Shear-Induced Unfolding Activates von Willebrand Factor A2 Domain for Proteolysis

TL;DR

This study uses molecular dynamics simulations to show how shear flow induces unfolding of the VWF A2 domain, exposing it for cleavage by ADAMTS13, revealing a force-sensing mechanism crucial for regulating VWF activity and size.

Contribution

The paper provides molecular-level evidence of shear-induced unfolding of VWF A2 and introduces a mutant that resists cleavage, advancing understanding of VWF regulation and disease mutations.

Findings

Shear flow causes stepwise unfolding of VWF A2 domain exposing the cleavage site.

Disulfide bonds in A1 and A3 domains prevent their unfolding under shear.

A mutant with a disulfide bond in A2 resists ADAMTS13 cleavage in vitro.

Abstract

To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13-mediated proteolysis in a shear flow dependent manner. We examined if tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. We find this mutant to feature ADAMTS13-resistant behavior in vitro. Our results yield molecular-detail evidence for the force-sensoring function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleavage while keeping the folded remainder of A2 intact and functional. We find the unconventional knotted Rossman fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we can explain the pathomechanism of some natural mutations in the VWF A2 domain that significantly increase the cleavage by ADAMTS13 without shearing or chemical denaturation, and provide with the cleavage-activated A2 conformation a structural basis for the design of inhibitors for VWF type 2 diseases.