Background
Von Willebrand’s Disease is characterised by an absence or reduction of plasma Von Willebrand Factor levels or reduced protein function. While the spectrum of causative Von Willebrand’s Disease (VWD) mutations is vast, there has been limited characterisation of variants occurring within the D4-C6 domains of VWF that comprise the C-terminal portion of the molecule.
Objectives
In the present study we investigated the impact of nine putative low frequency VWD causing variants on VWF function.
Methods
Variants were generated by site directed mutagenesis and expressed in HEK293(T) cells and analysed for expression, intracellular storage and multimeric profile. The ability of Arg2379Cys to form dimers was assessed using an A2-CK fragment of VWF.
Results
Arg2379Cys, Ser2497Pro and Cys2639Tyr had significantly reduced secretion from HEK293T cells, while the other mutations all failed to be secreted. While co-transfection with wtVWF appeared to rescue expression, co-transfection with a deletion A1 construct demonstrated that only the Gly2044Asp, Glu2343Val, Ser2497Pro and Cys2693Tyr variants could be rescued. All the variants failed to form appreciable pseudo-Webiel-Palade bodies in HEK293 cells and showed abnormal multimers in cell lysates. The Arg2379Cys variant could be overexpressed by only formed monomers and some dimers. Analysis with a VWF-A2CK protein demonstrated that in the homozygous state Arg2379Cys behaves likes a type 2A variant, while it is likely to be a type 1 variant in the heterozygous state.
Conclusion
This data shows that varaints within the C-terminal region of VWF can dramatically impact proper VWF expression and can different impacts on VWF depending on homo or heterozygosity.