The Significance of the Amino Acid at Position 2561 in the C4 Domain of Von Willebrand Factor

The VWF Phe2561Tyr variant has been previously shown to exhibit gain-of-function like activity and increase the risk of repeated MI in patients below 55 years of age. It was hypothesised that altered stem dynamics enhanced the responsiveness of the molecule to shear stress. In this study we investigated the evolutionary significance of the amino acid at position 2561 and functional impacts of variants at this site. Variants at 2561 identified by protein sequence alignments were generated by mutagenesis and expressed in HEK293T cells and function investigated using static and flow-based assays and biophysical methods. Phe2561 was heavily conserved in mammals, however, non-mammals occupied the site with either tyrosine, threonine or serine. Recombinant expression of Phe2561Tyr/Thr/Ser showed no effects on expression, multimers, collagen or GPIIbIIIa binding, but Ser2561 and Thr2561 demonstrated enhanced binding to gain-of-function GPIbα. While none of the variants, including Tyr2561, enhanced VWF mediated platelet capture to collagen at 1500 or 5000s-1, the Tyr2561, Ser2561 and Thr2561 variants all exhibited enhanced formation of rolling VWF-platelet aggregates, with Ser2561 demonstrating the greatest gain-of-function activity. Optical tweezer pulling experiments using VWF-D-CK dimers revealed altered unfolding properties of the Tyr, Thr and Ser2561 protein and AFM imaging of VWF dimeric stems demonstrated that Tyr, Thr and Ser2561 all favoured an more open stem state suggesting this is responsible for the enhanced response to shear stress. Together this study indicates that the amino acid at 2561 is crucial for modulating VWF function by helping to mediate stem dynamics.