D4-mediated regulation of VWF by ADAMTS13 - Project Summary: When damage to a blood vessel occurs from injury, hemostasis and thrombosis are initiated to prevent blood loss. This process is largely driven by von Willebrand factor (VWF), a multimeric blood glycoprotein. Following endothelial injury, VWF is activated by increased shear levels in the blood and initiates hemostasis by recruiting platelets to the injury site. VWF is therefore a first responder in bleeding prevention and a primary actor driving thrombus formation. VWF activity is tightly regulated by a disintegrin and metalloproteinase with a thrombospondin type 1 motif member 13 (ADAMTS13), a blood metalloprotease that cleaves VWF to reduce its activity. Therefore, loss of ADAMTS13 activity can lead to over-activation of VWF and increases the risk for microangiopathies, stroke and other thrombotic diseases. Notably, thrombotic thrombocytopenic purpura (TTP) is an immuno-thrombotic disease characterized by decreased ADAMTS13 levels. To understand the disease pathogenesis arising from dysregulation of the VWF-ADAMTS13 axis, we need to have a fundamental understanding of the molecular mechanisms governing VWF regulation by ADAMTS13. ADAMTS13 is recruited to the VWF D4 domain through its CUBs domains and subsequently cleaves VWF to reduce its activity. Under low shear, plasma VWF is thought to exist in a globular form with its D4 domain exposed. However, ADAMTS13 is constitutively associated with VWF despite D4’s presumed accessibility with very low affinity. It remains unknown how increases in shear lead to enhanced ADAMTS13 recruitment to D4. Therefore, we seek to investigate the D4-CUBs binding interface at the molecular level and understand how the D4-CUBs interaction regulates VWF function in response to shear. We hypothesize that the D4-CUBs binding site is protected in VWF under low shear. In Specific Aim 1, I will define the role of VWF D4-mediated dimerization in shielding the D4-CUBs binding site. In Specific Aim 2, I will determine the mechanism by which the D4-CUBs binding site is protected in the full-length VWF and how it dynamically responds to changes in shear levels. In Specific Aim 3, I will inhibit D4-CUBs binding in vivo and determine the downstream physiological effects of decreased VWF regulation by ADAMTS13. Overall, completion of the proposed studies will further our mechanistic understanding of VWF regulation by ADAMTS13 to help develop potential antithrombotic and antibleeding therapeutics.
