Mechanical regulation of von Willebrand factor

项目总结/摘要 在人体中，当血管损伤部位形成血栓时，出血就会停止。在快速流动下 条件下，血浆蛋白血管性血友病因子（VWF）在捕获两者中起着不可或缺的作用。 血小板和胶原蛋白在受损的血管壁上，允许血小板栓的形成。之间的粘附 VWF和血小板的相互作用是由VWF的A1结构域和血小板的Ib β链之间的相互作用介导的。 血小板受体GPIb-IX复合物。增强这种相互作用的A1中的功能获得性突变导致类型 2B型血管性血友病（VWD）。靶向A1与GPIb-IX的相互作用已成为一种新兴的治疗策略， 或预防出血和血栓性疾病，尽管这方面的成功非常有限。缺乏 这一进展在很大程度上是由于A1在血液循环中究竟是如何保持不活动的，以及它是如何在血液循环中保持不活动的。 在出血时立即被激活与GPIb-IX结合。我们最近发现了一个自我抑制模块 (AIM)由A1上的N-和C-末端侧翼区及其O-连接聚糖组成，对于 了解A1在出血过程中的机械活化。此外，AIM还可以通过拉伸拉力展开 8到20 pN。基于这些初步发现，我们假设O-连接聚糖结构， 特别是唾液酸，进一步稳定AIM并有助于A1 β GPIb-IX结合的机械调节 并且调节AIM的机械性质可用于治疗或预防血液疾病。我们提出 使用最先进的分析生物物理工具来测试这一潜在的范式转变假设，包括 单分子力谱、单分子荧光显微镜和全原子分子 动力学仿真三个具体目标将被追求来测试的假设。目的1是表征 AIM的结构和生物力学特性。目的2是确定O-连接的自抑制是如何调节的。 AIM中的糖基化。目的3探讨AIM在2B型VWD中的作用及其在治疗中的应用。 完成拟议的研究将确定关键的分子和生物物理机制， 目的机械调节VWF功能和血小板结合，将有助于设计新的治疗方法， 预防和治疗人类血液病的策略。

PROJECT SUMMARY/ABSTRACT In human bodies, bleeding is stopped when a clot is formed at the site of vascular damage. Under rapid flow conditions, the plasma protein von Willebrand factor (VWF) plays an indispensable role in capturing both platelets and collagen on damaged vessel walls, allowing the formation of platelet plugs. The adhesion between VWF and platelets is mediated by the interaction between the A1 domain of VWF and the Ib chain of the platelet receptor GPIb-IX complex. Gain-of-function mutations in A1 that enhance this interaction lead to type 2B von Willebrand disease (VWD). Targeting the A1GPIb-IX interaction has been an emerging strategy to treat or preempt bleeding and thrombotic disorders, though success in this area has been very limited. The lack of progress is due largely to the enigmatic nature of how exactly A1 remains inactive in blood circulation and how it is instantly activated to bind to GPIb-IX upon bleeding. Our recent identification of an autoinhibitory module (AIM), consisting of N- and C-terminal flanking regions on A1 and their O-linked glycans, is crucial for understanding A1 mechanoactivation during bleeding. In addition, AIM can be unfolded by a tensile pulling force of 8 to 20 pN. Based on these preliminary discoveries, we hypothesize that O-linked glycan structures, particularly sialic acids, further stabilize AIM and contribute to the mechanical regulation of A1GPIb-IX binding and that modulating AIM’s mechanical properties can be utilized to treat or preempt blood diseases. We propose to test this potentially paradigm-shifting hypothesis using state-of-the-art analytical biophysical tools, including single-molecule force spectroscopy, single-molecule fluorescence microscopy and all-atom molecular dynamics simulation. Three specific aims will be pursued to test the hypotheses. Aim 1 is to characterize the structure and biomechanical properties of AIM. Aim 2 is to determine how autoinhibition is regulated by O-linked glycosylation in AIM. And Aim 3 is to investigate the role of AIM in type 2B VWD and therapeutic applications. Completion of the proposed studies will identify the key molecular and biophysical mechanisms underlying how AIM mechanically regulates VWF function and platelet binding and will aid in devising novel therapeutic strategies for the prevention and treatment of human blood disease.