Unfolding, refolding and proteolysis of the von Willebrand Factor A2 domain under tensile force

Abstract

von Willebrand Factor (vWF) is a plasma protein essential to the early stages of blood coagulation. Shear induced proteolysis at the A2 domain of vWF is an important mechanism to convert the highly thrombogenic, ultra large vWF multimers to smaller multimeric forms and, consequently, to prevent overgrown thrombus. It has been hypothesized that the A2 domain undergoes conformational changes in response to tensile force and exposes its Tyr842-Met843 scissile bond for cleavage by ADAMTS13, a metalloprotease found in the circulating blood. In this work, the unfolding and folding kinetics of the A2 domain is studied using optical tweezers under pulling forces that mimicked the tensile forces exposed to vWF multimers in the vasculature. We demonstrate that A2 domain is unstable upon pulling and unfolds at between 7 to 14 pN at loading rates ranging from 0.3 to 300 pN/s. Once unfolded, stressfree refolding of A2 domain takes 1.9 seconds, increasing dramatically with tensile force. Unfolded A2 domain was cleaved by physiological concentration of ADAMTS13 with a catalytic rate constant of 0.14/s. The results suggest that the A2 domain is unraveled at physiological tensile forces in vivo and its slow refolding process ensures the enzymatic reaction of ADAMTS13. Hence, the A2 domain acts as a force sensor that triggers ADAMTS13 cleavage at the pico-newton force range.