Allosteric Regulation of Platelet Integrin Function

项目4：项目总结 当代观点表明，蛋白质是预先存在的种群的集合， 在室温下经历显著的结构波动。因此，变构效应器通过 改变这些构象和动态之间的平衡。与这些一致 观点，我们发现纯化的LLB3，介导血小板的纤维蛋白原的血小板受体 聚集，是活性和非活性分子的集合。此外，使用光学镊子来 测量纤维蛋白原与IIb 3结合和解离的纳米力学，我们发现 活性IIb 3至少以两种不同的可相互转换的构象存在 与纤维蛋白原的亲和力和与纤维蛋白原形成的络合物的机械稳定性。 因此，我们假设通过用变构抑制剂调节IIb3的活性状态， 它可以稳定在较低的亲和力构象中，从而破坏更多 在高切变区机械稳定的血栓，如狭窄动脉中出现的血栓， 但同时，保留足够的血小板功能以满足普通止血的需要。因此， 该项目的目标是将IIB 3结构与其动态行为联系起来，最终 开发新型变构IIb3抑制剂的目标是通过以下方式抑制血小板聚集 防止形成更高亲和力、更机械稳定的IIb 3-纤维蛋白原 复合体。该项目由两个具体目标组成。在具体目标1中，我们将确定 活性IIb 3区域参与其变构从低亲和力构象到 与纤维蛋白原形成更稳定的复合体的高亲和力构象 和纤维蛋白。为了确定变构抑制的靶点，这些区域的突变对 IIb 3-纤维蛋白原键的寿命和强度将在单分子上测量 使用光学镊子将其调平。然后我们将试验性地筛选化学库和 通过计算筛选分子数据库中潜在的变构抑制剂，其活性将 在体外使用光钳进行验证，在体内使用小鼠血栓模型进行验证。这个 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 阐述了蛋白质二硫键异构酶在变构IIb 3调节中的作用。在……里面 具体目标2，我们将研究IIb和3个胞浆结构域的动态行为，测试 细胞骨架蛋白talin-1和kindlin-3激活IIb3的假设是 合作事件；任一种蛋白与其3胞质结构域上的结合部位的结合 以破坏有利的3-膜结合为热力学代价 互动。将进行生物物理实验，以确定 Talin和kindlin结合，膜磷脂在这一过程中的作用，以及是否 研究IIb3的结果可以外推到其他受调控的整合素。

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.