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

血小板整合素 AlphaIIbBeta3 结构、激活和配体结合：纤维蛋白原、纤维蛋白、D-二聚体和血管性血友病因子

• 批准号: 10390812
• 负责人: Barry Coller
• 金额: $ 77.66万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-09-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10390812
• 关键词: Address; Affinity; Animals; Antibodies; Antiplatelet Drugs; Area; Binding; Binding Sites; Biological Assay; Biology; Blood Platelets; COVID-19; Cells; Chimeric Proteins; Clot retraction; Coagulation Process; Collaborations; Complement; Complex; Computer Simulation; Computing Methodologies; Cryoelectron Microscopy; Data; Data Set; Deuterium; Development; Disease; Docking; Drug Targeting; Electron Microscopy; FDA approved; Fab Immunoglobulins; Family; Fibrin; Fibrin fragment D; Fibrinogen; Fibrinolysis; Future; Goals; Grant; Hemostatic function; Hospitals; Human; Hydrogen; Impairment; Individual; Inflammation; Integrins; Leg; Length; Ligand Binding; Ligands; Mass Spectrum Analysis; Medical; Methods; Molecular; Molecular Conformation; Monitor; Monoclonal Antibodies; Mutation; Mutation Analysis; Myocardial Infarction; Natural Immunity; Nature; Neoplasm Metastasis; Pathologic; Pathology; Pathway interactions; Patients; Peptides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Platelet Glycoprotein GPIIb-IIIa Complex; Platelet aggregation; Plavix; Play; Polymers; Pre-Clinical Model; Preclinical Testing; Process; Proteins; RGD (sequence); Receptor Activation; Resistance; Resolution; Review Literature; Role; Solvents; Stroke; Structure; Testing; Therapeutic; Thrombosis; Thrombus; Time; VWF gene; X-Ray Crystallography; abciximab; antagonist; clopidogrel; computer studies; crosslink; design; high throughput screening; inhibitor; maltose-binding protein; monoclonal antibody production; mutant; nanodisk; novel; novel therapeutics; pre-clinical; protein structure; rational design; receptor; reconstitution; small molecule; thrombolysis; vascular injury; von Willebrand Factor; wound healing

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.

项目总结/摘要 血小板在止血和血栓形成中发挥关键作用，并促成COVID-19病理学。血小板 αIIbβ3是典型的整合素受体，也是一种经过验证的药物靶点，阿昔单抗就是在这种情况下开发的。 第一个获得FDA批准的αIIbβ3拮抗剂。在当前的资助期内，我们合理设计并 合成了新的αIIbβ3和αVβ3拮抗剂，将受体锁定在非活性构象， 赋予治疗益处。αIIbβ3拮抗剂（RUC-4;扎仑非班）目前正处于2期人体研究中， 心脏病发作的医院治疗和αVβ3拮抗剂正在开发用于临床前测试。我们也 开始研究我们对αIIbβ3：1的理解中的关键空白。结合其医学上重要的配体，包括 纤维蛋白原、聚合纤维蛋白、血管性血友病因子（vWf）和交联纤维蛋白。2.开始凝块收缩， 赋予对血栓溶解的抗性。3.从非活动状态转换到活动状态。因此，我们研究了 αIIbβ3与片段D-二聚体的相互作用，并使用一种新的功能分析来剖析αIIbβ3的相互作用 纤维蛋白聚合此外，我们获得了高分辨率的冷冻电子显微镜（EM）结构， αIIbβ3与阿昔单抗和活化单克隆抗体（mAb）PT 25 -2的复合物，提供了以下数据 每种抗体的激活机制。我们还启动了氢氘交换质谱 (HDX)αIIbβ3的研究，以提供配体结合和受体的肽水平动态结构信息 激活以补充冷冻EM数据。新的具体目标建立在PI的长期合作基础上 与计算方法专家玛尔塔菲利佐拉博士和冷冻电镜专家托马斯瓦尔兹博士一起。 具体目标1。A.确定复合物中D-二聚体和αIIbβ3的高分辨率冷冻EM结构 具有：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。为了得到肽- 通过HDX对单独的αIIbβ3和与配体复合的α IIbβ3的水平溶剂暴露面积数据。C.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， mAb的生产，以验证提出的结合机制，以及开发新的配体特异性 小分子拮抗剂。具体目标2。协调使用当前可用和未来的低温EM数据集 用计算和HDX数据来定义整合素腿部结构域沿着αIIbβ3的中间结构， 活化途径具体目标3。为了利用我们的新型功能性聚合纤维蛋白测定法， 表达天然和突变形式αIIbβ3的HEK 293细胞，与mAb和小分子αIIbβ3一致 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 抑制剂的A.生产抗D-二聚体的mAb，抑制血小板-纤维蛋白，但不抑制血小板-纤维蛋白原相互作用。B。 确定选择性损害纤维蛋白原、纤维蛋白或vWf结合的突变，并检查其对凝血的影响 收回C.为了测试来自mAb和小分子的结构和计算研究的预测， 分子配体特异性抑制剂作为动物和最终人类研究的前奏。