in VWD can disrupt this complex biosynthetic process at several steps to impair the assembly, intracellular targeting, or secretion of VWF multimers. Other VWD mutations impair the survival of VWF in plasma or the function of specific ligand binding sites. This growing body of information about VWF synthesis, structure, and function has allowed the reclassification of VWD based upon distinct pathophysiologic mechanisms that appear to correlate with clinical symptoms and the response to therapy.