Inhibiting biomechanical platelet aggregation to prevent arterial thrombosis without compromising hemostasis

项目概要/摘要 动脉血栓形成是指动脉中形成异常血凝块，是一种高度致命的疾病 每年夺去约 500,000 美国人生命的疾病。动脉血栓形成的一个标志性特征是血压升高 狭窄产生的剪切速率和剪切力，有利于血小板向血管聚集 闭塞。不幸的是，目前的治疗策略无法有效抑制血栓形成的影响 病理性血流，并有失血过多的重大风险。本提案的总体目标 就是建立一个机制驱动的策略来更好地治疗动脉血栓。我们实验室以前的工作和我的 研究发现，病理性血流的促血栓效应是由一种称为“病理性血流”的现象引起的。 “生物力学血小板聚集”，其中机械力驱动血小板交联。冯·维勒布兰德 因子 (VWF) 是一种血浆蛋白，通过与血小板受体 GPIbα 结合来介导血小板交联。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，抑制其聚集可抑制动脉血栓形成 不影响止血。我提出4个目标逐步建立抗血栓方法 针对生物力学血小板聚集。目标 1 将结合生物力学和血液学检测 研究 M13 抑制生物力学血小板聚集功能的机制，但没有 附着力。目标 2 将建立一种基于微流体的检测方法，同时评估血小板粘附和 剪切诱导的血栓形成，可筛选针对生物力学血小板的抗血栓药物 聚合。在目标 3 中，抗 VWF、NMC4 的单克隆抗体被鉴定为功能比对的 与我的抗血栓策略。我将与我的博士后实验室合作并使用 NMC4 作为原型 设计和生产抗血栓药物。将建立一个三步筛选程序来选择 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治疗 针对生物力学血小板聚集也很重要的另一种血小板受体的策略：整合素αIIbβ3。我会 探索抑制 VWF 和 αIIbβ3 介导的生物力学血小板聚集的有效性和安全性 治疗因糖尿病而加剧的动脉血栓形成。总体而言，这项研究将在 制定全面的职业发展计划，旨在帮助我实现作为一名员工的职业目标 血管生物学、机械生物学和生物工程跨学科领域的独立研究员。 在 K99 阶段，我将继续获得生化、临床前和转化方法方面的专业知识。 我的导师、合作者和顾问将共同指导我成功过渡到 授予期间的独立性。

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.