Mature Von Willebrand Factor Research Articles

Genotype/phenotype association in von Willebrand disease: is the glass half full or empty?

Since the original description of this disease in 1926, major advances have been made in our understanding of the pathogenetic mechanisms responsible for von Willebrand disease (VWD). We now recognize that this disease comprises a collection of diverse quantitative and qualitative abnormalities of the adhesive protein von Willebrand factor (VWF), the key protein involved in platelet adhesion, and the carrier protein for factor VIII (FVIII) in plasma. Since the mid-1970s there has been a growing appreciation of 'variant' forms of the disease and in the 20 years since the cloning of the VWF gene, much progress has been made in characterizing the molecular genetic pathology of type 3 VWD and the various type 2 variants, types 2A, 2B, 2M and 2N VWD. In all of these cases, mechanistic insights into VWF structure/function have been forthcoming. Most recently, preliminary results relating to the mutational landscape of type 1 disease have been published that highlight the complex pathogenic background of this mild/moderate quantitative trait.

Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia

Most cell types, including blood--and vascular cells, produce microparticles upon activation. Since cellular microparticles are known to be elevated in thromboembolic diseases, we hypothesized a role for microparticles in the pathogenesis of thrombosis in essential thrombocythemia. In plasma samples from 21 patients with essential thrombocythemia and ten healthy subjects, the levels and the cellular origin of microparticles were determined by flowcytometric analysis, while the microparticle-associated procoagulant activity was measured using a thrombin generation assay. Patients with essential thrombocythemia had significantly higher numbers of circulating annexin V-positive microparticles than controls (median 4500 vs. 2500x10(6) events/L; p=0.039), including significantly higher numbers of microparticles positive for the platelet marker CD61 (p=0.043), the endothelial markers CD62E (p=0.009) and CD144 (p=0.021), and for tissue factor (p=0.036). CD62E was co-expressed with the platelet marker CD41 on microparticles, suggesting a bilineage origin of such microparticles, which were observed only in patients with risk factors for thrombosis. Patients with essential thrombocythemia had higher plasma levels of mature von Willebrand factor (p=0.045) but similar propeptide levels compared to controls. In thrombin generation analyses, microparticle-rich plasma from patients with essential thrombocythemia had a shorter lag time (p=0.001) and higher peak height (p=0.038) than plasma from controls. Peak height correlated significantly with the total number of microparticles (R=0.634, p<0.001). Patients with essential thrombocythemia had higher number of circulating microparticles with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This was confirmed by an increased level of mature von Willebrand factor, an abnormal mature von Willebrand factor/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for microparticles in thrombosis in essential thrombocythemia.

An Arg545→ Cys545 substitution mutation of the von Willebrand factor in type IIB von Willebrand's disease

Type IIB is a special variant of von Willebrand's disease, characterized by an abnormal von Willebrand factor which shows an increased interaction with platelets. This interaction sometimes causes platelet aggregation and thrombocytopenia in vivo. It involves the glycoprotein-Ib (GPIb) receptor on platelets and corresponding GPIb-binding sites in the von Willebrand factor. We here demonstrate a C----T mutation at codon 1308 of the von Willebrand factor gene in 2 related patients with IIB von Willebrand's disease. The transition gives rise to a substitution of arginine by cysteine at position 545 of the mature von Willebrand factor subunit. This position is close to the GPIb- as well as the collagen- and heparin-binding domains of the von Willebrand factor. The mutation may change the conformation of the molecule in this region and activate the GPIb-binding domain, which is normally not exposed in the von Willebrand factor of circulating blood.

Genetic alteration of the D2 domain abolishes von Willebrand factor multimerization and trafficking into storage.

The large von Willebrand factor (VWF) propeptide (VWFpp) plays a critical role in the multimerization and regulated storage of the mature VWF protein. Although our laboratory and others have identified mutations in von Willebrand disease patients that disrupt VWF multimerization, little is known about the affect of mutations on the regulated storage of VWF. We identified a heterozygous 18 base pair, in-frame deletion in exon 12 of the VWF gene in a patient with an unusual, dimer-intense multimer pattern. This deletion results in loss of amino acids 436-442 of VWFpp, which include one cysteine. Through expression studies, we demonstrate reduced secretion, loss of VWF multimerization, and defective regulated storage of the variant VWF. The loss of VWF storage is secondary to loss of propeptide storage resulting from an apparently defective sorting signal on VWFpp. Suprisingly, coexpressed wild-type VWF or VWFpp functioned in trans to partially restore multimerization of VWF from the variant allele. The deletion of six amino acids in VWFpp results in defects in VWF processing, regulated storage, and function. Although VWFpp may usually function in a homotypic fashion, acting on its own mature VWF subunit, VWFpp may retain the ability to function in trans on VWF expressed from the variant allele.

Elevated Procoagulant Microparticles Expressing Endothelial and Platelet Markers in Essential Thrombocythemia

Background: Most cell types, including blood - and vascular cells, produce microparticles (MPs) upon activation. Since cellular MPs are known to be elevated in thromboembolic diseases, we hypothesized a role for MPs in the pathogenesis of thrombosis in Essential Thrombocythemia (ET).Design and methods: In plasma samples from 21 ET patients and 10 healthy subjects, the levels and the cellular origin of MPs were determined by flowcytometric analysis, while the MP-associated procoagulant activity was measured by the thrombin generation assay.Results: ET patients had significantly higher numbers of circulating AnnexinV-positive MPs than controls (median 4500 vs 2500×106 events/L; p=0.039), including significantly higher number of MPs positive for the platelet marker CD61 (median 4000 vs 2400×106/L; p=0.043) the endothelial marker CD62E (median 875 vs 14×106/L; p=0.009), and for Tissue Factor (median 1.8 vs 0.9×106/L; p=0.036). CD62E was co-expressed with the platelet marker CD41 on MPs, suggesting a bilineal origin of such MPs, that were observed only in patients with risk factors for thrombosis. ET patients had higher plasma mature von Willebrand factor (vWF) levels (median 50 vs 35 nM, p=0.045) but similar propeptide levels (median 7 vs 5 nM, p=0,07) compared to controls, indicating chronic endothelial activation. In thrombin generation analyses, MP rich plasma from ET patients had a shorter lag time (9.7 min, 95%CI: 8.7–10.7 versus 15.9 min, 95%CI: 10.9–20.9, p=0.001) and higher peak height (215 nM, 95%CI: 189–241 versus 142 nM, 95%CI: 87–189, p=0.038) than from controls. Peak height correlated significantly with the total number of MPs (R=0.634, p<0.001).Conclusions: ET patients showed higher number of circulating MPs with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This is confirmed by an increased mature vWF level and an abnormal mature vWF/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for MPs in thrombosis in ET.

Basal secretion of von Willebrand factor from human endothelial cells

AbstractEndothelial cells store the adhesive glycoprotein von Willebrand factor (VWF) in Weibel-Palade bodies (WPBs), distinctively shaped regulated secretory organelles that undergo exocytosis in response to secretagogue. A significant proportion of newly synthesized VWF is also secreted spontaneously from nonstimulated cells, through what is thought to be the constitutive secretory pathway. To learn more about VWF trafficking, we performed kinetic analyses of the storage and nonstimulated secretion of VWF in cultured human endothelial cells. We found that most VWF was secreted through a route that was significantly delayed compared with constitutive secretion, although this pathway was responsible for secretion of a small amount of uncleaved VWF precursor. Disruption of pH-dependent sorting processes with ammonium chloride converted the secretion kinetics of mature VWF to that of its precursor. Conversely, preventing constitutive secretion of nascent protein with brefeldin A had only a modest effect on the spontaneous release of VWF, showing that most VWF secreted by nonstimulated cells was not constitutive secretion but basal release of a post-Golgi storage organelle, presumably the WPB. These data suggest that VWF is sorted to the regulated secretory pathway in endothelial cells much more efficiently than previously reported.

The Effect of von Willebrand Factor Normandy Mutations on Intracellular Co-Trafficking of Factor VIII to Storage Organelles.

Von Willebrand Factor (VWF) serves a critical role in factor (F)VIII life-cycle. It is generally believed that FVIII and VWF assemble in the circulation after secretion from vascular endothelial cells (VWF) and hepatocytes (FVIII). However, during the last few years evidence has accumulated in favor of significant extra-hepatic FVIII sources that might be compatible with in vivo biosynthesis and co-storage of FVIII and VWF within the same cell. In agreement with this view, in vitro co-expression of FVIII and VWF results in the storage of FVIII in VWF-containing organelles. We have recently demonstrated that this intracellular co-trafficking is independent on Tyr1680 of the FVIII molecule which is required for extracellular high-affinity interaction to VWF (van den Biggelaar et al. JTH, in press). In the present study, we studied the requirements for intracellular co-trafficking of VWF and FVIII in more detail by addressing VWF variants which have been established as being deficient in extracellular FVIII binding (Normandy variants). To facilitate intracellular visualization, we made use of fluorescently tagged FVIII (FVIII-YFP) and VWF (VWF-CFP). Four different VWF-CFP Normandy variants (Thr791Met, Arg816Trp, Arg854Gln, Cys1060Arg) were created as well as a VWF-CFP variant containing an Arg763Gly replacement which prevents cleavage of the VWF propeptide from mature VWF. (Dr. C. Mazurier (Lille, France) is acknowledged for kindly providing DNA constructs containing the Thr791Met and Arg816Trp replacement). Constructs were expressed in HEK293 cells and purified from conditioned medium using immuno-affinity chromatography. Multimer analysis revealed similar high-molecular weight multimers for all Normandy variants. As expected, the Arg763Gly replacement resulted in an increased size of all multimers. Solid phase binding assays in combination with Surface Plasmon Resonance binding studies showed that binding of FVIII-YFP was strongly reduced for the variants carrying an Arg763Gly, Thr791Met and Arg816Trp replacement and only mildly reduced for the variants carrying an Arg854Gln or Cys1060Arg replacement. Confocal Laser Scanning Microscopy analysis demonstrated that expression of all variants induced the formation of pseudo-Weibel-Palade bodies in HEK293 cells. In addition, all variants were able to redirect FVIII from localization at the trans-Golgi network to VWF-containing storage organelles despite their evident reduction in affinity. These data indicate that mutations in the FVIII binding region of VWF do not affect FVIII/VWF intracellular co-trafficking. We therefore conclude that the requirements that determine extracellular complex assembly differ from those that determine intracellular co-trafficking. We propose that FVIII/VWF co-trafficking may be a result of low-affinity interactions or even indirect passive targeting. We further conclude that co-trafficking is independent of post-translational propeptide cleavage. Together with the observation that co-trafficking is independent on Tyr1680, this suggests that the interactions that determine co-trafficking take place prior to and independent of post-translational processing in the late secretory pathway. Analysis of intracellular trafficking of FVIII in VWF producing cells could prove essential in unraveling the putative role of FVIII and VWF co-storage in vivo. Knowledge of FVIII/VWF co-storage may be particularly relevant to gene therapy approaches that target the FVIII gene to VWF-producing cells.

Genome Wide Scan of Complete Blood Count (CBC) Measures Suggests Strong Linkage of Red Blood Cell (RBC) Count to Chromosome 4q25.

Previous reports suggest that peripheral blood counts are strongly influenced by environmental and genetic determinants; however few of the genetic factors that regulate these quantitative traits have been discovered. We analyzed CBC data from 395 samples collected from an 854-member Amish pedigree with von Willebrand disease. 71 individuals of the pedigree are heterozygous for a missense mutation at position 4120, represented by a single base substitution (C>T) that predicts an arginine to cysteine change at position 1374 (R1374C) in the A1 domain of the mature von Willebrand factor molecule. The detection of genetic signals is likely to be enhanced in groups that live in a more homogeneous environment like the Amish. Linear and quadratic age effect accounted for varying proportions of the gender-specific variation in the CBC measures (from 1% to 70%). The variance component associated with additive polygenic effects was estimated for each CBC phenotype using MENDEL to obtain estimates of heritability. Significant heritability was found for platelet (h2= 0.518, p <0.0005), white blood cell (WBC) (h2=0.395, p <0.0001), RBC counts (h2= 0.358, p <0.0025) and mean corpuscular hemoglobin concentration (MCHC) (h2= 0.547, p <0.0005). Lower heritability was found for red cell distribution width (RDW) (h2= 0.217, p <0.001) and mean corpuscular hemoglobin (MCH) (h2= 0.231, p < 0.02) hematocrit (Hct) (h2= 0.126, p <0.10) and hemoglobin (Hb) (h2= 0.055, p > 0.2). Interestingly since significant heritability for Hct and Hb was reported in other studies, and to rule out the significant effect of bleeding due to the VWF mutation, we estimated heritability of Hb excluding all members of the pedigree that exhibited the 4120 C>T mutation, h2= 0. 21 (p < 0.05). A primary genetic screen at a 10 cM average interval was performed on the entire Amish pedigree in collaboration with the Marshfield genotyping center. Standard Screening Set 16 with 400 short tandem repeat polymorphic (STRP) markers was utilized generating a total of approximately 160,000 genotypes on the 395 samples. Due to data complexity, the Markov Chain Monte Carlo (MCMC)-based program LOKI was used to conduct multipoint linkage analysis for eight CBC measures that were adjusted for age, sex and mutation status to control for potential confounding. The outcome of interest from the MCMC analysis is represented by the Bayes factor (BF) which examines the probability of linkage in complex pedigrees at every centimorgan (cM). A complete analysis of the genome scan with 1,000,000 iterations was performed. A quantitative trait locus (QTL) suggesting strong linkage was identified for the RBC measure at position 4q25 with a BF of 58.17 at 114.5 cM. This result was confirmed by splitting the pedigree into several smaller subsets and conducting traditional linkage analysis using MERLIN (all individuals in the pedigree were included in at least one subset) with a LOD score of 0.66 (p < 0.05). Interestingly, a previous twin study found evidence for linkage for RBC to 4q32. Similar to animal studies, no evidence of linkage was observed in chromosomal regions known to contain the genes that encode for the hemoglobin chains, erythropoietin or erythropoietin receptor. This analysis in a very large pedigree identified a region of strong linkage for RBC that will be analyzed at higher resolution for the presence of novel modifying genes and alleles that may potentially be important for our understanding of the control of erythropoiesis.

Clearance mechanisms of von Willebrand factor and factor VIII.

Clearance mechanisms of von Willebrand factor and factor VIII.

Variations in glycosylation of von Willebrand factor with O-linked sialylated T antigen are associated with its plasma levels

AbstractThe glycosylation profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarily consist of sialylated T antigen (NeuAc(α2-3)Gal-(β1-3)-[NeuAc(α2-6)]GalNAc). It is not yet known whether O-linked carbohydrates affect VWF levels. We developed an immunosorbent assay based on neuraminidase incubation allowing subsequent binding of peanut agglutinin (PNA) to desialylated O-linked T antigen on VWF. An inverse relation was found between PNA binding and VWF antigen levels in healthy individuals (n = 111; Pearson rank = −0.43; P < .001). A similar inverse association was observed in randomly selected plasma samples from our diagnostic laboratory: 252% ± 125% for VWF levels less than 0.5 U/mL (n = 15); 131% ± 36% for VWF levels between 0.5 and 1.5 U/mL (n = 32); and 92% ± 40% for VWF levels more than 1.5 U/mL (n = 19). Reduced or increased PNA binding was also observed in patients with increased (liver cirrhosis) or reduced (von Willebrand disease [VWD] type 1) VWF antigen levels, respectively. VWD type 1 patients further displayed increased ratios of propeptide over mature VWF antigen levels (0.38 ± 0.18 versus 0.17 ± 0.03 for patients and controls, respectively; P < .001), which is indicative of reduced VWF survival in these patients. Of interest, a linear relation between PNA binding and propeptide/VWF ratio was observed (Spearman rank = 0.47), suggesting a potential association between O-linked glycosylation and VWF survival. Finally, we detected a marked decrease in PNA binding in post-DDAVP (1-deamino-8-D-arginine vasopressin) samples from various patients, indicating that the O-linked glycosylation profile of VWF stored in endothelial storage organelles may differ from circulating VWF.

Cloning of the cDNA for murine von Willebrand factor and identification of orthologous genes reveals the extent of conservation among diverse species

Interaction of von Willebrand factor (VWF) with circulating platelets promotes hemostasis when a blood vessel is injured. The A1 domain of VWF is responsible for the initial interaction with platelets and is well conserved among species. Knowledge of the cDNA and genomic DNA sequences for human VWF allowed us to predict the cDNA sequence for murine VWF in silico and amplify its entire coding region by RT–PCR. The murine VWF cDNA has an open reading frame of 8,442 bp, encoding a protein of 2,813 amino acid residues with 83% identity to human pre-pro-VWF. The same strategy was used to predict in silico the cDNA sequence for the ortholog of VWF in a further six species. Many of these predictions diverged substantially from the putative Reference Sequences derived by ab initio methods. Our predicted sequences indicated that the VWF gene has a conserved structure of 52 exons in all seven mammalian species examined, as well as in the chicken. There is a minor structural variation in the pufferfish Takifugu rubripes insofar as the VWF gene in this species has 53 exons. Comparison of the translated amino acid sequences also revealed a high degree of conservation. In particular, the cysteine residues are conserved precisely throughout both the pro-peptide and the mature VWF sequence in all species, with a minor exception in the pufferfish VWF ortholog where two adjacent cysteine residues are omitted. The marked conservation of cysteine residues emphasizes the importance of the intricate pattern of disulfide bonds in governing the structure of pro-VWF and regulating the function of the mature VWF protein. It should also be emphasized that many of the conserved features of the VWF gene and protein were obscured when the comparison among species was based on the putative Reference Sequences instead of our predicted cDNA sequences.

Altered Von Willebrand Factor Subunit Proteolysis and Multimer Processing Associated with the Cys1599(2362)Phe Mutation in the B2 Domain.

Cysteine residues clustered in carboxyl- and amino-terminal domains mediate the assembly of von Willebrand factor (VWF) subunits into dimers and multimers, respectively. We have identified a patient with bleeding symptoms and very low plasma VWF antigen and ristocetin cofactor activity, compatible with clinically severe von Willebrand disease (VWD), whose abnormal plasma VWF multimers show a distinctly altered proteolytic processing. Analysis of plasma VWF multimers in low resolution agarose gels demonstrated a smearing extending to the origin of the separating gel in both the proband and her parents, suggestive of the presence of unusually large species with low electrophoretic mobility. In high resolution gels, these large multimers were stacked at the origin of the gel, clearly seen in a sample of proband's plasma obtained after DDAVP infusion even though the treatment caused only a modest increase in VWF:Ag and VWF:RCo (from 4.7 to 7.5 U/dL and from 5.2 to 8 U/dL, respectively). Individual VWF multimers in the proband's plasma comprised only one clearly visible band with different mobility as compared to the main band of normal multimers, and lacked the satellite bands that form the normal triplet structure. Analysis of the proband's plasma VWF subunit composition using anti M7 antibodies (which probe the aminoterminal region of VWF) revealed the apparent 225 kDa mature form and a single 205 kDa fragment, but notably absent was the 140 kDa aminoterminal fragment generated by ADAMTS-13 cleavage. Subunit analysis using anti-M31 antibodies (which probe the carboxyl-terminal region of VWF) revealed the 225 kDa intact subunit and the 205 kDa fragment, but notably absent were the fragments generated by ADAMTS-13 cleavage. Analysis with anti-22K antibodies, which react with one or more epitope(s) located in the extreme carboxyl terminal region of VWF downstream of Val1927, revealed the presence of only the intact subunit of 225kDa. Concentration of ADAMTS-13 antigen and VWF-cleaving activity in the patient plasma were within normal limits.The patient carried a homozygous Cys to Phe mutation at position 1599 in the mature VWF subunit B2 domain (Cys2362 of pre-pro-VWF). In her heterozygous parents, the 205 kDa fragment was distinctly visible along with the normal VWF cleavage products. Our finding indicate that a proper conformation of the B2 domain, dependent on critical Cys residues, may be required for normal proteolytic processing of VWF multimers. The association of these structural and functional abnormalities with a missense mutation in the B2 domain provides insights into the mechanism that may regulate the post-secretion processing of VWF multimers, and, thus, their prothrombotic potential.

A Genome-Wide Linkage Scan of a Large Amish Pedigree with Von Willebrand Disease (VWD) Identified Several Chromosomal Regions That May Contain Potential Modifiers of Von Willebrand Factor (VWF) Levels and Disease Variability.

VWD is a common and highly variable bleeding disorder with incomplete penetrance and variable expressivity. The ABO blood group is the only well-characterized modifier of VWF with a contribution of up to 30% of the genetic component. The remainder of the genetic influence has yet to be elucidated. Through linkage mapping, using large pedigrees, we aim to discover genes that exert a modifier effect on VWF levels and bleeding severity and that would be candidates for other disorders of hemostasis. We identified and characterized an extensive Amish pedigree from Indiana which is composed of 758 individuals of whom 395 have provided blood samples. Phenotypic characterization of the pedigree included VWF antigen levels (VWF: Ag), VWF Ristocetin cofactor activity (VWF: RCo), molecular ABO typing, CBCs and a clinical bleeding phenotype determined by a validated bleeding score. We used a value of 3 or greater to differentiate bleeders (≥ 3) from non-bleeders (< 3). Sequence from exon 28 of the VWF gene demonstrated a missense mutation at position 4120, represented by a single base substitution (C>T) that predicts an arginine to cysteine change at position 1374 (R1374C) in the A1 domain of the mature VWF molecule. All 71 individuals heterozygous for the 4120 C>T mutations have VWF: RCo levels ≤ 25, indicating 100% penetrance with the low VWF: RCo level phenotype. However the difference in factor levels between affected individuals is as high as 3-fold. Thus the genetic homogeneity of this family provides a large sample in which to study the genetic and/or environmental contributors to the remaining variability observed.A primary genetic screen at a 10 cM average interval was performed on the entire Amish pedigree in collaboration with the Marshfield genotyping center. Standard Screening Set 16 with 400 short tandem repeat polymorphic (STRP) markers was utilized generating a total of approximately 160,000 genotypes on the 395 samples. When the phenotype was defined as VWF: RCo levels ≤ 25 IU/dL and utilizing an autosomal dominant model (disease frequency = 0.1, penetrance = 0.6, no phenocopies), the highest LOD score (16.34) corresponded to marker GATA49D12 located on chromosome 12 p13.3 where VWF has been physically mapped. Despite the impressive effect of the 4120C>T mutation on this particular phenotype, three other regions of linkage were also identified. The first region, with a LOD score of 1.32 at 9q34 harbors several genes of interest including ABO. Two other regions linked to this phenotype exhibited a LOD score of 1.03 and 1.30 and are located on 9q22.3 and 21q22 respectively. When the bleeding scores (BS) were used as phenotypes several regions of linkage were identified. Two regions (19p13.2 and 12p33) showed suggestive LOD scores (1.09 and 1.1) with a phenotype of BS ≥ 3. Four regions with LOD scores greater than 1 were identified with the more stringent phenotype of BS ≥ 4: 6p23, 8p23, 10q26 and 18q12. Only two regions suggested linkage with both bleeding score phenotypes: 7q32, with LOD scores of 2.52 and 1.52 and 16q12 with LOD scores of 1.22 and 1.48 for the BS ≥ 3 and BS ≥ 4 respectively. This analysis in a very large pedigree with a homogeneous primary cause for VWD has identified several regions that will be investigated at higher resolution for the presence of modifier genes and alleles.

Von Willebrand factor and its propeptide: the influence of secretion and clearance on protein levels and the risk of venous thrombosis.

Elevated levels of factor (F)VIII are associated with an increased risk of thrombosis. FVIII levels are determined mainly by von Willebrand factor (VWF). We have investigated the contribution of secretion and clearance rates to the elevated VWF antigen (VWF:Ag) and to the risk of thrombosis. VWF is secreted in equimolar amounts with its propeptide, which has a shorter half-life. VWF propeptide can be used as a measure of VWF secretion and allows estimation of the VWF half-life. We have measured VWF propeptide, VWF:Ag, FVIII:Ag and FVIII activity (FVIII:C) in the Leiden Thrombophilia Study. In controls, high VWF propeptide was associated with high VWF:Ag, FVIII:Ag and FVIII:C. In contrast to mature VWF:Ag, VWF propeptide was not influenced by blood groups. Using an ELISA-based assay we have shown that VWF propeptide lacks ABO antigens. Levels were higher in men and increased with age. A long VWF half-life was also associated with high VWF:Ag, FVIII:Ag and FVIII:C. The VWF half-life was influenced by blood group (10 h in O vs. 12 h in non-O individuals), but not by sex, and only slightly by age. VWF propeptide was higher in thrombosis patients than in controls. The VWF half-life was similar in patients and controls (11.4 and 11.1 h, respectively). Both secretion and clearance rates are important determinants of VWF and FVIII levels. However, mainly high VWF and FVIII levels caused by increased secretion seem to be associated with thrombosis. ABO blood group influences the clearance rates of VWF rather than VWF secretion rates.

Von Willebrand factor, endothelial dysfunction, and cardiovascular disease.

von Willebrand factor (VWF), a glycoprotein involved in arterial thrombus formation, is released into the circulation by secretion from endothelial cells. Plasma VWF levels are determined by genetic factors including ABO blood groups and VWF mutations, and by non-genetic factors including aging, impaired nitric oxide production, inflammation, free radical production and diabetes. Plasma VWF levels have been proposed as a risk factor for cardiovascular events. Although they are only weakly associated with the risk of coronary heart disease (CHD) in the general population, they are a more promising CHD risk factor in high-risk populations with previous cardiovascular events, diabetes or old age. However, is it still unclear whether VWF levels directly determine the rate and severity of arterial thrombus formation or whether they merely reflect alteration in other endothelial functions. The future status of VWF levels as a cardiovascular risk factor depends on additional studies on the genetic determinants of both VWF levels and cardiovascular outcomes. Further studies on VWF levels as a predictor of the risk of stroke (rather than CHD) in elderly or other high-risk population are also promising. Such studies could lead to the clinical use of plasma VWF levels to refine the estimation of the cardiovascular risk and of the expected benefit of antithrombotic agents.

Shielding of the A1 Domain by the D′D3 Domains of von Willebrand Factor Modulates Its Interaction with Platelet Glycoprotein Ib-IX-V

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibalpha and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbalpha binding and recognizes an epitope in the amino acids 764-1035 region in the N-terminal D'D3 domains. In this study we demonstrated that the D'D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D'D3 region in VWF augmented binding to GPIbalpha, suggesting an inhibitory role for this region, (ii) the isolated D'D3 region inhibited the GPIbalpha interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D'D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbalpha. Our results support a model of domain translocation in VWF that allows interaction with GPIbalpha. The suggested shielding interaction of the A1 domain by the D'D3 region then becomes disrupted by VWF immobilization.

The Physiological Function of von Willebrand's Factor Depends on Its Tubular Storage in Endothelial Weibel-Palade Bodies

Weibel-Palade bodies are the 1-5 microm long rod-shaped storage organelles of endothelial cells. We have investigated the determinants and functional significance of this shape. We find that the folding of the hemostatic protein von Willebrand's factor (VWF) into tubules underpins the rod-like shape of Weibel-Palade bodies. Further, while the propeptide and the N-terminal domains of mature VWF are sufficient to form tubules, their maintenance relies on a pH-dependent interaction between the two. We show that the tubular conformation of VWF is essential for a rapid unfurling of 100 microm long, platelet-catching VWF filaments when exposed to neutral pH after exocytosis in cell culture and in living blood vessels. If tubules are disassembled prior to exocytosis, then short or tangled filaments are released and platelet recruitment is reduced. Thus, a 100-fold compaction of VWF into tubules determines the unique shape of Weibel-Palade bodies and is critical to this protein's hemostatic function.

Type 2N von Willebrand disease due to compound heterozygosity for R854Q and a novel R763G mutation at the cleavage site of von Willebrand factor propeptide

Type 2N von Willebrand disease (VWD) is characterized by a markedly decreased affinity of von Willebrand factor (VWF) for factorVIII (FVIII) and is caused by mutations in the D' or D3 domain of mature VWF. We now report a French patient with an atypical 2N VWD phenotype associating FVIII deficiency with plasmaVWF unable to bind FVIII (undetectableVWF:FVIIIB) but with an abnormal multimeric profile. This patient is heterozygous for both the frequent R854Q type 2NVWD mutation and a novel R763G mutation at the cleavage site between VWF propeptide and mature VWF. Four children of the patient displayed moderately decreased VWF:FVIIIB of plasma VWF and were heterozygous for either the R763G or the R854Q mutation. Children with the R763G mutation displayed the same abnormal multimeric profile as their father. Recombinant VWF (rVWF) expression studies performed in COS-7 cells showed that the R763G mutation subtly affects its multimeric profile and dramatically impairs its FVIII binding function. Furthermore, the characteristics of hybrid G763/Q854 rVWF resulting from cotransfection experiments were in agreement with the type 2N VWD diagnosis of the patient. We conclude that R763G is a new type 2N VWD mutation located in the VWF propeptide which alters the proteolytic processing of VWF and consequently its binding to FVIII.

Von Willebrand Factor Propeptide: Response to 1-Deamino-8-D-Arginine Vasopressin and Investigation of Relationship with Plasma Clearance of Von Willebrand Factor Antigen in Type 1 Von Willebrand Disease.

The propeptide of von Willebrand factor (VWFpp) has been demonstrated to be critical for multimerization and intracellular storage of the mature von Willebrand factor (VWF) protein. Although VWFpp and VWF have been shown to circulate at a distinct ratio in plasma, they are stored in endothelial cells in equimolar amounts and stimulation of the endothelium results in acute simultaneous release of these polypeptides in equimolar concentrations. This includes following DDAVP administration to healthy individuals. An increased ratio of plasma VWFpp to VWF antigen (VWF:Ag) may reflect increased clearance of VWF:Ag or reduced clearance of VWFpp. We have quantitated plasma VWFpp levels and the VWFpp to VWF:Ag ratio in patients with type 1 VWD and examined their relationship with the plasma clearance of VWF:Ag and VWFpp. We have also examined the endothelial release of VWFpp and VWF:Ag following DDAVP stimulation in these patients. Plasma molar VWF:Ag and VWFpp concentrations were determined by immunosorbent assay1 in 27 patients with type 1 VWD and 21 normal controls. In the VWD patients VWF:Ag and VWFpp levels were quantitated over 6 hours following the administration of intravenous DDAVP. The half-lives of VWF:Ag (VWF:Ag t1/2) and VWFpp (VWFpp t1/2) were calculated by determining the first-order rate constant for the elimination phase of the respective proteins.2 To determine the molar ratio of VWFpp and VWF:Ag released following DDAVP, values at 30 mins were extrapolated from semi-log plots of VWFpp and VWF:Ag against time. Median concentrations of VWFpp and VWF:Ag were 2.9nmol/L (range, 1.4–10.9) and 24.5nmol/L (2.5–42) pre-DDAVP in VWD group and 9.7nmol/L (5.8–18.1) and 51.5nmol/L (32–98.5) at baseline in normal subjects. The median VWFpp/VWF:Ag ratio was 0.13 (0.06–1.5) in the VWD group as compared to 0.23 (0.08–0.34) in the normals. Median VWF:Ag t1/2 and VWFpp t1/2 values in the VWD patients were 4 hours (0.9–8.7) and 2.5 hours (1.7–8) respectively. VWF:Ag normally has a half-life of 8–12 hours in plasma,3 while that of VWFpp is 2–3 hours.1 In the patient group, inverse correlation was found between VWFpp/VWF:Ag ratio and VWF:Ag t1/2, r=−0.48, p=0.01. There was also significant inverse correlation between VWFpp and VWF:Ag t1/2, r=−0.53, p<0.005. In contrast, there was no correlation between VWFpp/VWF:Ag ratio and VWFpp t1/2, r=−0.29, p>0.1. The values for VWFpp and VWF:Ag extrapolated 30 mins post DDAVP were corrected for baseline values and plotted to give a slope of 0.48, representing release of two moles of VWF per mole VWFpp in the patient group. The range of the plasma VWFpp/VWF:Ag ratio is wide in type 1 VWD patients and this may reflect heterogeneity in the pathophysiological processes, including post secretion events such as increased clearance of VWF:Ag, that contribute towards disease phenotype. The finding of inverse correlation between VWF:Ag t1/2 and the ratio of VWFpp to VWF:Ag indicates that increased plasma clearance of VWF:Ag is not associated with an increased VWFpp clearance in some patients with type 1 VWD. In addition, we have not demonstrated equimolar release of VWFpp and mature VWF in patients with type 1 VWD.

A Novel Mutation in the von Willebrand Factor Propeptide Causes Defective Regulated Storage in Platelets and Weibel-Palade Bodies.

Two children from a Turkish consanguineous family suffering from significant mucocutaneous bleeding episodes were diagnosed with von Willebrand disease type 2A. The relative reduction of large plasma von Willebrand factor (VWF) multimers together with the absent triplet structure was consistent with type 2A (phenotype IIC) VWD. Surprisingly, platelet VWF was completely deficient of multimers beyond the VWF protomer, suggesting defective α-granular storage of larger multimers. The two patients were nearly unresponsive to DDAVP, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage in endothelial cells as well as in platelets. We identified the homozygous mutation in these patients, N528S, in the D2 domain of the VWF propeptide (VWFpp). To examine the effect of the N528S mutation on VWF regulated storage, a VWF expression vector containing the mutation was constructed and expressed in AtT-20 cells that contain an intact regulated storage pathway. Transfected cells were immunofluorescently labeled and the intracellular localization of VWFpp and VWF were examined by confocal microscopy. In cells expressing N528S-VWF, VWF was not stored in granules but was localized to the endoplasmic reticulum (ER). Our previous studies have shown that VWF granular storage is initiated by VWFpp: VWF is trafficked to storage granules through its non-covalent association with VWFpp. To determine if the loss of granular storage of N528S-VWF was due to defective VWFpp trafficking, an N528S-VWFpp expression vector was constructed and expressed in AtT-20 cells. The N528S-VWFpp was found to be colocalized with endogenous ACTH-containing storage granules, demonstrating normal granular trafficking of the variant VWFpp. The N528S-VWFpp was co-expressed with propeptide-deleted, mature VWF (Δpro) to examine regulated storage when the two proteins are expressed in trans. In the majority of transfected AtT-20 cells, N528S-VWFpp was trafficked to granules while the coexpressed Δpro appeared to be ER-localized, consistent with a loss of non-covalent association between VWFpp and VWF. In a subset of cells neither VWFpp nor VWF were stored in granules. In contrast, when N528S-VWF was expressed as a full-length construct (in cis), VWFpp granular storage was observed only in a subset of cells and the majority of transfected cells demonstrated ER-localization of both proteins, suggesting the loss of regulated storage of N528S-VWF is not due to defective VWFpp sorting, but rather an aberrant interaction of VWFpp with the mature VWF protein. Together these data indicate that the phenotype observed in patients with N528S variant VWF is result of defective multimerization, storage, and secretion due to an aberrant VWFpp/VWF interaction.