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.
Keywords: blood coagulation factors; missense; mutation; protein multimerization; von Willebrand diseases; von Willebrand factor.
© 2022 International Society on Thrombosis and Haemostasis.