von Willebrand disease type 2A phenotypes IIC, IID and IIE: A day in the life of shear-stressed mutant von Willebrand factor.

The bleeding disorder von Willebrand disease (VWD) is caused by mutations of von Willebrand factor (VWF), a multimeric glycoprotein essential for platelet-dependent primary haemostasis. VWD type 2A-associated mutations each disrupt VWF biosynthesis and function at different stages, depending on the VWF domain altered by the mutation. These effects cause considerable heterogeneity in phenotypes and symptoms. To characterise the molecular mechanisms underlying the specific VWF deficiencies in VWD 2A/IIC, IID and IIE, we investigated VWF variants with patient-derived mutations either in the VWF pro-peptide or in domains D3 or CK. Additionally to static assays and molecular dynamics (MD) simulations we used microfluidic approaches to perform a detailed investigation of the shear-dependent function of VWD 2A mutants. For each group, we found distinct characteristics in their intracellular localisation visualising specific defects in biosynthesis which are correlated to respective multimer patterns. Using microfluidic assays we further determined shear flow-dependent characteristics in polymer-platelet-aggregate formation, platelet binding and string formation for all mutants. The phenotypes observed under flow conditions were not related to the mutated VWF domain. By MD simulations we further investigated how VWD 2A/IID mutations might alter the ability of VWF to form carboxy-terminal dimers. In conclusion, our study offers a comprehensive picture of shear-dependent and shear-independent dysfunction of VWD type 2A mutants. Furthermore, our microfluidic assay might open new possibilities for diagnosis of new VWD phenotypes and treatment choice for VWD patients with shear-dependent VWF dysfunctions that are currently not detectable by static tests.

Shear-induced unfolding activates von Willebrand factor A2 domain for proteolysis.

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 'knotted' 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.