Von Willebrand Factor (VWF) is a multimeric plasma glycoprotein that is critical for both hemostasis and thrombosis. Normal VWF function depends on its size distribution in plasma, regulated via proteolytic cleavage by ADAMTS13. On the one hand, insufficient VWF cleavage leads to thrombotic thrombocytopenic purpura, a disease characterized by microvascular thrombosis;on the other hand, excessive cleavage of VWF leads to Von Willebrand disease, a potentially-fatal bleeding disorder manifested by lack of large VWF multimers in plasma. VWF cleavage by ADAMTS13 is regulated by many factors including denaturing agents like urea, shear stress in blood, VWF glycosylation state, ST2 (a two-domain fragment of ADAMTS13), and VWF domains like A1. It has been found that a single recombinant A2 domain can be unfolded by an increasing pulling force and the unfolding force depends on the force loading rate. A2 unfolding facilitates its cleavage by ADAMTS13. However, mechanics of A2 unfolding was rarely studied in the context of a VWF multimer. How ST2, A1, and glycosylation state regulate A2 unfolding and cleavage has never been examined from a mechanical perspective. Therefore, our overall goal of this project is to characterize force-induced A2 unfolding and its relationship with VWF cleavage by ADAMTS13, as well as how this relationship is influenced by ST2, A1, and VWF glycosylation state.
Our specific aims are: 1) determine whether A2 is unfolded first among 14 VWF domains when a VWF multimer is stretched by force, 2) determine whether A1 stabilizes A2 mechanically, manifested by larger unfolding force and less A2 cleavage in the presence of A1, and 3) determine whether ST2 and deglycosylation affect A2 unfolding and cleavage in an additive fashion, manifested by additive A2 unfolding forces. These studies will be carried out mainly with the optical trap and the recombinant protein technology. The successful completion of these studies will improve our understanding of how VWF mechanics is related to VWF cleavage. It will also provide a platform for our multidisciplinary effort of exploring novel methodologies for screening drugs and designing effective interventions for VWF-related diseases.
This research project investigates how the A2 domain of von Willebrand factor is unfolded under a pulling force and how A2 unfolding is related to VWF cleavage by ADAMTS13. Excessive VWF cleavage results in von Willebrand disease (VWD), whereas insufficient VWF cleavage results in thrombotic thrombocytopenic purpura (TTP), a disease characterized by microvascular thrombosis.
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