Molecular mechanisms of platelet activation and adhesion

人血小板质膜含有数百种不同的蛋白质，这些蛋白质控制着重要的功能，包括与细胞外基质的黏附、信号转导、血小板聚集和凝块回缩。血小板相互黏附和与细胞外基质黏附能力的核心是丰富的细胞表面黏附分子的供应，包括整合素家族的成员，它们以不同的激活状态存在。这些细胞黏附受体反过来将信号传递到细胞内部，并对来自细胞内部的信号做出反应。然而，调控整合素并最终激活血小板的信号通路的分子细节仍然不完全清楚。因此，这个项目的目标是检查血小板的激活，然而，仍然是血小板的激活和粘连。提出了三个具体目标。为了更好地了解整合素基因多态性影响与血小板相关的病理生理状况的可能性，如冠状动脉疾病、高血压和中风，特殊目的1试图调查新发现的具有结构性活性的整合素形式影响血小板功能的能力。表达野生型和激活型主要血小板整合素αIIbeta3的转基因细胞株将被检测其附着和扩散到细胞外基质蛋白上的能力，并将信号传导到细胞内。选择的整合素激活突变对血小板功能的影响也将在体内使用转基因小鼠进行检测，转基因小鼠的血小板已经被设计成表达活化形式的AlphaIIbbeta3。第二个和第三个特定目标将探索新发现的抑制性受体家族成员PECAM-1调节血小板激活和整合功能的能力。特定目的2提出了一种假设，即由PECAM-1和蛋白酪氨酸磷酸酶SHP-2组成的信号复合体调节信号转导、整合素激活和由主要的血小板Fc受体FcGammaRIIa启动的血小板聚集。我们将使用人血小板、稳定表达FcGammaRIIa+/-野生型和突变形式的PECAM-1的细胞系以及来自PECAM-1缺陷小鼠的小鼠血小板来检测PECAM-1减少或抑制FcGammaRIIa介导的细胞激活的能力。了解FcGammaRIIa的开启和关闭具有重要的生理和临床意义，因为免疫复合物激活FcGammaRIIa及其随后的细胞内信号转导是导致许多血小板免疫紊乱的原因，如肝素诱导的血小板减少和血栓形成。最后，特定目标3验证了这一假设，即PECAM-1/SHP-2复合体选择性地减弱了最近发现的胶原受体GPVI/FcRGamma链复合体介导的血小板对胶原的反应。总而言之，这些研究代表了一系列协调一致的研究，旨在促进我们对血小板生理学的了解，并在输血治疗、血小板储存以及血小板功能和免疫紊乱的管理方面取得改进。

The human platelet plasma membrane contains several hundred different proteins that control crucial functions, including adhesion to extracellular matrix, signal transduction, platelet aggregation, and clot retraction. Central to the ability of platelets to adhere to each other and to extracellular matrix is an abundant supply of cell surface adhesion molecules, including members of the integrin family, that exist in varying states of activation. These cell adhesion receptors, in turn, transmit signals into, and respond to signals from, the cell interior. The molecular details of the signaling pathways that regulate integrin, and ultimately platelet, activation, however, remain incompletely understood. The goal of this project, therefore, is to examine platelet, activation, however, remain platelet activation and adhesion. Three Specific Aims are proposed. To better understand the potential for integrin polymorphisms to affect platelet-related pathophysiological conditions such as coronary artery disease, hypertension, and stroke, Specific Aim 1 seeks to investigate the ability of newly identified forms of constitutively active integrins to affect platelet function. Transfected cell lines expressing wild-type versus activated forms of the major platelet integrin, alphaIIbeta3, will be examined for their ability to adhere to and spread on extracellular matrix proteins and transduce signals into the cell. The effects of selected integrin-activated mutations on platelet function will also be examined in vivo using transgenic mice whose platelets have been engineered to express activated forms of alphaIIbbeta3. The second and third Specific Aims will explore the ability of a newly identified members of the Inhibitory Receptor family, PECAM-1, to modulate platelet activations and integration function. Specific Aim 2 proposes to test the hypothesis that a signaling complex consisting of PECAM-1 and the protein-tyrosine phosphatase, SHP-2, regulates signal transduction, integrin activation, and platelet aggregation initiated by the major platelet Fc receptor for IgG, FcgammaRIIa. We will examine the ability of PECAM-1 to reduce or inhibit FcgammaRIIa-mediated cellular activation using human platelets, stably-transfected cell lines expressing FcgammaRIIa +/- wild-type and mutant forms of PECAM-1, and murine platelets derived from PECAM-1 deficient mice. Understanding how FcgammaRIIa is turned on and off is both physiologically and clinically important, since its activation by immune complexes and its subsequent intracellular signaling is responsible for a number of platelet immunological disorders such as heparin-induce thrombocytopenia and thrombosis, Finally, Specific Aim 3 tests the hypothesis that the PECAM- 1/SHP-2 complex selectively attenuates platelet responses to collagen mediated by a recently identified collagen receptor, the GPVI/FcRgamma-chain complex. Together, these studies represent a coordinated line of investigation designed to advance our understanding of platelet physiology and lead to improvements in transfusion therapy, platelet storage, and management of platelet functional and immunological disorders.