Mechanoregulatory mechanisms of von Willebrand disease and thrombosis

项目摘要 血小板膜受体的粘附是止血和血栓形成的关键起始步骤 糖蛋白Ibα（GPIbα）转化为血管性血友病因子（VWF）， 糖蛋白VWF促进血小板GPIbα与其A1结构域粘附的确切机制 只是在止血或血栓形成，而不是在正常的循环，目前尚不清楚。了解这些 机制对于开发更有效的血管血栓诊断和治疗方法至关重要 以及最常见的遗传性出血性疾病，血管性血友病（VWD）。申请人博士 Hongxia Fu将开发创新的单分子方法来研究全长VWF串联体 在导师Timothy Springer博士的实验室中进行止血功能研究。该系统可用于 同时监测分子内VWF构象转换和GPIbα结合， 结合快速气压驱动剪切流与全内荧光显微镜（TIRF）。 利用这个系统，傅博士将测试流体动力学流动直接诱导构象的假设。 在VWF多联体中从紧凑形式转变为伸长形式，从而暴露高亲和力，力- 依赖性结合位点募集GPIbα（血小板粘附）和额外的VWF分子（VWF自身）。 协会）。为了扩展该系统，以包括生理和病理生理的复杂特征， 她将进一步开发一种新的基于荧光的检测方法，用于检测原液中VWF的功能 以及其在具有或不具有VWD相关突变的内皮细胞内的韦伯-帕拉德小体中的表达。 这项工作将提供直接洞察的监管机制，管理初级止血， 血栓形成和出血性疾病，建立了受体-配体的机械感觉控制的范例 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业知识。傅医生会把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 VWF功能和VWF相关疾病的定量分析，从单分子到细胞 规模，提供了一个坚实的基础，继续在这一领域的研究和职业发展， 生物医学的独立研究者阶段。

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.