(19d) Dual Role of VWF A2 Domain in Regulating VWF Molecular Size and Thrombus Growth in Circulation

Von Willebrand Factor (VWF) is a large multimeric blood protein (~0.5-10MDa) that aids platelet recruitment, adhesion and thrombus growth at sites of vascular injury. It is an essential protein regulating thrombosis and hemostasis, particularly in the arterial circulation. Larger VWF multimers are more bioactive, with the size of the protein being regulated by two opposing fluid shear dependent processes: proteolysis by the constitutively-active plasma ADAMTS13 which reduces molecular mass, and VWF self-association which enhances protein size. Von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. This paper demonstrates that the shear-dependent unfolding of the VWF A2-domain is a major regulator of protein self-association. This mechanism controls self-association on the platelet GpIbα receptor, on collagen substrates and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain-unfolding due to disulfide bridging of N- and C-terminal residues (‘Lock-VWF’) reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and two mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD Type 2A mutation) enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain (‘ΔA1-proteins’) suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified HDL (high density lipoprotein) and ApoA-I (Apolipoprotein A1) partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13 mediated proteolysis, with low calcium and A2 disease-mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved two balancing shear-dependent molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation under hydrodynamic flow conditions: ADAMTS13 mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.