Allosteric Regulation of Platelet Integrin Function

血小板整合素功能的变构调节

• 批准号: 8909173
• 负责人: Joel S. Bennett
• 金额: $ 43.22万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8909173
• 关键词: Address; Adhesions; Adhesives; Affinity; Agonist; Allosteric Regulation; Arteries; Atherosclerosis; Attenuated; Behavior; Binding; Binding Sites; Blood Platelets; Blood Vessels; Collagen; Complex; Cytoplasmic Tail; Cytoskeletal Proteins; Cytosol; Databases; Deuterium; Dissociation; Distal; Doctor of Medicine; Environment; Equilibrium; Event; Excess Mortality; Fibrin; Fibrinogen; Fibrinogen Receptors; Genetic; Goals; Hemorrhage; Hemostatic function; Hydrogen; In Vitro; Integrins; Intravenous; Measurement; Measures; Mechanics; Mediating; Medicine; Membrane; Modeling; Molecular; Molecular Conformation; Mus; Mutation; Myocardial Infarction; Oral; Pathogenesis; Pennsylvania; Pharmacology; Phospholipids; Physiology; Platelet aggregation; Play; Population; Principal Investigator; Process; Protein Binding; Protein Disulfide Isomerase; Proteins; Regulation; Relative (related person); Rest; Role; Sampling; Signal Transduction; Site-Directed Mutagenesis; Spectrometry; Spectrum Analysis; Stroke; Structure; Tail; Talin; Temperature; Testing; Thermodynamics; Thrombosis; Thrombus; Time; Transmembrane Domain; Trauma; Universities; base; clinical application; cohesion; design; in vivo; inhibitor/antagonist; laser tweezer; medical schools; nanomechanics; novel; prevent; professor; research study; single molecule; small molecule libraries; von Willebrand Factor

Project 4: Project Summary Contemporary views indicate that proteins are ensembles of pre-existing populations that undergo significant structural fluctuation at room temperature. Thus, allosteric effectors act by shifting the equilibrium between these conformational and dynamic states. Consistent with these views, we found that purified llb 3, the platelet receptor for fibrinogen that mediates platelet aggregation, is an ensemble of active and inactive molecules. Further, using optical tweezers to measure the nanomechanics of fibrinogen binding to and unbinding from IIb 3, we found that active IIb 3 is present in a minimum of two inter-convertible conformations that differ in their affinity for fibrinogen and in the mechanical stability of the complexes they form with fibrinogen. Accordingly, we postulate that by modulating the active state of IIb 3 with allosteric inhibitors, it can be stabilized in its lower affinity conformation, impairing the formation of more mechanically-stable thrombi in regions of high shear, such as those present in stenotic arteries, but at the same time, preserving sufficient platelet function for ordinary hemostasis. Thus, the objectives of this project are to relate IIb 3 structure to its dynamic behavior, with the ultimate goal of developing novel allosteric IIb 3 inhibitors that attenuate platelet aggregation by preventing the formation of higher affinity, more mechanically stable, IIb 3-fibrinogen complexes. The Project consists of two Specific Aims. In Specific Aim 1, we will identify the regions of active IIb 3 involved in its allosteric conversion from a lower affinity conformation to the higher affinity conformation that forms more mechanically stable complexes with fibrinogen and fibrin. To identify targets for allosteric inhibition, the effect of mutations in these regions on the lifetime and strength of IIb 3-fibrinogen bonds will be measured at the single molecule level using optical tweezers. We will then experimentally screen chemical libraries and computationally screen molecular data bases for potential allosteric inhibitors whose activity will be verified in vitro using the optical tweezers and in vivo using mouse thrombosis models. The relative contributions of fibrin and fibrinogen to the formation of platelet thrombi will also be addressed, as will the role of protein disulfide isomerase in allosteric IIb 3 regulation. In Specific Aim 2, we will study the dynamic behavior of the IIb and 3 cytosolic domains, testing the hypothesis that IIb 3 activation by the cytoskeletal proteins talin-1 and kindlin-3 is a cooperative event; the association of either protein with its binding site on 3 cytosolic domain occurs at the thermodynamic expense of disrupting favorable 3-membrane binding interactions. Biophysical experiments will be performed to determine the order and synergy of talin and kindlin binding, the role of membrane phospholipids in the process, and whether the result obtained by studying IIb 3 can be extrapolated to other regulated integrins.

项目4：项目总结 当代的观点表明，蛋白质是预先存在的群体的集合， 在室温下经历显著的结构波动。因此，变构效应物通过 改变这些构象和动态之间的平衡。符合这些 我们发现，纯化的LLB 3，纤维蛋白原的血小板受体，介导血小板 聚集体是活性和非活性分子的集合。此外，使用光学镊子， 通过测量纤维蛋白原与IIb 3结合和不结合的纳米力学，我们发现， 活性IIb 3以至少两种相互转化的构象存在， 对纤维蛋白原的亲和力以及它们与纤维蛋白原形成的复合物的机械稳定性。 因此，我们假设通过用变构抑制剂调节IIb 3的活性状态， 它可以稳定在其较低的亲和力构象，削弱更多的形成， 高剪切区域中的机械稳定血栓，例如存在于狭窄动脉中的血栓， 但同时保留了足够的血小板功能用于普通止血。因此 本项目的目标是将IIb 3结构与其动态行为联系起来， 目的是开发新型变构IIb 3抑制剂， 阻止形成更高亲和力、更机械稳定的IIb 3-纤维蛋白原 配合物该项目有两个具体目标。在具体目标1中，我们将确定 活性IIb 3的区域参与其从低亲和力构象到低亲和力构象的变构转化， 与纤维蛋白原形成更机械稳定复合物的更高亲和力构象 和纤维蛋白。为了确定变构抑制的靶点，研究了这些区域中突变对 IIb 3-纤维蛋白原键的寿命和强度将在单个分子处测量， 水平使用光学镊子。然后，我们将实验性地筛选化学文库， 计算筛选潜在的变构抑制剂的分子数据库， 在体外使用光镊和在体内使用小鼠血栓形成模型进行验证。的 纤维蛋白和纤维蛋白原对血小板血栓形成的相对贡献也将被 解决，将蛋白质二硫键异构酶在变构IIb 3调节的作用。在 具体目标2，我们将研究IIb和3胞质结构域的动态行为，测试 细胞骨架蛋白talin-1和kindlin-3激活IIb 3的假设是一种 协同事件;任一蛋白质与其3个胞质结构域上的结合位点的结合 以破坏有利的3-膜结合的热力学代价发生 交互.将进行生物物理实验，以确定以下顺序和协同作用： talin和kindlin的结合，膜磷脂在此过程中的作用，以及是否 通过研究IIb 3获得的结果可以外推到其他受调节的整合素。