OC 13.4 - Von Willebrand Factor C1-domain Mutations Promote Gain-of-function Activity

Background: Recently characterised mutations F2561Y and P2555R in the Von Willebrand Factor (VWF) C4-domain confer gain-of-function like activity on the molecule, increasing responsiveness to shear stress, resulting in enhanced platelet capture. This implies that the C-domains, particularly the C4-domain, plays a crucial role in modulating VWF function.

Aims: To investigate how reported mutations in C-terminal domains impact VWF function, specifically under conditions of shear stress.

Methods: A panel of uncharacterised C-domain mutations were selected from the Leiden Open Variation Database and generated in a full-length VWF expression vector using site-directed mutagenesis and subsequently expressed in HEK293T cells. VWF protein expression and function was analysed by ELISA, multimer composition, collagen binding and in-vitro flow-based assays. The biophysical properties of the variants were characterised by single-molecular technology, optical tweezers (OT).

Results: Generated mutations with >25% expression, normal multimeric pattern and collagen binding were further analysed under flow conditions. Interestingly, two variants, R2287W and R2313H, located in the C1-domain demonstrated enhanced VWF-mediated platelet capture to collagen under shear at both high and pathological shear rates. Moreover, further flow-based analysis revealed that both these variants displayed enhanced responsiveness to shear stress with the formation of rolling VWF-platelet aggregates at lower shear thresholds and larger aggregates compared to wild-type VWF (wtVWF). Molecular modelling suggested that the R2287W variant disrupts a putative interaction with the adjacent D4 domain, while R2313H alters the flexibility of the C1-domain. These findings were further supported by using single-molecule OT. The results demonstrated that D’-CK dimers of both variants exhibited more frequent unfolding events and longer unfolding extensions compared to wtVWF under tensile forces, indicating the variants are less stable.

Conclusion(s): Together our data demonstrates that mutations in the C1-domain can render the VWF molecule more responsive to shear stress, therefore, enhancing VWF platelet capture function.