PB0812 - The Importance of the Amino-acid Residue at Position 2561 in the C4-Domain of Von Willebrand Factor

Background: Von Willebrand Factor (VWF) plays a critical role in haemostasis by mediating platelet capture at sites of vascular injury. Recently it has been demonstrated that variant F2561Y in the VWF C4-domain is associated with a “gain-of-function” phenotype being more responsive to shear forces leading to enhanced platelet capture function and is associated with an increased risk of thrombotic disease.

Aims: To investigate the importance of the amino acid (aa.) residue at position 2561 for VWF function.

Methods: VWF protein sequence alignment of various species demonstrated the conservation of phenylalanine at position 2561 in mammals and tyrosine/threonine/serine in non-mammals. Variants F2561Y, F2561S and F2561T were generated by site-directed mutagenesis in full-length VWF and expressed in HEK293T cells. Protein expression and function was analysed by ELISA and flow-based assays. Single-molecule experiments, optical tweezers (OT), were conducted to characterise the dynamics of unfolding events in the protein.

Results: The variants had little to no effect on expression, multimeric pattern or collagen binding. Interestingly, VWF-mediated platelet capture at 1500s-1 was similar between all variants. However, at 5000s-1 significantly increased platelet capture and larger thrombi were observed with the F2561T molecule. In-keeping with previous reports, the F2561Y variant was more responsive to shear stress with platelet VWF aggregates forming at a lower shear threshold compared to wtVWF; however, the F2561T and F2561S proteins demonstrated enhanced responsiveness to shear with larger aggregates formed at lower shear rates. The OT experiments demonstrated that D’-CK dimers of F2561T had more frequent unfolding events, associated with longer unfolding extensions, compared to wtVWF, indicating less stability of the molecule.

Conclusion(s): Together this data confirms the importance of the aa. at position 2561 for controlling VWF activity and indicate that serine, threonine or tyrosine at this position may have provided alternative adaptive qualities which are required for effective platelet capture in non-mammalian species.