Identification of missense variants in the C-domains of von Willebrand factor that cause gain-of-function–like activity

ABSTRACT: Recently characterized mutations Phe2561Tyr, Pro2555Arg in the von Willebrand factor (VWF) C4 domain, and Gly2705Arg in the C6 domain reportedly confer gain-of-function-like activity on the molecule, increasing responsiveness to shear stress, resulting in enhanced platelet capture. This implies that the C-terminal stem, comprised of the D4-C6 domains, plays a crucial role in modulating VWF function. To further investigate this, we used site-directed mutagenesis to generate a panel of uncharacterized C-domain variants. VWF expression and function were analyzed under static and shear stress conditions, and the biophysical properties of the variants were characterized by single-molecule optical tweezers (OT), and atomic force microscopy (AFM) imaging. All the expressed variants exhibited normal function in static assays, except the C1 domain Arg2287Trp variant that demonstrated increased binding to GPIbα. Under flow conditions at 1500 per second, all the variants had normal VWF-mediated platelet capture to collagen; however, at 5000 per second Arg2287Trp demonstrated enhanced platelet capture, whereas Asn2636Tyr (C5 domain), Thr2647Met (C6 domain), and Gly2705Arg showed reduced activity. Further analysis of the formation of rolling VWF-platelet aggregates over a VWF surface demonstrated an enhanced response to shear stress for the Arg2287Trp and Arg2384Trp (C2 domain) variants. OT analysis identified novel extension events exclusive to the C-terminal domains and more frequent unfolding events and longer unfolding extensions for Arg2287Trp and Arg2384Trp, consistent with increased flexibility and stem opening. AFM imaging confirmed that both variants favored the open stem conformation. Together, we demonstrate that mutations in the C1 and C2 domains alter stem dynamics, rendering VWF more responsive to shear stress.