Mechanoregulatory mechanisms of von Willebrand disease and thrombosis

项目总结 血小板膜受体黏附是止血和血栓形成的关键起始步骤 糖蛋白Ibα(GPIBα)到血管性血友病因子(VWF)，一种大的、多结构域的聚合血液 糖蛋白。血管性血友病因子促进血小板GPIBα与其A1结构域黏附的精确机制 只是在止血或血栓形成期间，而不是在正常循环中，目前还不清楚。了解这些 机制是开发更有效的血管血栓诊断和治疗方法的关键 以及最常见的遗传性出血性疾病，von Willebrand病(VWD)。申请者是Dr。 傅红霞将开发创新的单分子方法来研究全长VWF级联子 导师蒂莫西·斯普林格博士的实验室中的止血功能。该系统可用于 通过以下方式同时监控分子内vwf构象转变和GPIBα结合 将快速气压驱动剪切流与全内荧光显微镜(TIRF)相结合。 利用这个系统，傅博士将检验流体动力流动直接导致构象变化的假设。 在VWF级联体中，从紧凑到拉长的形式的转变，从而暴露出高亲和力，力- 依赖结合部位招募GPIBα(血小板黏附)和额外的vwf分子(vwf自身 协会)。将这一系统扩展到包括复杂的生理和病理生理学特征 她将进一步开发一种新的基于荧光的分析方法，用于检测原液中的VWF功能 在有或无VWD相关突变的血管内皮细胞内表达的Webel-Palade小体。 这项工作将提供对初级止血的调控机制的直接洞察， 血栓形成和出血障碍，建立了受体-配体的机械感觉控制范例 结合亲和力。它还将为傅博士提供细胞生物学、基因组编辑、干细胞、 和生物医学，补充了她在定量科学方面的专长。傅博士将把她100%的时间 在斯普林格博士的直接指导下进行研究。傅博士的研究计划将建立新的 从单分子到细胞的VWF功能和VWF相关疾病的定量检测 规模，为这一领域的继续研究和职业发展提供了坚实的基础 生物医学领域的独立研究员阶段。

PROJECT SUMMARY A critical initiating step in hemostasis and thrombosis is adhesion of platelet membrane receptor glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF), a large, multidomain, polymeric blood glycoprotein. The precise mechanisms whereby VWF promotes platelet GPIbα 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 GPIbα 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 GPIbα (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.