Mechanical regulation of von Willebrand factor

项目摘要/摘要 在人体内，当血管损伤处形成凝块时，出血就会停止。在急流下 在此条件下，血浆蛋白von Willebrand因子(Vwf)在捕获两者中起着不可或缺的作用。 受损血管壁上的血小板和胶原蛋白，允许形成血小板塞。它们之间的粘附力 VWF和血小板之间的相互作用是通过VWF的A1结构域和VWF的Ib链之间的相互作用来实现的 血小板受体GPIb-IX复合体。A1中增强这种相互作用的功能获得突变导致类型 2B型血管性血友病(VWD)。以A1GPIB-IX相互作用为目标已经成为一种新兴的治疗策略 或者预防出血和血栓性疾病，尽管在这一领域取得的成功非常有限。缺乏 这一进展在很大程度上是由于A1到底是如何在血液循环中保持不活跃以及它是如何保持不活跃的这一谜团。 在出血时立即被激活以与GPIB-IX结合。我们最近发现了一种自我抑制模块 (AIM)由A1上的N-末端和C-末端侧翼区域及其O-连接的多糖组成，对 了解出血过程中A1的机械激活。此外，AIM可以通过拉伸拉力展开 8~20pN。基于这些初步发现，我们假设O-连接的多糖结构， 特别是唾液酸，进一步稳定AIM，并有助于A1GPIB-IX结合的机械调节 调节AIM的力学性能可用于治疗或预防血液病。我们建议 要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换假说，包括 单分子力谱、单分子荧光显微镜和全原子分子 动力学模拟。将追求三个具体目标来检验这些假说。目标1是描述 AIM的结构和生物力学性能。目的2是确定自体抑制是如何由O-连接调节的 AIM中的糖基化作用。目的3是研究AIM在2B型VWD和治疗应用中的作用。 拟议研究的完成将确定关键的分子和生物物理机制 AIM通过机械调节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战略。

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.