Removal of the vicinal disulfide enhances the platelet capturing function of Von Willebrand Factor.

A redox autoinhibitory mechanism has previously been proposed in which the reduced state of the vicinal disulfide bond in the von Willebrand factor (VWF) A2 domain allows A2 to bind A1 and inhibit platelet adhesion to the A1-domain.1 The VWF A1A2A3 tri-domain was expressed with and without the vicinal disulfide in A2 (C1669S/C1670S) via atomic replacement of Sulfur for Oxygen to test the relevance of the vicinal disulfide to the physiological platelet function of VWF under shear flow. A comparative study of the shear-dependent platelet translocation dynamics on these tri-domain variants reveals that reduction of the vicinal disulfide moderately increases the platelet-capturing function of A1, an observation counter to the proposed hypothesis. Surface plasmon resonance spectroscopy confirms that C1669S/C1670S slightly increases the affinity of A1A2A3 binding to GPIbα. Differential scanning calorimetry and Hydrogen-Deuterium exchange mass spectrometry demonstrate that reduction of the vicinal disulfide destabilizes the A2 domain which consequently disrupts interactions between the A1, A2, and A3 domains and enhances the conformational dynamics of A1-domain secondary structures known to regulate the strength of platelet adhesion to VWF. This study clarifies that the reduced state of the A2 vicinal disulfide is not inhibitory, but rather slightly activating.