PB1433 - Analysis of uncharacterised Von Willebrand's Disease causing mutations located in the D4 and C-domains: identification of a novel type 2A variant and gain-of-function VWF

Background: Von Willebrand's disease is a common autosomal inherited bleeding disorder characterised by a quantitative (type 1 and type 3) or qualitative (type 2) deficiency of Von Willebrand Factor (VWF). While most of the repeating domains in the VWF molecule have ascribed roles, the functional role of the D4 and C-domains remain poorly understood, although we have previously shown that deletion of various C-domain regions abolishes VWF secretion. Analysis of the VWD database highlighted several putative type 1 and type 3 mutations in the D4 and C-domains that are yet to be characterised.

Aims: To characterise the impact of ten D4 and C-domains VWD mutations; C1950Y, G2044D, C2174G, E2233Q, R2287W, C2340R, G2343V, R2379C, S2497P and C2693Y

Methods: Mutations generated by site-directed mutagenesis were expressed in HEK293/T cells alone or alongside wild-type (wt) VWF or VWF lacking the A1 domain. Expression was analysed by ELISA and multimer gels and cellular retention investigated by endo H digestion and immunofluorescence staining. Flow assays were performed to determine protein function.

Results: Homozygous transfections resulted in abolished or reduced secretion for all mutants which was rescued by co-transfection with wtVWF; however, for mutations C1950Y, C2174G, E2233Q, C2340R and R2379C the mutant monomer was not expressed. Endo-H digests confirmed endoplasmic-reticulum processing and immunofluorescent imaging showed most mutants formed pseudo Weibel-Palade bodies, albeit to a lesser extent than wtVWF. The R2379C mutation was shown to prevent dimer and subsequent multimer formation and accordingly could not support platelet capture under shear stress. Interestingly, although the R2287W mutation showed significantly reduced expression, its platelet capture function was enhanced under shear stress indicating gain-of-function activity.

Conclusion(s): Mutations in the D4 and C-domain affect VWF secretion indicating a critical role for the D4 and C-domains for correct VWF folding and multimer formation, however mutations in this region can also enhance function under flow conditions.