Molecular mechanisms of platelet activation and adhesion

人血小板质膜包含数百种不同的蛋白质，这些蛋白质控制着细胞外基质的粘附、信号转导、血小板聚集和凝块缩回等关键功能。血小板相互粘附和细胞外基质的能力的核心是细胞表面粘附分子的丰富供应，包括整合素家族的成员，它们以不同的激活状态存在。这些细胞粘附受体依次向细胞内部传递信号，并对来自细胞内部的信号作出反应。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不完全清楚。因此，本项目的目标是检查血小板的活化，然而，仍然是血小板的活化和粘附。提出了三个具体目标。为了更好地了解整合素多态性影响血小板相关病理生理状况（如冠状动脉疾病、高血压和中风）的潜力，Specific Aim 1旨在研究新发现的组成活性整合素影响血小板功能的能力。转染的细胞系表达野生型与活化型的主要血小板整合素α iibeta3，将检查它们粘附和扩散到细胞外基质蛋白以及将信号转导到细胞内的能力。选择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，这些小鼠的血小板已被改造成表达活化形式的alphaIIbbeta3。第二和第三个特定目标将探索抑制受体家族新发现的成员PECAM-1调节血小板激活和整合功能的能力。特异性目的2提出验证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、整合素激活和主要血小板Fc受体IgG FcgammaRIIa引发的血小板聚集的假设。我们将使用人血小板、表达FcgammaRIIa +/-野生型和突变型PECAM-1的稳定转染细胞系以及PECAM-1缺陷小鼠的小鼠血小板来检测PECAM-1降低或抑制FcgammaRIIa介导的细胞激活的能力。了解FcgammaRIIa如何开启和关闭在生理学和临床上都很重要，因为它被免疫复合物激活及其随后的细胞内信号传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症和血栓形成。最后，特异性目的3验证了PECAM- 1/SHP-2复合物选择性地减弱血小板对胶原蛋白的反应的假设，这种反应是由最近发现的胶原受体介导的。GPVI/ fγ链复合物。总之，这些研究代表了一条协调的调查路线，旨在促进我们对血小板生理学的理解，并导致输血治疗，血小板储存以及血小板功能和免疫疾病管理的改进。

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.