Mechanosensitive Von Willebrand Factor Protein-Protein Interactions Regulate Hemostasis

Abstract

A recurring theme of mechanosensitive proteins is a cryptic binding site that gets uncovered by an external force. Here, we test the role of such mechanism in the adhesion of platelets at sites of vascular injury, a key process mediated by the von Willebrand factor (VWF). Our data from atomistic simulations, atomic force microscopy (AFM), and microfluidic experiments demonstrate that the VWF A2 domain binds to the VWF A1 domain, such that it buries the platelet binding site located at A1. This implies inactivation of VWF for the binding of platelets by a direct protein-protein interaction between the VWF A1 and A2 domains. During force-probe simulations and AFM experiments, a stretching force uncovered the platelet binding site, by dissociating the A1-A2 complex. This process was accompanied with only a partial unfolding of the A2 domain causing minor exposure of its cleavage site. Our data thus suggest that activation for platelet binding and degradation by cleavage are coupled through the interaction of A1 with A2, and force guarantees that VWF gets ready for activation before cleavage. Microfluidic experiments with an A2-deletion VWF mutant corroborate the critical inactivation role of the A2 domain in vitro. Overall, inactivation of VWF by force-dependent inter-domain A1-A2 interactions answers the question of how platelets are prevented to bind to VWF under equilibrium conditions. The notion of a cryptic protein binding site uncovered by force does not only provide the molecular basis for the long-standing observation of shear-dependent platelet binding during blood clotting, but it might also help to explain shear-induced VWF self-assembly.