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

Abstract

Summary. Background: 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. Patients/Methods: 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. Results: 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. Conclusions: 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.

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

“…40 The D3 domain is important in VWF multimerization, which could impact VWF tertiary structure and ultimately circulating levels of VWF and its clearance. 39 It is also interesting to note that the rate of VWF clearance is found to be accelerated in patients with VWD Vicenza, which often have mutations, including intronic, in the D domains [41][42][43] (we did not find positive SNPs in the exon encoding D4 and flanking intronic sequence). However, extensive biologic experiments are required to establish a mechanistic link between positive SNPs and VWF expression in general and the putative role of this 50-kb region in regulating VWF expression in particular.…”

“…[36][37][38] Mutations in the VWF propeptide, specifically in the D2 domain, have been shown to result in decreased VWF storage, multimerization, and secretion. 39 They also impact the ratio of VWF propeptide to mature VWF, which in turn affects the clearance of VWF in the circulation. 40 The D3 domain is important in VWF multimerization, which could impact VWF tertiary structure and ultimately circulating levels of VWF and its clearance.…”

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

“…26 To generate point mutations in human WT-pCIneo and WT-mycHis, the QuikChange II XL site-directed mutagenesis kit (Stratagene) was used according to the manufacturer's instructions. Primer sequences are available on request.…”

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.…”