Functional Characterization of the Type 1 VWD Candidate VWF Gene Variants: P.M771I, p.L881R, p.P1413L, and p.Q1475X

Abstract 97

Von Willebrand Factor (VWF) is a multimeric glycoprotein that maintains hemostasis in the vascular system. VWF is secreted mainly from endothelial cells. During its biosynthesis VWF undergoes a series of post-translational modifications and some of the newly synthesized protein is released constitutively. The remainder of the protein is stored for regulated release from cytoplasmic storage granules, Weibel Palade Bodies. Abnormalities in the biosynthetic pathway or increased clearance of plasma VWF are likely to contribute to decreased plasma VWF levels. Inherited partial deficiency of VWF is classified as Type I von Willebrand disease (VWD). Clinical diagnosis of Type I VWD is complicated due to incomplete penetrance and variable expressivity of the abnormal VWF phenotype. The molecular basis of Type 1 VWD remains incompletely understood. The purpose of this study is to explore the effect of four VWF missense mutations (p.M771I, p.L881R, p.P1413L, p.Q1475X), that were reported as candidate mutations in Type I VWD patients in the ISTH SSC VWF Database. The focus of these studies is on the intracellular biosynthetic processing and localization of VWF in a heterologous cell system. For this purpose, selected VWF gene variations were generated in a VWF cDNA expression vector by in vitro site directed mutagenesis. The effect of the candidate mutations on the intracellular localization were analyzed in HEK293 cells by immunoflourescence antibody staining and confocal microscopy. In addition, the effect of the mutations on the biosynthesis of the recombinant VWF was analyzed in COS-7 cells The expression vectors WT-cDNA (wild type) and the expression vector carrying the candidate mutations were transiently transfected into COS-7 cells alone or together to generate wild-type, mutant homozygous and heterozygous genotypes, the latter situation mimicking the status in type 1 VWD patients. To determine whether the mutant recombinant protein was retained within the cell or was efficiently secreted, VWF:Ag levels were assayed in cell lysates and in the conditioned media using ELISA.

Confocal analysis of the transfected HEK293 cells demonstrated impaired intracellular localization of recombinant VWF having p.M771I and p.Q1475X variations. On the other hand, a normal pattern of intracellular storage was observed for the VWF variants having p.L881R and p.P1413L changes.

Expression studies for the p.P1413L and p.Q1475X mutations are currently in progress. Transient transfection of COS-7 cells with the VWF expression vector carrying p.M771I mutation revealed that secretion of the recombinant VWF protein was decreased by 73% (P=0.013, n=3) in the homozygous state, and by 47% (P=0.013, n=3) in the heterozygous state relative to the secretion of wild-type recombinant VWF protein. Intracellular levels of the mutant recombinant protein was increased by 212% in the homozygous state (P=0.005, n=3) and by 69% in the heterozygous state (P=0.255, not significant, n=3). This study demonstrated that p.M771I mutation causes increased intracellular retention of the protein and it has a dominant negative effect on the processing of the wild type protein.

Transient transfection of COS-7 cells with the VWF expression vector carrying p.L881R mutation revealed that secretion of the recombinant VWF protein was decreased by 10% (P=0.61, not significant, n=3) in the homozygous state, and increased by 47% (P=0.36, not significant, n=3) in the heterozygous state relative to the secretion of wild-type recombinant VWF protein. Intracellular levels of the mutant recombinant protein were increased by 58% in the homozygous state (P=0.925, not significant, n=3,) and decreased by 35% in the heterozygous state (P=0.405, not significant, n=3). This study demonstrates that in contrast to the findings with the p.M771I mutant, the p.L881R mutation has little effect on VWF biosynthesis and secretion and does not show a dominant negative effect on the wild type VWF. The pathogenetic mechanism responsible for the type 1 VWD phenotype associated with the p.L881R mutation requires additional evaluation but may involve enhanced protein clearance.