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

Kutzki, F.; Butera, D.; Lay, A. J.; Maag, D. M.; Chiu, J.; Woon, H.-G.; Kubar, T.; Elstner, M.; Aponte-Santamaria, C.; Hogg, P.; Graeter, F.

doi:10.1101/2022.08.20.504523

bioRxiv · 10.1101/2022.08.20.504523

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

Kutzki, F.·Butera, D.·Lay, A. J.·Maag, D. M.·Chiu, J.·Woon, H.-G.·Kubar, T.·Elstner, M.·Aponte-Santamaria, C.·Hogg, P.·Graeter, F.

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Abstract

BackgroundThe 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 C4s disulfide topologies and their implication in vWFs 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. EssentialsO_LIPlatelet integrins interact with the disulfide-bonded C4 domain of von Willebrand Factor C_LIO_LIThe redox state of vWF-C4s disulfide bonds is studied by molecular simulations and experiments C_LIO_LITwo bonds are reduced causing C4 unfolding and disulfide swapping C_LIO_LIOpening of disulfide bonds impairs integrin-mediated platelet binding C_LI

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Kutzki, F., Butera, D., Lay, A. J., Maag, D. M., Chiu, J., Woon, H.-G., Kubar, T., Elstner, M., Aponte-Santamaria, C., Hogg, P., Graeter, F.. 2022-08-21. Dynamic Disulfide Bond Topologies in von-Willebrand-Factor's C4-Domain Undermine Platelet Binding. https://doi.org/10.1101/2022.08.20.504523

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