Shear‐induced unfolding activates von Willebrand factor A2 domain for proteolysis

Abstract

Summary. Background: 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. Objective and methods: We examined whether tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. 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. Results: 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 find this mutant A2 (N1493C and C1670S) to feature ADAMTS13-resistant behavior in vitro. Conclusions: Our results yield molecular-detail evidence for the force-sensing 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 ÔknottedÕ Rossmann fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we discuss 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.

Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism

“…Remarkably, the knotted C-terminal part of the protein (residue index < 1580) presented small RMSFs both for the wild-type and the mutant. This implies a high structural stability for this region and is in concordance with previous computational studies (7).…”

“…Enhanced proteolysis has been attributed to mutationinduced structural destabilization of the A2 domain, which promotes its unfolding and thereby facilitates exposure of the ADAMTS13 cleavage site (7,15,16). In a broad study, Hassenpflug et al investigated the impact of 13 different clinically relevant VWD type 2A mutations on ADAMTS13-dependent proteolysis (17).…”

Early Diagnosis of Acquired von Willebrand Syndrome (AVWS) is Elementary for Clinical Practice in Patients Treated with ECMO Therapy

“…AVWS results from increased shear stress in VADs and the tubes that connect the VAD to the heart and great vessels. Artificial surfaces and differences in velocity may enhance shear stress, which subsequently leads to the unfolding of Von Willebrand factor (VWF) and cleavage by the protease ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin motif ) [9][10][11] . This process induces the loss of high-molecular weight (HMW) multimers, thus resulting in the decreased binding of VWF to collagen.…”

Anticoagulation management of left ventricular assist devices

“…Loss of high molecular weight (HMW) vWF multimers is believed to result from high shear stress effects on vWF multimers; this hypothesis is supported by in vitro data [10][11][12][13][14]. High shear stress changes the conformation of the large vWF multimer from a globular mass to a more linear shape, resulting in exposure of ADAMTS13 binding and cleavage sites in the A2 domain [10] Proteolysis of the vWF multimers by ADAMTS13 yields significantly smaller multimers that provide less hemostasis.…”