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

Fabian Kutzki, Diego Butera, Angelina J. Lay, Denis Maag, Joyce Chiu, Heng-Giap Woon, Tomáš Kubař, Marcus Elstner, Camilo Aponte-Santamaría, Philip J. Hogg, Frauke Gräter
2022 · doi:10.1101/2022.08.20.504523
preprint OA: closed CC-BY-ND-4.0
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Abstract

Summary Background 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 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. Objective To quantitatively determine C4’s disulfide topologies and their implication in vWF’s platelet-binding function via integrin. Methods We employed a combination of classical Molecular Dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. Results We 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. Conclusion Overall, 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. Essentials Platelet integrins interact with the disulfide-bonded C4 domain of von Willebrand Factor The redox state of vWF-C4’s disulfide bonds is 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

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License: CC-BY-ND-4.0