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 ␣-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. (Blood. 2010;115(22): 4580-4587)

Intersection of mechanisms of type 2A VWD through defects in VWF multimerization, secretion, ADAMTS-13 susceptibility, and regulated storage

“…This suggests that the primary processing defect may lie upstream at the point of transcription, as reported previously by Nichols et al, and may be similar to the previously reported type 2A variant N528S-VWF. 29,31 In contrast, several variants were secreted efficiently, but had various degrees of multimer abnormalities, including C1099P, C1190S, C1272S, and C1272Y. These variants were placed in an "unclassified" group because they did not seem to fit into either of the 2 traditional groups for type 2A VWD.…”

“…22,24,33 The impact of mutations in VWF on formation of storage granules has been investigated for few variants. 17,26,29,30,[34][35][36] A loss of regulated storage was characteristic of the "classic" group 1 variants, which did not form Weibel-Palade body-like storage granules and had only a diffuse staining pattern in HEK293 cells. The group 2 variants G1579R and I1568N formed storage granules.…”

“…2,12 VWF expressed in HEK293 cells can form Weibel-Palade body-like granules. 29,30 The 2A variants were expressed in HEK293 cells, immunostained, and examined by confocal microscopy ( Figure 4A). Only C1099P, C1190S (data not shown), C1272Y, I1568N, and G1579R (Figure We also investigated the impact of heterozygosity on regulated storage.…”

Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease

“…Studies in these cell lines have provided insights into the effects of VWF mutations on the storage and secretion of VWF. [11][12][13][14][15][16][17] In contrast, the use of nonendothelial cell systems has obvious limitations: The possible gross overexpression of recombinant VWF on transfection may influence interpretation of the data, and the exocytotic machinery is probably different from that of endothelial cells. Mimicking the heterozygous state of VWF mutations by cotransfection of wild-type and mutant VWF is potentially also problematic.…”

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

“…The phenotype of this mutation was defective multimerization, storage and secretion. The introduction of a new glycosylation site could potentially affect the interaction of the propeptide with mature VWF or disrupt the nearby CGLC disulphide isomerase sequence, that is implicated in VWF multimerization 38 As well as the specific effect on disulphide bond formation in the CK domain, all the glycan mutants presented an overall increased free thiol reactivity compared with wtVWF. Removal of N-linked glycan chains may therefore lead to a more open VWF conformation allowing exposure and detection of previously buried cysteine residues.…”