Inhibiting biomechanical platelet aggregation to prevent arterial thrombosis without compromising hemostasis

项目总结/摘要 动脉血栓形成，描述了动脉中异常血块的形成，是一种高度致命的疾病， 每年夺去约50万美国人生命的疾病。动脉血栓形成的一个象征性特征是动脉壁增厚 狭窄产生剪切速率和剪切力，促进血小板向血管聚集 闭塞不幸的是，目前的治疗策略在抑制血栓形成前作用方面是无效的。 病理性血流，并有大量出血的重大风险。本提案的总体目标是 是建立一种机制驱动的策略来更好地治疗动脉血栓形成。我们实验室以前的工作和我 研究表明，病理性血流的促血栓形成作用是由一种称为 “生物力学血小板聚集”，其中机械力驱动血小板交联。血管性血友病 血管性血友病因子（VWF）是一种血浆蛋白，通过与血小板受体GPIbα结合介导血小板交联。在 在我的初步研究中，我研究了一种人工设计的三残基VWF突变，名为“M13”， 抑制VWF介导的生物力学血小板聚集和剪切诱导的血栓形成，但不 影响血小板粘附或止血。基于这些发现，我假设：生物力学血小板 聚集是动脉血栓形成的关键因素，抑制聚集可以抑制动脉血栓形成 而不影响止血。我提出了4个目标，逐步建立抗血栓形成的方法 靶向生物力学血小板聚集。Aim 1将结合联合收割机生物力学和血液学分析， 研究M13抑制生物力学血小板聚集的作用机制， 粘连目标2将建立一种基于微流控的检测方法，同时评估血小板粘附和 剪切诱导的血栓形成，其允许筛选靶向生物力学血小板的抗血栓药物 聚合来在目标3中，鉴定了抗VWF的单克隆抗体NMC 4，其在功能上与 我的抗血栓策略我将与我的博士后实验室合作，并使用NMC 4作为原型， 设计和生产抗血栓药物。将建立一个3步筛选程序， 具有最佳功能性能的候选代理。最后，在目标4中，我将扩展我的抗血栓药物， 另一种血小板受体整合素αIIbβ3对生物力学血小板聚集也很重要。我会 探讨抑制VWF和αIIbβ3介导的生物力学血小板聚集的有效性和安全性 治疗糖尿病导致的动脉血栓总的来说，这项研究将在 一个全面的职业发展计划，旨在帮助我实现我的职业目标， 他是血管生物学、机械生物学和生物工程学跨学科领域的独立研究员。 在K99阶段，我将继续获得生物化学，临床前和转化方法的专业知识。 我的导师、合作者和顾问将共同指导我成功过渡到 在授标期间保持独立性。

Project Summary/Abstract Arterial thrombosis, which describes the formation of abnormal blood clots in the artery, is a highly fatal disease that claims ~500,000 American lives per year. A symbolic feature of arterial thrombosis is the elevated shear rate and shear force generated by stenosis, which facilitates platelet aggregation towards vessel occlusion. Unfortunately, current therapeutic strategies are ineffective in inhibiting the prothrombotic effects of the pathological blood flow, and have a major risk of excessive bleeding. The overall objective of this proposal is to establish a mechanism-driven strategy to better treat arterial thrombosis. Our lab's previous works and my research identified that the prothrombotic effects of pathological blood flow result from a phenomenon called “biomechanical platelet aggregation”, in which mechanical force drives platelets to crosslink. Von Willebrand factor (VWF) is a plasma protein that mediates platelet crosslinking by binding to platelet receptor GPIbα. In my preliminary study, I worked on an artificially designed triple-residue VWF mutation named `M13', which inhibited VWF in mediating biomechanical platelet aggregation and shear-induced thrombogenesis, but did not affect platelet adhesion or hemostasis. Based on these findings, I hypothesize that: biomechanical platelet aggregation is a key factor to arterial thrombosis, and its inhibition can suppress arterial thrombosis without compromising hemostasis. I propose 4 aims to step-by-step establish anti-thrombotic approaches targeting the biomechanical platelet aggregation. Aim 1 will combine biomechanics and hematology assays to study the mechanism underlying M13's function in inhibiting biomechanical platelet aggregation but not adhesion. Aim 2 will establish a microfluidic-based assay that concurrently assesses platelet adhesion and shear-induced thrombogenesis, which allows the screening of anti-thrombotics targeting biomechanical platelet aggregation. In Aim 3, a monoclonal antibody against VWF, NMC4, was identified to be functionally aligned with my anti-thrombotic strategy. I will collaborate with my postdoctoral lab and use NMC4 as a prototype to design and produce anti-thrombotic agents. A 3-step screening procedure will be established to select candidate agents with the best functional performance. Lastly in Aim 4, I will expand my anti-thrombotic strategy to another platelet receptor also important to biomechanical platelet aggregation: integrin αIIbβ3. I will explore the efficacy and safety of inhibiting both VWF- and αIIbβ3-mediated biomechanical platelet aggregation in treating arterial thrombosis exacerbated by diabetes. Overall, this research will be accomplished in the setting of a comprehensive career development program designed to help me achieve my career goal as an independent researcher in the interdisciplinary field of vascular biology, mechanobiology and bioengineering. During the K99 phase, I will continue to gain expertise in biochemical, preclinical and translational approaches. My mentor, collaborators and consultants will together guide me in the steps towards successful transition to independence over the course of the award period.