Allosteric Regulation of Platelet Integrin Function

血小板整合素功能的变构调节

• 批准号: 9511908
• 负责人: Joel S. Bennett
• 金额: $ 45.09万
• 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/9511908
• 关键词: Address; Adhesions; Adhesives; Affinity; Agonist; Allosteric Regulation; Arteries; Atherosclerosis; Attenuated; Behavior; Binding; Binding Proteins; Binding Sites; Biophysics; 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; Impairment; 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 Disulfide Isomerase; Proteins; Regulation; 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; amphiphilicity; base; clinical application; cohesion; design; experimental study; in vivo; inhibitor/antagonist; laser tweezer; medical schools; nanomechanics; novel; prevent; professor; single molecule; small molecule libraries; synergism; 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：项目摘要 当代观点表明，蛋白质是现有种群的共同体 在室温下经历明显的结构波动。因此，变构效应子通过 改变这些构象和动态状态之间的平衡。与这些一致 视图，我们发现纯化的LLB 3，纤维蛋白原的血小板受体，可介导血小板 聚集是活性和非活性分子的合奏。此外，使用光学镊子 测量与IIB 3结合并解开纤维蛋白原的纳米力学，我们发现 活跃的IIB 3至少存在两个相互性构象，它们的不同 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，我们假设通过使用变构抑制剂调节IIB 3的活性状态， 它可以以较低的亲和力构象稳定，从而损害了更多的形成 高剪切区域的机械稳定的血栓，例如狭窄动脉中的那些 但与此同时，保留了足够的血小板功能来使普通止血。因此， 该项目的目标是将IIB 3结构与其动态行为联系起来，并将 开发新型的变构IIB 3抑制剂的目标，通过 防止形成较高的亲和力，更稳定，IIB 3-纤维蛋白原 复合物。该项目由两个具体目标组成。在特定目标1中，我们将确定 主动IIB 3的区域参与其变构转化从较低的亲和力构象到 较高的亲和力构象与纤维蛋白原形成更机械稳定的复合物 和纤维蛋白。为了识别变构抑制的靶标，这些区域中突变的影响 IIB 3-纤维蛋白原键的寿命和强度将在单分子下测量 使用光学镊子等级。然后，我们将通过实验筛选化学库和 计算筛选分子数据库的潜在变构抑制剂的活性将会 使用光学镊子和体内使用小鼠血栓形成模型在体外进行验证。这 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是 解决了蛋白质二硫异构酶在变构IIB 3调节中的作用。在 特定目标2，我们将研究IIB和3个胞质域的动态行为，测试 通过细胞骨架蛋白Talin-1和Kindlin-3激活IIB 3的假设是一个 合作活动；两种蛋白质与其结合位点在3个胞质结构域的关联 发生在破坏有利的3膜结合的热力学费用 互动。将进行生物物理实验，以确定 Talin和Kindlin结合，膜磷脂在此过程中的作用，以及是否是否 通过研究IIB 3获得的结果可以推断到其他调节的整合素。