Platelet Integrin AlphaIIbBeta3 Structure, Activation, and Ligand Binding: Fibrinogen, Fibrin, D-dimer, and von Willebrand Factor

项目摘要/摘要 血小板在止血和血栓形成中发挥关键作用，并有助于新冠肺炎的病理。血小板 αIIbβ3是一种聚集性整合素受体，在此基础上开发的阿昔单抗是一种有效的药物靶点 格兰特是美国食品和药物管理局批准的第一个αIIbβ3拮抗剂。在当前的资助期内，我们理性地设计和 合成了新的αIIbβ3和αVβ3拮抗剂，将受体锁定在非活性构象中，这可能 提供治疗上的好处。αIIbβ3拮抗剂(RUC-4；Zalunfiban)目前处于第二阶段的人体研究， 心脏病发作的医院疗法和αVβ3拮抗剂正在开发中，用于临床前测试。我们也 开始研究我们对αIIbβ3：1如何结合其医学上重要的配体的理解中的关键差距，包括 纤维蛋白原、聚合纤维蛋白、血管性血友病因子(VWF)和交联型纤维蛋白。2.启动血栓回缩至 使人对溶栓有抵抗力。3.从非活动状态转变为活动状态。因此，我们研究了 αIIbβ3与D-二聚体片段的相互作用及其与αIIbβ3相互作用的功能研究 通过聚合纤维蛋白。此外，我们还获得了高分辨率的低温电子显微镜(EM)结构 αIIbβ3与阿昔单抗和激活的单抗PT25-2的复合物，提供了关于 每种抗体的激活机制。我们还启动了氢-氚交换质谱学。 (Hdx)研究αIIbβ3，以提供关于配体结合和受体的多肽水平的动态结构信息 激活以补充冷冻-EM数据。新的具体目标建立在国际刑警组织长期合作的基础上 与计算方法专家Marta Filizola博士和低温EM专家Thomas Walz博士合作。 具体目标1.确定D-二聚体和αIIbβ3络合物的高分辨低温电子显微镜结构 含有：1)纤维蛋白原片段D100和纤维蛋白原γ-模块；2)VWF C4结构域；3)D-二聚体。B.获得多肽- 通过HDX单独在αIIbβ3上以及在与配体的络合物中计算溶剂暴露面积数据。C.为了获得 来自计算机模拟的补充结构和动力学信息，以指导突变分析和 单抗生产，以验证所提出的结合机制，以及开发新的配体特异性 小分子拮抗剂。具体目标2.协调利用现有的和未来的低温电磁数据集 用计算和HDX数据定义沿着αIIbβ3的整合素支链结构域的中间结构 激活途径。具体目的3.利用我们的新型功能聚合纤维蛋白检测方法，使用血小板和 表达天然和突变型αIIbβ3的HEK293细胞与单抗和小分子αIIbβ3的结合 拮抗剂，以确定独特的αIIbβ3配体特异性结合机制并开发配体特异性结合机制 抑制剂。A.产生抗D-二聚体的单抗，以抑制血小板-纤维蛋白，但不抑制血小板-纤维蛋白原的相互作用。胡麻B. 识别选择性损害纤维蛋白原、纤维蛋白或VWF结合的突变，并检测它们对血栓的影响 撤回。C.用单抗和小分子抗体检验结构和计算研究得出的预测 分子配体特异性抑制剂是动物研究的前奏，最终也是人类研究的前奏。

Project Summary/Abstract Platelets play a key role in both hemostasis and thrombosis and contribute to COVID-19 pathology. Platelet αIIbβ3 is the paradigmatic integrin receptor and a validated drug target, with abciximab, developed under this grant, the first FDA-approved αIIbβ3 antagonist. In the current grant period, we rationally designed and synthesized novel αIIbβ3 and αVβ3 antagonists that lock the receptor in the inactive conformation, which may confer therapeutic benefits. The αIIbβ3 antagonist (RUC-4; zalunfiban) is now in Phase 2 human studies for pre- hospital therapy of heart attacks and the αVβ3 antagonists are being developed for pre-clinical testing. We also began studying key gaps in our understanding of how αIIbβ3: 1. Binds its medically important ligands, including fibrinogen, polymerizing fibrin, von Willebrand factor (vWf), and cross-linked fibrin. 2. Initiates clot retraction to confer resistance to thrombolysis. 3. Transitions from its inactive to its active state. Thus, we studied the interaction of αIIbβ3 with fragment D-dimer and used a novel functional assay to dissect the interaction of αIIbβ3 with polymerizing fibrin. In addition, we obtained high-resolution cryo-electron microscopy (EM) structures of αIIbβ3 in complex with abciximab and with the activating monoclonal antibody (mAb) PT25-2, providing data on each antibody’s mechanism of activation. We also initiated hydrogen-deuterium exchange-mass spectroscopy (HDX) studies of αIIbβ3 to provide peptide-level dynamic structural information on ligand binding and receptor activation to complement cryo-EM data. The new Specific Aims build on the PI’s long-standing collaborations with Dr. Marta Filizola, an expert in computational methods, and Dr. Thomas Walz, an expert in cryo-EM. Specific Aim 1. A. To determine high-resolution cryo-EM structures of D-dimer and of αIIbβ3 in complex with: 1) fibrinogen fragment D100 and fibrinogen γ-module; 2) vWf C4 domain; 3) D-dimer. B. To obtain peptide- level solvent-exposed area data by HDX on αIIbβ3 alone and in complex with the ligands. C. To obtain complementary structural and dynamics information from computer simulations to guide mutational analysis and mAb production to validate the proposed mechanisms of binding, as well as to develop novel ligand-specific small-molecule antagonists. Specific Aim 2. To utilize currently available and future cryo-EM data sets in concert with computational and HDX data to define intermediate structures of the integrin leg domains along the αIIbβ3 activation pathway. Specific Aim 3. To utilize our novel functional polymerizing fibrin assays using platelets and HEK293 cells expressing native and mutant forms of αIIbβ3, in concert with mAbs and small-molecule αIIbβ3 antagonists, to define the unique αIIbβ3 ligand-specific binding mechanisms and develop ligand-specific inhibitors. A. To produce mAbs to D-dimer that inhibit platelet-fibrin, but not platelet-fibrinogen interactions. B. To identify mutations that selectively impair fibrinogen, fibrin, or vWf binding, and examine their impact on clot retraction. C. To test predictions derived from the structural and computational studies with mAb and small- molecule ligand-specific inhibitors as a prelude to animal and ultimately human studies.