Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding

The 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 that is 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. To determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF’s platelet binding function. We combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. We show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 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 integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal 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 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. Our data suggests 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. •Platelet integrins interact with the disulfide-bonded C4 domain of von Willebrand Factor (VWF).•The redox state of disulfide bonds in C4 domain of VWF was studied by molecular simulations and experiments.•Two bonds are reduced, causing C4 unfolding and disulfide swapping.•Opening of disulfide bonds impairs integrin-mediated platelet binding.