Inhibiting biomechanical platelet aggregation to prevent arterial thrombosis without compromising hemostasis

项目摘要/摘要 动脉血栓形成是指在动脉中形成异常的血块，是一种高度致命的疾病。 每年夺走约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 狭窄产生的剪切率和剪切力，促进血小板向血管聚集 遮挡。不幸的是，目前的治疗策略在抑制血栓前病变方面无效。 病理性血流过多，并有出血过多的重大风险。这项提案的总体目标是 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的 研究发现，病理性血流的血栓前效应是由一种现象引起的，这种现象称为 “生物力学中的血小板聚集”，指的是机械力驱使血小板发生交联化。冯·威勒布兰德 凝血因子(Vwf)是一种血浆蛋白，通过与血小板受体GPIbα结合来介导血小板的交联。在……里面 在我的初步研究中，我研究了一种人工设计的三个残基的VWF突变，命名为‘M13’，它 抑制VWF在介导生物力学血小板聚集和剪切诱导的血栓形成中的作用，但不能 影响血小板黏附或止血。基于这些发现，我假设：生物力学的血小板 凝集是动脉血栓形成的关键因素，其抑制作用可以抑制动脉血栓形成 而不影响止血效果。我提出4个目标，循序渐进地建立抗血栓方法 以生物力学的血小板聚集为靶点。目标1将结合生物力学和血液学分析 M13‘S抑制生物力学血小板聚集作用机制的研究 粘附力。AIM 2将建立一种基于微流控的分析方法，同时评估血小板粘附性和 剪切诱导血栓形成，这使得针对生物机械血小板的抗血栓药物的筛选成为可能 聚合。在目标3中，抗vwf的单抗nmc4被鉴定为功能配对。 用我的抗血栓策略。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。将建立一个三步筛选程序来选择 功能表现最佳的候选代理。最后，在目标4中，我将扩大我的抗血栓药物 另一种对生物力学血小板聚集也很重要的血小板受体的策略：整合素αIIbβ3。 联合抑制VWF和αIIbβ3介导的生物力学血小板聚集的有效性和安全性 治疗糖尿病加重的动脉血栓形成。总体而言，这项研究将在 设置一个全面的职业发展计划，旨在帮助我实现我作为一名 血管生物学、机械生物学和生物工程交叉学科领域的独立研究员。 在K99阶段，我将继续获得生化、临床前和翻译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 在获奖期内保持独立性。

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.