Dynamic Disulfide Bond Topologies in von-Willebrand-Factor’s C4-Domain Undermine Platelet Binding

Abstract

SummaryBackgroundThe von Willebrand Factor (vWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large multi-domain mechano-sensitive protein stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the vWF-C4 domain which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed.ObjectiveTo quantitatively determine C4’s disulfide topologies and their implication in vWF’s platelet-binding function via integrin.MethodsWe employed a combination of classical Molecular Dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays.ResultsWe quantitatively show that two disulfide bonds in the vWF-C4 domain, namely the two major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the RGD-integrin binding motif, and thereby impair integrin-mediated platelet binding. Our combined approach also reveals that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all six vWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme.ConclusionOverall, our data put forward a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of vWF with integrin and potentially other partners, thereby critically influencing its hemostatic function.EssentialsPlatelet integrins interact with the disulfide-bonded C4 domain of von Willebrand FactorThe redox state of vWF-C4’s disulfide bonds is studied by molecular simulations and experimentsTwo bonds are reduced causing C4 unfolding and disulfide swappingOpening of disulfide bonds impairs integrin-mediated platelet binding