Reduced Von Willebrand Factor Secretion Is Associated with Loss of Weibel-Palade Formation

Abstract

AbstractAbstract 541 Background.Von Willebrand Disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes patients with quantitative plasma VWF deficiency with normal VWF structure and function. Aim of the study.We report three different novel type 1 VWF mutations (A1716P, C2190Y and R2663C) which although located in different VWF domains are associated with reduced secretion and lack of formation of Weibel-Palade body-like granules. Methods.Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding, and GpIb binding assessed in comparison to wild-type VWF. Furthermore, expression was also examined in HEK293 cells that form Weibel-Palade body (WPB)-like granules when transfected with wt VWF. Results.The multimer analysis of plasma VWF was compatible with type 1 VWD. The results of 3 different expression experiments showed a slightly reduced VWF synthesis and drastically impaired secretion into the medium with homozygous expression.MutationVWF:Ag Lysate (%)VWF:Ag Medium (%)GpIb binding (%)GpIb binding/VWF:Ag MediumVWF:CB (%)VWF:CB/VWF:Ag MediumA1716P homo62 ± 305 ± 0.22 ± 0.10.480.2 ± 0.010.05A1716P hetero56 ± 145 ± 0.937 ± 0.40.8226 ± 0.20.58C2190Y homo82 ± 1311 ± 0.23 ± 0.10.312 ± 0.10.19C2190Y hetero73 ± 1242 ± 0.539 ± 0.40.9230 ± 0.40.72R2663C homo73 ± 219 ± 0.52 ± 0.10.221 ± 0.10.1R2663C hetero68 ± 145 ± 0.128 ± 0.10.6316 ± 0.80.35In HEK293 cells, homozygous A1716P and C2190Y VWF variants failed to form WPB-like granules, while R2663C was capable of forming granules, but had fewer cells with granules and more with ER-localized VWF. Heterozygous expression of A1716P and C2160Y VWF variants had a negative impact on wild-type VWF and WPB-like granules were observed in transfected cells. Conclusions.Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations can be associated with inability to form endothelial Weibel-Palade-like granules and mutations in different VWF domains can affect the formation of these organelles. Disclosures:No relevant conflicts of interest to declare.