VWF‐Gly2752Ser, a novel non‐cysteine substitution variant in the CK domain, exhibits severe secretory impairment by hampering C‐terminal dimer formation

Abstract

Background Von Willebrand factor (VWF) is a multimeric glycoprotein that plays important roles in hemostasis and thrombosis. C‐terminal interchain‐disulfide bonds in the cystine knot (CK) domain are essential for VWF dimerization. Previous studies have reported that missense variants of cysteine in the CK domain disrupt the intrachain‐disulfide bond and cause type 3 von Willebrand disease (VWD). However, type 3 VWD‐associated noncysteine substitution variants in the CK domain have not been reported. Objective To investigate the molecular mechanism of a novel non‐cysteine variant in the CK domain, VWF c.8254 G>A (p.Gly2752Ser), which was identified in a patient with type 3 VWD as homozygous. Methods Genetic analysis was performed by whole exome sequencing and Sanger sequencing. VWF multimer analysis was performed using SDS‐agarose electrophoresis. VWF production and subcellular localization were analyzed using ex vivo endothelial colony forming cells (ECFCs) and an in vitro recombinant VWF (rVWF) expression system. Results The patient was homozygous for VWF‐Gly2752Ser. Plasma VWF enzyme‐linked immunosorbent assay showed that the VWF antigen level of the patient was 1.2% compared with healthy subjects. A tiny amount of VWF was identified in the patient's ECFC. Multimer analysis revealed that the circulating VWF‐Gly2752Ser presented only low molecular weight multimers. Subcellular localization analysis of VWF‐Gly2752Ser‐transfected cell lines showed that rVWF‐Gly2752Ser was severely impaired in its ER‐to‐Golgi trafficking. Conclusion VWF‐Gly2752Ser causes severe secretory impairment because of its dimerization failure. This is the first report of a VWF variant with a noncysteine substitution in the CK domain that causes type 3 VWD.

Potentials of Endothelial Colony-Forming Cells: Applications in Hemostasis and Thrombosis Disorders, from Unveiling Disease Pathophysiology to Cell Therapy

“…The data obtained from these studies led to the discovery the novel pathogenesis mechanisms leading to VWF deficiencies in VWD patients and clarifying the effect of VWF variants on intracellular processing of VWF, including multimerization, storage/formation of WPBs, and secretion. 22,[44][45][46][47][48][49][50][51] The use of the patient-derived ECFCs emphasized the significance of the exonic synonymous and deep intronic variants on VWF splicing and their contribution to the pathogenesis of VWD. [44][45][46][47]51 Furthermore, we inspected the gene expression profile and trafficking of the inflammatory/angiogenesis proteins Ang2 and P-selectin, co-stored with VWF in WPBs, in the patient-and healthy control-derived ECFCs.…”