Molecular mechanisms of platelet activation and adhesion

人血小板质膜含有数百种不同的蛋白质，控制关键功能，包括粘附到细胞外基质，信号转导，血小板聚集和凝块收缩。血小板相互粘附和粘附于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，包括以不同活化状态存在的整联蛋白家族的成员。反过来，这些细胞粘附受体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，调节整合素并最终调节血小板活化的信号通路的分子细节仍不完全清楚。因此，本项目的目的是检测血小板的活化，但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的病理生理条件，如冠状动脉疾病，高血压和中风的潜力，具体目标1旨在研究新发现的组成型活性整合素影响血小板功能的能力。将检查表达主要血小板整联蛋白α II β 3的野生型与活化形式的转染细胞系粘附和在细胞外基质蛋白上扩散的能力以及向细胞内传递信号的能力。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查，所述转基因小鼠的血小板已被工程化以表达活化形式的α IIb β 3。第二个和第三个特定目的将探索抑制性受体家族新鉴定的成员PECAM-1调节血小板活化和整合功能的能力。具体目的2提出检验以下假设：由PECAM-1和蛋白酪氨酸磷酸酶SHP-2组成的信号传导复合物调节信号转导、整联蛋白活化和由IgG的主要血小板Fc受体Fc γ RIIa引发的血小板聚集。我们将使用人血小板、表达Fc γ RIIa +/-野生型和突变型PECAM-1的稳定转染细胞系以及来源于PECAM-1缺陷小鼠的鼠血小板，检查PECAM-1降低或抑制Fc γ RIIa介导的细胞活化的能力。理解Fc γ RIIa如何开启和关闭在生理学和临床上都是重要的，因为其通过免疫复合物的活化及其随后的细胞内信号传导是许多血小板免疫学病症如肝素诱导的血小板减少症和血栓形成的原因。最后，具体目标3测试了PECAM- 1/SHP-1的假设。2复合物选择性地减弱血小板对胶原蛋白的反应，该胶原蛋白由最近鉴定的胶原蛋白受体GPVI/FcR γ链复合物介导。总之，这些研究代表了一个协调的调查路线，旨在促进我们对血小板生理学的理解，并导致输血治疗，血小板储存和血小板功能和免疫疾病的管理的改善。

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.