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

Abstract We 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.

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

Blood ◽

10.1182/blood.v106.11.1007.1007 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1007-1007

Author(s):

Sandra L. Haberichter ◽

Ulrich Budde ◽

Elke Drewke ◽

Tobias Obser ◽

Cornelia Wermes ◽

...

Keyword(s):

Von Willebrand Factor ◽

Expression Vector ◽

Intracellular Localization ◽

Von Willebrand Disease ◽

Relative Reduction ◽

Homozygous Mutation ◽

Transfected Cells ◽

Storage Granules ◽

Von Willebrand ◽

Willebrand Factor

Abstract 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.

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ADAMTS13 In 4 Different VWF/VIII Concentrates and Its Impact on Therapy.

Blood ◽

10.1182/blood.v116.21.3677.3677 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3677-3677

Author(s):

Mirjeta Qorraj ◽

Tanja Falter ◽

Sarah Steinemann ◽

Thomas Vigh ◽

Inge Scharrer

Keyword(s):

Von Willebrand Factor ◽

Gel Electrophoresis ◽

Conflicts Of Interest ◽

Von Willebrand Disease ◽

Relative Reduction ◽

Hemorrhagic Diathesis ◽

Adamts13 Activity ◽

Kinetic Measurements ◽

Von Willebrand ◽

Willebrand Factor

Abstract Abstract 3677 Introduction: The hemostatic activity of von Willebrand Factor (VWF) is mainly controlled by the plasma metalloprotease ADAMTS13, which cleaves ultralarge VWF multimers. A qualitative or quantitative deficiency of VWF induces the most common hemorrhagic diathesis, the von Willebrand Disease (VWD). The current classification graduates the VWD in three major types. Depending on severity and the type of VWD the treatment with VWF/FVIII concentrates may by necessary. The commercially available VWF/FVIII concentrates differ in their multimer structure and furthermore also in their pharmacokinetics. We investigated commercial VWF concentrates with respect to their ADAMTS 13 activity and antigen levels with the newest available methods. Moreover, to detect a possible correlation, we analysed the VWF multimer structure of the concentrates. Methods: We analysed 4 human derived VWF/VIII-concentrates (over all 7charges) after reconstitution according to the manufacturer's instructions in different dilutions. Following methods were used: BCS Method according to Böhm detects the capacity of the concentrates for autoproteolysis. The VWF solutions were diluted with 5mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples: pool plasma; plasma from patients with TTP with neutralizing ADAMTS13 auto-antibodies; plasma from patients with TTP without auto-antibodies. The residual VWF:Ristocetin Cofactor (VWF:RCo) activity was subsequently measured using the BC von Willebrand Reagent from Dade Behring. ELISA Technozym®ADAMTS13 and Actifluor TM ADAMTS13 are based on the kinetic measurements of the activity with fluorescence resonance energy transfer (FRET). ADAMTS13 antigen was measured by use of the Technozym ELISA kit. SDS-Gel electrophoresis in 1% Agarose Gel was used to investigate the structure of VWF multimers. Results: The BCS Method according to Böhm is an indirect measurement for endogenous ADAMTS13 activity in the investigated concentrate. Important is the loss of the residual VWF:RCo in the concentrates in presence of TTP-plasma without antibodies and pool plasma compared to the residual VWF:RCo in presence of TTP-plasma with antibodies. All concentrates show some ADAMTS13 activity, however product 1 contains more ADAMTS13 than the other concentrates. The results of the two FRETS-assays correspond very well to the BCS-method results; in addition the assays detect directly the ADAMTS13 activity also in very low measurement range. In a dilution of 16U VWF per ml concentrate the ADAMTS13 activity in product 1 with 4.3% was the highest compared to product 2: 3.2%, product 3: 2.6% and product 4: 2%. The great variability of the test results in higher concentrations may be caused by interferences between some constituents of the concentrates and the analysis. In the same sample set and dilution the ADAMTS13 antigen values correlate very well with ADAMTS13 activity values. The SDS gel electrophoresis reveals the different VWF structure of product1; it has less large and ultralarge multimers. There could be a correlation to the relatively higher ADAMTS13 activity and antigen level. Conclusion: All the investigated VWF/VIII concentrates contain some ADAMTS13 activity and antigen. This was found especially by FRETs assay due to the high sensitivity. Because of the correlation between ADAMTS13 activity and modified VWF multimer structure we like to conclude that ADAMTS13 has influence on stability and therefore also on quality of the concentrates. This might have a therapeutic consequence especially for VWD type 2A. Type 2A is characterized by a relative reduction of intermediate and large VWF multimer. The multimeric abnormalities are commonly the result of in vivo proteolytic degradation of the von Willebrand factor caused by ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

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Genetic determinants of plasma von Willebrand factor antigen levels: a target gene SNP and haplotype analysis of ARIC cohort

Blood ◽

10.1182/blood-2010-08-300152 ◽

2011 ◽

Vol 117 (19) ◽

pp. 5224-5230 ◽

Cited By ~ 32

Author(s):

Marco Campos ◽

Wei Sun ◽

Fuli Yu ◽

Maja Barbalic ◽

Weihong Tang ◽

...

Keyword(s):

Von Willebrand Factor ◽

Von Willebrand Disease ◽

Blood Type ◽

Nucleotide Polymorphisms ◽

Genetic Determinants ◽

Single Nucleotide ◽

Distinct Haplotype ◽

Von Willebrand ◽

And Storage ◽

Willebrand Factor

Abstractvon Willebrand factor (VWF) is an essential component of hemostasis and has been implicated in thrombosis. Multimer size and the amount of circulating VWF are known to impact hemostatic function. We associated 78 VWF single nucleotide polymorphisms (SNPs) and haplotypes constructed from those SNPs with VWF antigen level in 7856 subjects of European descent. Among the nongenomic factors, age and body mass index contributed 4.8% and 1.6% of VWF variation, respectively. The SNP rs514659 (tags O blood type) contributed 15.4% of the variance. Among the VWF SNPs, we identified 18 SNPs that are associated with levels of VWF. The correlative SNPs are either intronic (89%) or silent exonic (11%). Although SNPs examined are distributed throughout the entire VWF gene without apparent cluster, all the positive SNPs are located in a 50-kb region. Exons in this region encode for VWF D2, D′, and D3 domains that are known to regulate VWF multimerization and storage. Mutations in the D3 domain are also associated with von Willebrand disease. Fifteen of these 18 correlative SNPs are in 2 distinct haplotype blocks. In summary, we identified a cluster of intronic VWF SNPs that associate with plasma levels of VWF, individually or additively, in a large cohort of healthy subjects.

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An Arg760Cys mutation in the consensus sequence of the von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant

Blood ◽

10.1182/blood-2002-04-1046 ◽

2003 ◽

Vol 101 (1) ◽

pp. 151-156 ◽

Cited By ~ 22

Author(s):

Alessandra Casonato ◽

Francesca Sartorello ◽

Maria Grazia Cattini ◽

Elena Pontara ◽

Carmen Soldera ◽

...

Keyword(s):

Von Willebrand Factor ◽

Cleavage Site ◽

Binding Capacity ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Von Willebrand Disease ◽

Plasma Factor ◽

Von Willebrand ◽

Willebrand Factor

Abstract We describe a von Willebrand disease (VWD) variant characterized by the persistence of von Willebrand factor (VWF) propeptide as a result of a C>T transition at nucleotide 2527 in exon 17 of the VWF gene. This mutation, which was present in the proband and his father, predicts the substitution of Cys for Arg at position 760 of pre–pro-VWF, 4 residues before the propeptide cleavage site belonging to a consensus sequence for substrate recognition by the processing enzyme paired dibasic amino acid–cleaving enzyme (PACE)/furin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) documented the presence of both processed and unprocessed VWF in the patient's plasma, with unprocessed VWF relatively less represented. The patient's hemostatic phenotype was characterized by a mild decrease in plasma factor VIII (FVIII) and VWF, a decrease in plasma VWF multimers, and a mild reduction in the FVIII binding capacity of VWF. The FVIII binding defect was more pronounced in the proband than in the father because he also inherited the type 2N Arg91Gln mutation from his mother. The persistence of VWF propeptide did not impair VWF synthesis because platelet VWF content was normal, nor did it compromise VWF storage in endothelial cells, because of the normal post–1-deamino-8-D-arginine vasopressin (DDAVP) increase in plasma VWF. Coexpression of wild-type and Arg760Cys VWF into a Furin-producing BHK cell line resulted in decreased VWF secretion and a defect in the FVIII binding capacity of VWF, together with the persistence of VWF propeptide. These findings confirm that a normal consensus sequence for VWF propeptide cleavage and efficient cleavage are required in vivo for normal FVIII binding capacity of VWF.

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The Interaction of Botrocetin with Normal or Variant von Willebrand Factor (Types IIA and IIB) and Its Inhibition by Monoclonal Antibodies that Block Receptor Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646298 ◽

1992 ◽

Vol 68 (04) ◽

pp. 464-469 ◽

Cited By ~ 9

Author(s):

Y Fujimura ◽

S Miyata ◽

S Nishida ◽

S Miura ◽

M Kaneda ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Von Willebrand Factor ◽

Binding Activity ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Normal Individuals ◽

Rag 1 ◽

The One ◽

Von Willebrand ◽

Willebrand Factor

SummaryWe have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca2+-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957–64).Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an “activated complex” formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GPIb, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

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Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649724 ◽

1993 ◽

Vol 70 (06) ◽

pp. 1053-1057 ◽

Cited By ~ 32

Author(s):

Agnès M Journet ◽

Simin Saffaripour ◽

Denisa D Wagner

Keyword(s):

Endothelial Cells ◽

Endoplasmic Reticulum ◽

Von Willebrand Factor ◽

Deletion Mutants ◽

Relative Importance ◽

D2 Domain ◽

Constitutive Secretion ◽

Von Willebrand ◽

And Storage ◽

Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

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The Genetic Defect of Type I von Willebrand Disease “Vicenza” Is Linked to the von Willebrand Factor Gene

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651575 ◽

1993 ◽

Vol 69 (02) ◽

pp. 173-176 ◽

Cited By ~ 5

Author(s):

Anna M Randi ◽

Elisabetta Sacchi ◽

Gian Carlo Castaman ◽

Francesco Rodeghiero ◽

Pier Mannuccio Mannucci

Keyword(s):

Von Willebrand Factor ◽

Autosomal Dominant ◽

Genetic Defect ◽

Von Willebrand Disease ◽

Type I ◽

Bleeding Tendency ◽

Factor Gene ◽

Low Levels ◽

Von Willebrand ◽

Willebrand Factor

SummaryType I von Willebrand disease (vWD) Vicenza is a rare variant with autosomal dominant transmission, characterized by the presence of supranormal von Willebrand factor (vWF) multimers in plasma, similar to those normally found in endothelial cells and megakaryocytes. The patients have very low levels of plasma vWF contrasting with a mild bleeding tendency. The pathophysiology of this subtype is still unknown. The presence of supranormal multimers in the patients’ plasma could be due to a mutation in the vWF molecule which affects post-translational processing, or to a defect in the cells’ processing machinery, independent of the vWF molecule. In order to determne if type I vWD Vicenza is linked to the vWF gene, we studied six polymorphic systems identified within the vWF gene in two apparently unrelated families with type I vWD Vicenza. The results of this study indicate a linkage between vWF gene and the type I vWD Vicenza trait. This strongly suggests that type I vWD Vicenza is due to a mutation in one of the vWF alleles, which results in an abnormal vWF molecule that is processed to a lesser extent than normal vWF.

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Characterization of Partial Gene Deletions in Type III von Willebrand Disease with Alloantibody Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648835 ◽

1994 ◽

Vol 72 (02) ◽

pp. 180-185 ◽

Cited By ~ 22

Author(s):

David J Mancuso ◽

Elodee A Tuley ◽

Ricardo Castillo ◽

Norma de Bosch ◽

Pler M Mannucci ◽

...

Keyword(s):

Von Willebrand Factor ◽

Von Willebrand Disease ◽

Pcr Analysis ◽

Gene Deletions ◽

Factor Gene ◽

Type Iii ◽

Large Deletions ◽

Von Willebrand ◽

Willebrand Factor

Summaryvon Willebrand factor gene deletions were characterized in four patients with severe type III von Willebrand disease and alloantibodies to von Willebrand factor. A PCR-based strategy was used to characterize the boundaries of the deletions. Identical 30 kb von Willebrand factor gene deletions which include exons 33 through 38 were identified in two siblings of one family by this method. A small 5 base pair insertion (CCTGG) was sequenced at the deletion breakpoint. PCR analysis was used to detect the deletion in three generations of the family, including two family members who are heterozygous for the deletion. In a second family, two type III vWD patients, who are distant cousins, share an -56 kb deletion of exons 22 through 43. The identification and characterization of large vWF gene deletions in these type III vWD patients provides further support for the association between large deletions in both von Willebrand factor alleles and the development of inhibitory alloantibodies.

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Autoantibody Selectively Inhibits Binding of von Willebrand Factor to Glycoprotein Ib. Recognition Site Is Located in the A1 Loop of von Willebrand Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656047 ◽

1997 ◽

Vol 77 (04) ◽

pp. 760-766 ◽

Cited By ~ 4

Author(s):

Hiroshi Mohri ◽

Etsuko Yamazaki ◽

Zekou Suzuki ◽

Toshikuni Takano ◽

Shumpei Yokota ◽

...

Keyword(s):

Von Willebrand Factor ◽

Significant Inhibition ◽

Von Willebrand Disease ◽

Recognition Site ◽

Severe Bleeding ◽

Bleeding Tendency ◽

Virtual Absence ◽

Heavy Chains ◽

Von Willebrand ◽

Willebrand Factor

SummaryA 20-year-old man with severe von Willebrand disease recently presented a progressive bleeding tendency, characterized recurrent subcutaneous hemorrhages and cerebral hemorrhage. Mixing and infusion studies suggested the presence of an inhibitor directed against vWF:RCo activity of von Willebrand factor (vWF) without significant inhibition of the FVIII:C. The inhibitor was identified as an antibody of IgG class. The inhibitor inhibited the interaction of vWF in the presence of ristocetin and that of asialo-vWF with GPIb while it partially blocked botrocetin-mediated interaction of vWF to GPIb. The inhibitor reacted with native vWF, the 39/34kDa fragment (amino acids [aa] 480/ 481-718) and the recombinant vWF fragment (MalE-rvWF508-704), but not with Fragment III-T2 (heavy chains, aa 273-511; light chains, aa 674-728). A synthetic peptide (aa 514-542) did not inhibit vWF-inhibitor complex formation. We conclude that this is the first autoantibody of class IgG from human origin that recognizes the sequence in the A1 loop of vWF, resulting in a virtual absence of functional vWF and a concomitant severe bleeding tendency although recognition site is different from the residues 514-542 which is crucial for vWF-GPIb interaction.

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