Mature Von Willebrand Factor Research Articles

The severe von Willebrand disease variant p.M771V leads to impaired anterograde trafficking of von Willebrand factor in patient-derived and base-edited endothelial colony-forming cells

Backgroundvon Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects in von Willebrand factor (VWF). The p.M771V VWF variant leads to a severe bleeding phenotype in homozygous patients. However, the exact molecular mechanism remains unclear, which prevents personalized treatment of those VWD patients. ObjectivesThis study aimed to characterize the underlying molecular mechanisms of the p.M771V variant in multiple representative ex vivo cell models. MethodsEndothelial colony-forming cells (ECFCs) were isolated from venous blood of VWD patients from the Willebrand in the Netherlands cohort carrying homozygous and heterozygous p.M771V VWF variants. The p.M771V variant was also introduced in cord blood-derived ECFCs (CB-ECFCs) through adenine base editing and was overexpressed in HEK293 cells. Biosynthesis, storage, and secretion of VWF was studied using biochemical methods and confocal microscopy. ResultsTwo unrelated homozygous p.M771V patients presented with very low VWF activity and antigen levels in plasma. Patient ECFCs showed impaired uncleaved VWF processing into mature VWF, with secreted VWF being severely reduced when compared to ECFCs of healthy donors. Multimer analysis of p.M771V ECFCs showed a deficiency of high molecular weight VWF multimers. Immunofluorescent staining revealed VWF retention in the endoplasmic reticulum; this was confirmed in various populations of base-edited CB-ECFCs harboring the p.M771V variant. ConclusionThe severe endothelial phenotype observed in patient-derived p.M771V ECFCs, HEK293 cells, and an original base-edited CB-ECFC modeling system show that endoplasmic reticulum retention of VWF and failure to undergo subsequent proteolytic processing underpins the severe bleeding phenotype of patients with homozygous variants at M771.

Preparation and Application of Monoclonal Antibody Against Human von Willebrand Factor Propeptide

To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.

Functional Roles of the von Willebrand Factor Propeptide.

The primary polypeptide sequence of von Willebrand factor (VWF) includes an N-terminal 741-amino acid VWF propeptide (VWFpp). In cells expressing VWF, the VWFpp performs two critical functions. In the Golgi, VWFpp mediates the intermolecular disulfide linkages that generate high-molecular-weight VWF multimers. Subsequently, the VWFpp, which is proteolytically cleaved from mature VWF by furin, functions to generate the endothelial storage organelles (Weibel-Palade bodies) in which VWF and a distinct collection of proteins are stored, and from where they undergo regulated secretion from the endothelium. The VWFpp is secreted from endothelial cells as dimers and circulates in plasma with at least some of the dimers associating with a noncovalent manner with the D'D3 domain of mature VWF. The VWFpp has a half-life of 2 to 3 hours in plasma, but to date no extracellular function has been determined for the molecule. Nevertheless, its large size and several biologically interesting structural features (two sets of vicinal cysteines and an RGD sequence) suggest that there may be roles that the VWFpp plays in hemostasis or associated physiological processes such as angiogenesis or wound repair.

Von Willebrand factor propeptide (VWFpp) — potential biomarker in inherited von Willebrand disease and acquired von Willebrand syndrome

Von Willebrand factor propeptide (VWFpp) is a fragment of a new synthesized VWF molecule that plays an important part in the biosynthesis of this protein. After completion of multimerisation of von Willebrand factor (VWF) dimers in the Golgi apparatus, as result of furin proteolysis VWFpp is truncated/disconnected from the mature VWF molecule to form a non-covalent complex with VWF. The VWFpp-VWF complex is stored in endothelial and platelet storage granules and released into the bloodstream where it dissociates into VWFpp and VWF. Plasma VWFpp level and ratio of plasma VWF propeptide (VWFpp) to VWF antigen (VWF:Ag) i.e VWFpp /VWF:Ag ratio are important biomarkers of VWF synthesis/release/clearance. These biomarkers have distinct therapeutic utility as they help to identify patients with von Willebrand disease (VWD) in whom DDAVP (desmopressin 1-desamino-8-D-arginine vasopressin) is ineffective due to rapid VWF clearance. They also have significant diagnostic value because they help to differentiate between congenital VWD subtypes as well as between congenital VWD and acquired von Willebrand Syndrome (AVWS). The purpose of the study was to determine the VWFpp level and the VWFpp/VWF: Ag ratio in patients with VWD and AVWS and to assess the significance of these biomarkers for diagnosis and management of congenital and acquired von Willebrand disease. Our study involved 120 VWD patients; 21 with AVWS and 111 healthy controls. Study results confirm that VWFpp level and the VWFpp/VWF: Ag ratio have significant value fordiscrimination between severe type 1 VWD and type 3 VWD as well as between acquired and congenital VWD which has serious implications for therapy. The biomarkers are also useful for identification of patients in whom DDAVP treatment may prove ineffective. In type 1 VWD (< 30 IU/ml) the VWFpp/VWF: Ag ratio was elevated in 57% of patients while in the group of patients with threshold VWF values (‘Low VWF’, 30–50 IU/dL) the ratio was within normal, which may be suggestive of other underlying causes of VWF deficiency. Assays in patients with non-neutralizing anti-VWF antibodies (AVWS) have shown that the VWFpp/VWF: Ag ratio can have significant impact on monitoring therapy and assessment of remission in patients with anti VWF antibodies.

The von Willebrand factor D′D3 assembly and structural principles for factor VIII binding and concatemer biogenesis

AbstractD assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D′D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D′D3 forms the binding site for factor VIII. The crystal structure of monomeric D′D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D′ with its TIL′ and E′ modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL′ domain of D′ distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.

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

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

Kif5b Modulates Processing and Secretion of von Willebrand Factor in Endothelial Cells and Mice

Von Willebrand factor (VWF) secreted by Weibel-Palade bodies within endothelial cells is critical to maintain normal platelet adhesion during vascular arrest. Because the transportation of VWF is microtubule-dependent partly via kinesin activity, suppression of kinesin could affect intracellular trafficking of VWF. In this study, we investigated the role of Kif5b, the key member of the kinesin superfamily, in the processing and secretion of VWF. A hypothetical interaction between VWF and Kif5b was confirmed and the tail domain of Kif5b was identified as VWF binding region. Knocking-down Kif5b in human umbilical vein endothelial cells led to significantly increased non-stimulated VWF secretion, considerably higher ratio of pro-VWF to mature VWF, and shorter VWF length. Consistent to the in vitro assay, Kif5b knockdown mice demonstrated dramatically increased VWF secretion after epinephrine stimulation. Significantly prolonged bleeding time was observed in these Kif5b knockdown mice as well, which was further elucidated by the decreased basal/regulated VWF secretion in Kif5b knockdown mice comparing to their wild-type littermates. Taken together, these findings suggest that Kif5b modulates processing and secretion of VWF in vitro and in vivo.

Genome‐wide studies of von Willebrand factor propeptide identify loci contributing to variation in propeptide levels and von Willebrand factor clearance

Essentials Variants at ABO, von Willebrand Factor (VWF) and 2q12 contribute to the variation in plasma in VWF. We performed a genome-wide association study of plasma VWF propeptide in 3,238 individuals. ABO, VWF and 2q12 loci had weak or no association or linkage with plasma VWFpp levels. VWF associated variants at ABO, VWF and 2q12 loci primarily affect VWF clearance rates. Background Previous studies identified common variants at the ABO and VWF loci and unknown variants in a chromosome 2q12 linkage interval that contributed to the variation in plasma von Willebrand factor (VWF) levels. Whereas the association with ABO haplotypes can be explained by differential VWF clearance, little is known about the mechanisms underlying the association with VWF single-nucleotide polymorphisms (SNPs) or with variants in the chromosome 2 linkage interval. VWF propeptide (VWFpp) and mature VWF are encoded by the VWF gene and secreted at the same rate, but have different plasma half-lives. Therefore, comparison of VWFpp and VWF association signals can be used to assess whether the variants are primarily affecting synthesis/secretion or clearance. Methods We measured plasma VWFpp levels and performed genome-wide linkage and association studies in 3238 young and healthy individuals for whom VWF levels had been analyzed previously. Results and conclusions Common variants in an intergenic region on chromosome 7q11 were associated with VWFpp levels. We found that ABO serotype-specific SNPs were associated with VWFpp levels in the same direction as for VWF, but with a much lower effect size. Neither the association at VWF nor the linkage on chromosome 2 previously reported for VWF was observed for VWFpp. Taken together, these results suggest that the major genetic factors affecting plasma VWF levels, i.e. variants at ABO, VWF and a locus on chromosome 2, operate primarily through their effects on VWF clearance.

Identification and characterization of the elusive mutation causing the historical von Willebrand Disease type IIC Miami

Essentials Von Willebrand disease IIC Miami features high von Willebrand factor (VWF) with reduced function. We aimed to identify and characterize the elusive underlying mutation in the original family. An inframe duplication of VWF exons 9-10 was identified and characterized. The mutation causes a defect in VWF multimerization and decreased VWF clearance from the circulation. Background A variant of von Willebrand disease (VWD) type 2A, phenotype IIC (VWD2AIIC), is characterized by recessive inheritance, low von Willebrand factor antigen (VWF:Ag), lack of VWF high-molecular-weight multimers, absence of VWF proteolytic fragments and mutations in the VWF propeptide. A family with dominantly inherited VWD2AIIC but markedly elevated VWF:Ag of > 2 U L(-1) was described as VWD type IIC Miami (VWD2AIIC-Miami) in 1993; however, the molecular defect remained elusive. Objectives To identify the molecular mechanism underlying the phenotype of the original VWD2AIIC-Miami. Patients and Methods We studied the original family with VWD2AIIC-Miami phenotypically and by genotyping. The identified mutation was recombinantly expressed and characterized by standard techniques, confocal imaging and in a mouse model, respectively. Results By Multiplex ligation-dependent probe amplification we identified an in-frame duplication of VWF exons 9-10 (c.998_1156dup; p.Glu333_385dup) in all patients. Recombinant mutant (rm)VWF only presented as a dimer. Co-expressed with wild-type VWF, the multimer pattern was indistinguishable from patients' plasma VWF. Immunofluorescence studies indicated retention of rmVWF in unusually large intracellular granules in the endoplasmic reticulum. ADAMTS-13 proteolysis of rmVWF under denaturing conditions was normal; however, an aberrant proteolytic fragment was apparent. A decreased ratio of VWF propeptide to VWF:Ag and a 1-desamino-8-d-arginine vasopressin (DDAVP) test in one patient indicated delayed VWF clearance, which was supported by clearance data after infusion of rmVWF into VWF(-/-) mice. Conclusion The unique phenotype of VWD2 type IIC-Miami results from dominant impairment of multimer assembly, an aberrant structure of mutant mature VWF and reduced clearance in vivo.

Mutations in the D1 Domain Inhibit Propeptide-Dependent Multimerization, Trafficking and Regulated Secretion of Von Willebrand Factor in VWD

Gene mutations play an important role in the pathogenesis of von Willebrand disease (VWD), resulting in the qualitative defect or quantitative deficit of von Willebrand factor (VWF). VWF propeptide is composed of D1 and D2 domain, which acts as a covalent oxidoreductase in the multimerization. In addition, the propeptide is necessary for the transport from endoplasmic reticulum (ER) to Golgi-apparatus, basal secretion and regulated secretion of VWF. However, the mechanisms of the mutation in D1 domain impairing VWF multimerization and causing low VWF levels in patients remain unknown. Herein we identified four mutations in the D1 domain from VWD patients, and assessed the effect of these mutations on the function of propeptide.We identified p.G39R, p.D141N, p.K157E, and p.C379G in three VWD patients. To characterize the roles of four mutations in propeptide-depend multimerization, we generated several truncated-VWFs, D1D2D¡¯D3 (residues 1-1241), including G39R, K157E, D141N, C379G and wide type (WT). These constructs were then expressed in HEK 293 cells, and were evaluated the D¡¯D3-dimer formation of mutations and WT. Full-length VWF comprising mutations and WT were also restructured and were transfected in HEK 293 cells. We then analyzed the VWF multimer distribution and VWF antigen in the cell supernatant and cell lysate. We also assessed VWF retention in ER and the stimulation secretion by phorbol-12-myristate-13- acetate (PMA) of mutations and those of WT.Two type 3 and 1 type 1 patients were enrolled in our study. VWF antigen were 3, 1 and 8 IU/dL, and VWF:Rco were 2.1, 2.3 and 5.6 IU/dL respectively in three patients. VWF multimer distribution exhibited none in two type 3 patients and normal-like multimer pattern in the type 1. Sequence analysis of VWF gene showed two heterozygous mutations (p.G39R and D141N) in D1 domain of one type 3 patient, and a heterozygous K157E in propeptide and a heterozygous C1165R in D3 domain of the other patient with type 3. Type 1 patients had a heterozygous C379G mutation. Among these four mutations in D1 domain, p.G39R, p.K157E, and p.C379G were novel.In the supernatant of transfected cell, dimerization of D¡¯D3 was absent in truncated-G39R. Compared with that of truncated-WT, decreased but detectable dimerizations were detected in K157E, D141N, and C379G. Similar results were also observed in multimerization of full-length constructs. The multimer assembly was too low to visualize in G39R, whereas decreased medium and absent large VWF multimers were seen in K157E, D141N, and C379G.By immunofluorescence imaging, all full-length VWF variants were detained in ER in different degrees (fig 1). The basal secretions of G39R, K157E, D141N, and C379G were (2.7¡À0. 3)%, (2.5¡À0.2)%, (26.0¡À4.1)%, and (22.4¡À3.8)% of WT respectively. However, VWF antigens in the lysate of transfected cells were (116.5¡À5.4)%, (90.9¡À3.0)%, (91.7¡À0.4)% and (113.8¡À2.9)% of WT for G39R, K157E, D141N, and C379G. No detectable secretion increases of mutant VWF induced by PMA were observed in the transfected cells, while WT-VWF with PMA exhibited increased secretion from 0.69% to 1.66%. [Display omitted] These four mutations in D1 domain downgraded the activity of propeptide as the covalent oxidoreductase, and impaired the muiltimeriztion induced by propeptide. They also interfered with VWF transport from ER to Golgi-apparatus and caused the VWF retentions in ER. Therefore, they further reduced the basal secretion and regulated secretion of mature VWF, which could explain the possible pathogenesis of quantitative deficit of VWF in VWD. DisclosuresNo relevant conflicts of interest to declare.

Platelet-Type von Willebrand Disease: Toward an Improved Understanding of the “Sticky Situation”

We are pleased to highlight in this issue of Seminars in Thrombosis & Hemostasis, the study by Wood et al,1 wherein the authors describe a novel mutation in the platelet GP1BA gene creating a hyperresponsive GPIbα protein—a receptor for von Willebrand factor (VWF)—and causing platelet-type von Willebrand disease (PT-VWD). This is a new naturally occurring mutation in a 23-year-old male patient and is considered the sixth reported mutation thus far in patients described with this disease worldwide. Despite being a rare bleeding disorder, PT-VWD represents a significant challenging clinical problem as it may cause life-threatening bleeding, if not appropriately treated, particularly in situations related to surgeries and childbirth. The clinical diagnosis challenge stems from the close similarity of PT-VWD to the more common bleeding disorder, type 2BVWD.2,3 The discrimination and correct diagnosis can only be made after carefully assessing less commonly performed laboratory tests,3–8 and confirmed only after DNA analysis of the binding regions in the two genes VWF and GP1BA9 has been performed. The report by Woods et al1 highlights some important issues that add to our understanding of this rare but potentially life-threatening bleeding disorder. First, the patient presented with severe bleeding symptoms (rather than mild/moderate bleeding symptoms, and unlike previously reported cases) while also showing other typical laboratory phenotypic data known in this disease such as macrothrombocytopenia, mild spontaneous platelet aggregation, absence from plasma of high-molecular-weight VWF multimers, positive ristocetin-induced platelet aggregation (RIPA) at 0.3 and 0.4 mg/mL, VWF ristocetin cofactor (VWF:RCo) T GP1BA gene mutation that predicted the protein change Try246Leu. This mutation was absent in the unaffected mother and also in 100 healthy control subjects. Second, in that report,1 an attempt was made to quantify the bleeding symptoms in a patient with PT-VWD (bleeding score of 13). The knowledge about variation in bleeding symptoms and the ability to objectively assess these symptoms have proven to be important inmanagement of bleeding disorders and can help predict disease outcome and also aid in treatment.10 The only other such attempt was in a small case series reported recently. This series showed considerable variability in bleeding severity among PT-VWD that is independent of age or gender. The phenotypic variability in type 2B VWD has been reviewed in relation to various mutations and different patients’ cohorts.12 However, a systematic analysis of PT-VWD phenotype and the genotype–phenotype relationship remains to be investigated. Third, for the first time, the level of VWF propeptide (VWFpp) and VWFpp/VWF:Ag ratio was reported in a patient with PT-VWD.1 VWFpp is a 741 amino acid portion that gets cleaved from mature VWF by proteolysis. After cleavage, the VWFpp remains in noncovalent association with the VWF multimers, and both are stored together in the α-granules (megakaryocytes/platelets) or Weibel–Palade bodies (endothelial cells). Upon release and under physiologic pH, the VWF multimers and VWFpp dissociate and are secreted in 1:1 stoichiometric amounts.13 Studies have shown a regulatory role for VWFpp as an intramolecular chaperone for the mature VWF protein and an aid to its storage and multimerization.14,15 The VWFpp circulates in the plasma for a short time, with a half-life of approximately 2 to 3 hours and plasma levels of approximately 1 μg/mL, whereas multimeric VWF circulates with a half-life of approximately 8 to 12 hours and plasma levels of approximately 10 μg/mL.16,17 Recent

Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.

Partial in Vivo FVIII Stabilization by VWF Fragments

Abstract 15Plasma factor VIII (fVIII) circulates in complex with von Willebrand factor (VWF) and is rapidly cleared in the absence of VWF. Previous in vitro studies have 1) localized the fVIII-binding region of VWF to the N-terminus, comprised of the contiguous D’ and D3 domains and 2) observed reduced affinity for fVIII upon alterations to the tertiary structure of VWF. To gain insight into the structure-function of VWF for fVIII stabilization, we tested VWF fragments for in vivo fVIII stabilization and investigated the architecture of a VWF fragment in complex with fVIII. For the in vivo study, fragments of murine Vwf cDNA were cloned into the hepatic-specific expression vector, pLIVE, and modified to fuse tandem E and FLAG tags to the C-terminus. The following VWF fragments were expressed in vivo by hydrodynamic tail vein injection into Vwf−/− mice: 1) a monomer of the VWF D’D3 domains (monoD’D3; M1-C22, S764-P1274); 2) a truncation of monomeric D’D3 (truncD’D3; M1-C22, S764-R1035); 3) dimers of D’D3 (diD’D3; M1-P1274); 4) multimers of D’D3 (multiD’D3; M1-P1274, G2713-K2813); 5) dimers of mature VWF subunits (DPro, M1-C22, S764-K2813); or 6) full length, multimeric VWF (wtVWF, M1-K2813). Expression of all VWF fragments persisted throughout the period of observation (4 weeks) with peak antigenic levels at 1 or 3 days post-injection. Prolonged elevation of plasma fVIII activity (fVIIIa) from ∼10% to ∼50–200% were observed (100% defined as the fVIIIa level of pooled platelet poor plasma from 10 wild type C57BL/6 mice) for all but the truncated monomer of D’D3 (Figure 1). Significantly increased fVIIIa levels (p<0.05, relative to pre-injection) were first observed at 1 day, peaked at 3 days, and persisted for the duration of observation. A minimal VWF fragment (S764-R1035, truncD’D3) reported to bind fVIII in vitro significantly increased plasma fVIIIa to 34% only at 3 days post-injection. Clearance of VWF fragments from circulation were determined from injections of pooled platelet poor plasma containing recombinant VWF fragments derived from hydrodynamically injected mice into naïve Vwf−/− mice. Nonlinear regression estimated the half-life for monoD’D3 (3.4hr), diD’D3 (2.1hr), multiD’D3 (2.3hr), DPro (2.8hr), and wtVWF (3.5hr). To examine how dimers of D’D3 bind fVIII, diD’D3 from HepG2 conditioned media was purified either alone or with recombinant fVIII, and negative stained samples were visualized by electron microscopy (EM). Single-particle EM analysis revealed that each subunit of the dimer binds 1 fVIII molecule. 3D EM reconstructions indicate that the light chain of fVIII directly interacts with, and potentially induces torsion in the flexible D’D3 domains of VWF. Together, these results emphasize the importance of VWF’s tertiary structure in fVIII stabilization and that the N-terminal D’D3 alone is sufficient to support fVIII survival in vivo. These findings could lead to improved methods of recombinant fVIII production and the development of novel approaches to treatment for hemophilia and von Willebrand disease. [Display omitted] Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Von Willebrand factor (VWF) propeptide binding to VWF D′D3 domain attenuates platelet activation and adhesion

AbstractNoncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D′D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D′D3 domain. At pH 6.2 and 10mM Ca2+, conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (KD = 0.2nM, koff = 8 × 10−5 s−1). Significant, albeit weaker, binding (KD = 25nM, koff = 4 × 10−3 s−1) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca2+. This interaction was also observed in human plasma (KD = 50nM). The addition of recombinant VWFpp in both flow-chamber–based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D′D3 mAb DD3.1, which blocks VWFpp binding to VWF-D′D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.

The N-Terminal Amino Acid Residues Ser764 and Lys773 of the D' Domain of Von Willebrand Factor Contribute to Factor VIII Binding

Abstract 2248Factor VIII (FVIII) circulates in a tight complex with its carrier protein von Willebrand factor (VWF). Activation of FVIII results in the dissociation of the FVIII-VWF complex after which FVIII can perform its role in the coagulation cascade. In the complex with VWF, FVIII is protected from rapid clearance from the circulation. Individuals with a mutation in VWF that impairs the ability of VWF to bind FVIII can therefore have a bleeding disorder caused by a low plasma level of FVIII. Mature VWF contains multiple domains of which the N-terminal D’-D3 domains have been shown to comprise the FVIII binding site. Detailed information about amino acid regions in VWF that contribute to the direct interaction with FVIII is, however, lacking. In the present study we have employed a chemical footprinting approach to identify amino acid regions of VWF that are involved in binding FVIII. To this end, the lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII. VWF was subsequently cleaved into peptides employing chymotrypsin. The identity of the peptides and whether or not they contained a modified lysine residue was assessed by nanoLC mass spectrometry. The results showed that the lysine residues of almost all identified peptides were modified to the same extent upon incubation of VWF with FVIII or activated FVIII. However, lysine residue 773 in the N-terminal peptide comprising the residues 766-SCRPPMVKL-774 was protected from chemical modification in the presence of FVIII. In addition, a peptide was identified in which the free amine group of serine 764 at the start of the D’ domain was also differentially modified in the presence of FVIII or activated FVIII. We next studied the structure of a molecular model of the D’ domain that was obtained by comparative homology modeling. Structure analysis revealed that the N-terminal region 764–773 is situated at the tip of the D’ domain and that the amino acid residues Ser764 and Lys773 are in close proximity. This observation combined with the results obtained with the chemical footprinting approach implies that the residues Ser764 and Lys773 at the N-terminus of VWF are directly involved in the FVIII-VWF complex formation. Alternatively, upon binding of FVIII, there is a conformational change in this N-terminal region resulting into a differential accessibility of these residues for chemical modification. To further investigate on this issue, we constructed recombinant VWF variants in which the lysine residue 773 and the serine residue at position 764 were replaced by alanines. The variants Ser764Ala, Lys773Ala and WT-VWF were expressed in 293 cells and purified. The binding of Ser764Ala and Lys773Ala to FVIII was evaluated employing surface plasmon resonance (SPR) analysis. The data revealed that the N-terminal region of the VWF D’ domain modulates the interaction with FVIII. The contribution of Ser764 and Lys733 was mainly reflected in the dissociation kinetics of the complex. We also assessed the association of the VWF variants to FVIII in a solid phase binding assay. In addition, we evaluated to what extent the VWF variants can compete with WT-VWF for binding FVIII. The results were in agreement with the findings obtained with SPR analysis, and demonstrated a modulatory role of the residues 764 and 773. Taken together, our data reveal that the residues Ser764 and Lys773 at the N-terminus of mature VWF contribute to the affinity of the FVIII-VWF complex. 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

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. [Display omitted] 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.

Chronic renal failure is accompanied by endothelial activation and a large increase in microparticle numbers with reduced procoagulant capacity

In patients with chronic renal failure (CRF), cardiovascular disease is the leading cause of increased morbidity and mortality. We hypothesized a role for endothelial activation and microparticle (MP) numbers and procoagulant activity in the pre-thrombotic state of these patients. We analysed blood samples of 27 patients with CRF [8 chronic kidney disease Stage 4 (CKD4), 9 peritoneal dialysis (PD) and 10 haemodialysis (HD), samples taken before and after HD] and 10 controls. Degree and nature of endothelial activation were assessed by measuring mature von Willebrand factor (vWF) and vWF propeptide levels. Cellular MPs were characterized by flow cytometry and MP-specific thrombin generation (TG) measurements. CRF was accompanied by chronic (CKD4 and PD) or acute (HD) endothelial activation. Patients with CRF had substantially higher MP numbers than controls (median 9400 versus 4350×10(6)/L, P=0.001), without significant differences between the treatment subgroups or between pre- and post-HD. The vast majority of MPs were platelet derived. Of the minor populations, endothelial MPs and tissue factor-bearing MPs were more abundant in CRF. MPs were procoagulant, but the increase in numbers was not reflected in a proportional increase in MP-specific TG. Renal failure is accompanied by endothelial activation of a different nature in CKD4 and PD patients compared to HD patients, and results in all subgroups in an increase of mainly platelet-derived MPs that appear to be less procoagulant than in other disease states, possibly because of the uraemic functional defect of their cellular source.

Quantitative Trait Locus (QTL) Linkage Analysis of a Large Pedigree with Von Willebrand Disease Identifies Novel Modifier Loci of Von Willebrand Factor Levels and Confirms Some Previously Reported Modifier Loci

AbstractAbstract 240Von Willebrand disease (VWD) is the most common inherited bleeding disorder with a prevalence ranging from 0.8 to 1.3%. VWD is characterized by incomplete penetrance and variable expressivity, even in families with a unifying VWF mutation, suggesting the presence of modifier genes. We diagnosed and characterized a 1500-member multigenerational Amish family in which 132 individuals exhibit a single autosomal-dominant C4120T mutation (R1374C) in the A1 domain of the mature von Willebrand Factor (VWF) molecule. When accounting for the C4120T mutation, analyses of coagulation factor VIII levels (FVIII:C) and log transformed, sex- and age-adjusted VWF levels in SOLAR estimated a heritability (h2) of 0.301 for VWF antigen levels (VWF:Ag; p=2.7×10−6), 0.386 for VWF ristocetin cofactor activity (VWF:RCo; p=1.16×10−8), and 0.280 for FVIII:C (p=1.52×10−5). These results demonstrate strong evidence of residual heritability of the traits despite the obvious effect of the 4120 mutation. Among individuals with the wild-type VWF CC4120 genotype, ABO genotype, the only known major modifier of VWF levels, explained 6.5, 10.0 and 4.2% of the variability in VWF:Ag, VWF:RCo and FVIII:C levels, respectively, suggesting that a significant genetic component of the variability of these traits remains unaccounted for.In order to search for modifier loci that contribute to the observed variability we then performed a primary genetic screen at an average 10 cM interval using a standard short tandem repeat polymorphism mapping set in a 384-member subset of the pedigree. Quantitative trait locus (QTL) linkage analysis was performed using SOLAR for VWF:Ag, VWF:RCo and FVIII:C. The highest LOD score generated demonstrated linkage of VWF:RCo to region 12p13 where VWF has been physically mapped (LOD=64.73). When accounting for the C4120T mutation, five other chromosomal regions were identified with LOD scores above 2.0 with linkage to FVIII:C, VWF:Ag, and/or VWF:RCo: 2q14 (LOD=2.48), 6p12 (LOD=2.22), 9q33 (LOD=2.84), 10q26 (LOD=2.29), and 16q11 (LOD=2.66). Notably, the ABO locus maps to chromosome 9q33, demonstrating by linkage the known effect of ABO on VWF levels.Five chromosomal regions demonstrated LOD scores between 1.5 and 2.0 for the above phenotypes: 3p14 (LOD=1.56), 4q34 (LOD=1.64), 5q33 (LOD=1.8373), 15q14 (LOD=1.78), and 21q21 (LOD=1.85). Comparison to other linkage and association studies such as the Genetic Analysis of Inherited Thrombophilia (GAIT) and the Framingham Study identified several concurrent peaks in our analysis. Specifically, 22q11and 9q34 were reported in the GAIT study as chromosomal regions linked to VWF:Ag and 6p22 was reported in the Framingham study as well as in the GAIT study as linked to the same phenotype. Furthermore the 22q11 and 21q21 signals display orthology to Mvwf6, a murine modifier region of VWF levels recently reported. Two other signals, 5q33 and 6p12 also display orthology with regions closely syntenic to Mvwf4, another murine modifier. These comparisons strengthen the validity of this multigenerational pedigree as a tool to identify genetic modifiers of the phenotypic variability of VWF levels and provide the basis to which chromosomal regions will be followed-up to identify causative alleles. While genome-wide association studies (GWAS) are characterized by the identification of common variants of relatively modest effect, linkage studies such as the one we present here may have the ability to identify rare variants as well as allelic heterogeneity at a given modifier locus, which may explain a significant portion of the observed variability and likely account for a portion of the genetic component not identified by GWAS analyses. Disclosures:No relevant conflicts of interest to declare.

The mutation N528S in the von Willebrand factor (VWF) propeptide causes defective multimerization and storage of VWF

AbstractWe characterized a consanguineous Turkish family suffering from von Willebrand disease (VWD) with significant mucocutaneous and joint bleeding. The relative reduction of large plasma von Willebrand factor (VWF) multimers and the absent VWF 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. Patients were nearly unresponsive to desmopressin acetate, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage also in endothelial cells. We identified an N528S homozygous mutation in the VWF propeptide D2 domain, predicting the introduction of an additional N-glycosylation site at amino acid 526 in close vicinity to a “CGLC” disulphide isomerase consensus sequence. Expression studies in mammalian cells demonstrated that N528S-VWF was neither normally multimerized nor trafficked to storage granules. However, propeptide containing the N528S mutation trafficked normally to storage granules. Our data indicate that the patients' phenotype is the result of defective multimerization, storage, and secretion. In addition, we have identified a potentially novel pathogenic mechanism of VWD, namely a transportation and storage defect of mature VWF due to defective interaction with its transporter, the mutant propeptide.

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.