A critical initiating step in hemostasis and thrombosis is adhesion of platelet membrane receptor glycoprotein Ib? (GPIb?) to von Willebrand factor (VWF), a large, multidomain, polymeric blood glycoprotein. The precise mechanisms whereby VWF promotes platelet GPIb? adhesion to its A1 domain only during hemostasis or thrombosis, but not in normal circulation, are not yet clear. Understanding these mechanisms is essential for developing more effective diagnostics and therapeutics for vascular thrombosis and the most common hereditary bleeding disorder, von Willebrand disease (VWD). The applicant, Dr. Hongxia Fu, will develop innovative single-molecule approaches to study full-length VWF concatemers hemostatic function in the laboratory of the mentor, Dr. Timothy Springer. This system can be utilized to monitor both intramolecular VWF conformational transitions and GPIb? binding simultaneously by combining rapid air pressure-actuated shear flow with total internal fluorescence microscopy (TIRF). Utilizing this system, Dr. Fu will test the hypothesis that hydrodynamic flow directly induces a conformational transition in VWF concatemers from a compact to an elongated form, thereby exposing high-affinity, force- dependent binding sites to recruit both GPIb? (platelet adhesion) and additional VWF molecules (VWF self- association). To expand this system to include complex features of physiological and pathophysiological blood flow, she will furthermore develop a new fluorescence-based assay for VWF function in bulk solutions and its expression in Weibel-Palade bodies inside endothelial cells with or without VWD-relevant mutations. This work will provide direct insight into the regulatory mechanisms governing primary hemostasis, thrombosis, and bleeding disorder, establishing a paradigm for mechanosensory control of receptor-ligand binding affinity. It also will provide Dr. Fu with additional training in cell biology, genome editing, stem cells, and biomedicine, complementing her expertise in quantitative sciences. Dr. Fu will devote 100 % of her time to research under the direct mentorship of Dr. Springer. Dr. Fu's research program will establish new quantitative assays for VWF function and VWF-related diseases, from the single molecule to the cellular scale, providing a firm foundation for continued research in this area and career development to the independent investigator stage in biomedicine.
The blood is constantly under force, and dysregulation of blood clotting can be disastrous, causing either bleeding disorder or thrombosis, in which a critical step is activation of von Willebrand factor (VWF), a component of the blood that is theorized to be a `force sensor' and change shape when subjected to suddent changes in blood flow. We will develop multidisciplinary approaches to directly observe this flow- induced shape change in single molecules of VWF, and determine the factors that regulate VWF and how mutations cause bleeding disorders and thrombosis in patients. This work will establish much-needed laboratory models for human bleeding disorders and improve our fundamenal understanding of how our bodies sense the physical world and respond in specific ways to prevent disease.
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