Research Project
8253695
DESCRIPTION (provided by applicant): Von Willebrand factor (VWF) is a concatameric plasma glycoprotein that has multiple functions in hemostasis. Mutations in VWF in von Willebrand disease (VWD) are the most common cause of heritable bleeding disorders. The length, or number of monomers per concatamer, and flow in blood vessels regulate the hemostatic and thrombotic potency of VWF. VWF acts as a flow sensor, and elevated flow at sites of bleeding in arterioles, and binding to subendothelial collagen, activate VWF. The A1 domain in VWF binds to glycoprotein Ib1 on platelets to form a hemostatic plug. ADAMTS13 is an enzyme that cleaves the A2 domain of VWF, and by cleaving only after unfolding of the A2 domain by hydrodynamic force, regulates its size distribution after secretion in an ultralarge form from endothelial cells and platelets. Inherited or acquired deficiency of ADAMTS13 causes life-threatening thrombotic thrombocytopenic purpura (TTP). Here, we study the A1 and A2 domains, which are particularly important in the shear sensor function of VWF, mutations of which cause qualitative defects in VWD type 2B and 2A, respectively. To appreciate the functions of these domains in the physiologic setting in which tensile force is applied them in vivo, we apply tensile force to the isolated domains using laser tweezers and novel constructs in which the domains are linked to beads using DNA handles. Selected type 2A mutations in the A2 domain will be used to test the hypothesis that there is heterogeneity among patients in whether the A2 domain unfolds at lower force, is more mechanically sensitive, or refolds more slowly, increasing sensitivity to ADAMTS13. Experiments on larger constructs will test the role of domains that neighbor A2 in force-resistance. A Receptor and Ligand in a Single Molecule (ReaLiSM) will be used to characterize the physiologically-relevant force-dependence of the A1-GPIb1 receptor-ligand bond, and how VWD type 2B mutations enhance this bond. ReaLISM is a fusion protein that consists from N to C- terminus of the A1 domain, a polypeptide linker, and the leucine-rich repeat domain of GPIb1. We have found that the receptor-ligand complex acts as a flex-bond, with one state (state 1, flexed) at low force, and a stronger state with lower koff0 at high force (state 2, extended). Experiments on the healthy receptor-ligand complex will further characterize the fine structure and kinetics of this bond. Experiments on gain of function type 2B and platelet-type VWD mutations will test the hypothesis that they stabilize switching to state 2. The use of ReaLiSM for the first time allows us to measure the force dependence of the on-rate for receptor binding to ligand. We will test the hypothesis that tensile force slows the conversion of encounter complexes to the bound complex. Furthermore, we will examine whether after dissociation from state 2, the conformation of the receptor or ligand in state 2 can persist, and enhance the rate of bond formation. The results will be relevant to our long-term goal of improving the diagnosis and treatment of VWD and thrombotic thrombocytopenic purpura, and developing antagonists of the A1-GPIb1 complex to prevent thrombosis. The results will also be of wide significance for understanding the specializations of receptor-ligand bonds that enable cell adhesion in the vasculature to resist strong hydrodynamic forces. 1 PUBLIC HEALTH RELEVANCE: von Willebrand factor (VWF) is a large plasma glycoprotein that crosslinks platelets to stop bleeding from arterioles. It size and potency in blood clotting is regulated by the enzyme ADAMTS13. Deficiency of VWF is the most common heritable cause of bleeding disorders, and deficiency of ADAMTS13 causes life-threatening thrombosis. We will study the function, in the presence of forces that mimic those in blood vessels, of key regulatory domains in VWF.
