Magnetic Tweezers Experiments Reveal Increased Mechanical Sensitivity of the F2561Y Mutant in Von Willebrand Factor

The large glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis. By sensing hydrodynamic forces in the bloodstream, VWF elongates under elevated shear, increasing its adhesiveness to platelets. This mechanosensitive behavior arises from its multidomain architecture, where distinct structural elements respond at different force scales. The stem, formed by interactions between C-domains of adjacent monomers in VWF dimers, opens at relatively low forces (~1 pN), representing an early step in force activation. The F2561Y mutation in the C4 domain is a gain-of-function mutation associated with increased shear sensitivity and myocardial infarction, yet its effect on VWF mechanics in the physiologically relevant low-force regime remains unresolved. Here, we combine single-molecule magnetic tweezers with AFM imaging to investigate how F2561Y alters VWF stem dynamics. Magnetic tweezers measurements reveal that A2 domain unfolding remains unchanged in the mutant, whereas stem opening transitions occur at significantly lower forces compared to wildtype. The midpoint force for stem opening is reduced by approximately one order of magnitude, indicating a pronounced destabilization of the compact stem conformation. AFM imaging independently confirms a higher population of open stem states in the mutant, even without applied force. Our findings demonstrate that F2561Y selectively destabilizes the VWF stem and increases its force sensitivity without perturbing global domain stability, providing a direct molecular explanation for its enhanced shear responsiveness and prothrombotic phenotype. More broadly, our work establishes magnetic tweezers as a powerful approach to resolve low-force conformational equilibria in mechanosensitive proteins and to dissect the mechanical impact of disease-associated mutations.