Significance of the amino acid at position 2561 in the C4 domain of von Willebrand factor.

Background: The von Willebrand factor (VWF) Phe2561Tyr variant has been previously shown to exhibit gain-of-function-like activity and increase the risk of repeated myocardial infarction in patients aged <55 years. It was hypothesized that altered stem dynamics enhanced the responsiveness of the molecule to shear stress.

Objectives: In this study, we investigated the evolutionary significance of the amino acid at position 2561 and functional impacts of variants at this site.

Methods: 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.

Results: Phe2561 was heavily conserved in mammals; however, nonmammals occupied the site with tyrosine, threonine, or serine. Recombinant expression of Phe2561Tyr/Thr/Ser showed no effects on expression, multimers, collagen or glycoprotein IIbIIIa binding, but Ser2561 and Thr2561 demonstrated enhanced binding to gain-of-function glycoprotein Ibα. While none of the variants, including Tyr2561, enhanced VWF-mediated platelet capture to collagen at 1500/s or 5000/s, the Tyr2561, Ser2561, and Thr2561 variants 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 atomic force microscopy imaging of VWF dimeric stems demonstrated that Tyr, Thr, and Ser2561 favored a more open stem state, suggesting this is responsible for the enhanced response to shear stress.

Conclusion: This study indicates that the amino acid at 2561 is crucial for modulating VWF function by helping to mediate stem dynamics.