Platelet ADP Receptors

描述（由申请人提供）：ADP是血小板活化的重要激动剂，在止血和血栓形成中起主要作用。ADP使血小板改变形状，聚集，释放颗粒内容物，并产生另一种有效的血小板激动剂血栓素A2。ADP对血小板的生理作用和细胞内反应已被很好地表征，介导这些作用的受体已被克隆。然而，这些adp介导的生理过程的分子机制尚不清楚。在之前的资助期间，我们已经在血小板上证明了3种ADP受体亚型，P2Y1， P2Y12和P2X1，并阐明了血小板中的一些信号传导机制。在本次资助期间，我们建议通过阐明adp诱导血小板活化的分子机制来加强我们的理解，并评估P2Y受体对血栓形成的相对贡献。我们假设不同的酪氨酸激酶在不同的P2Y受体亚型下游被激活。我们在之前的研究中得出的结论是，Src家族激酶有助于纤维蛋白原受体的激活、血栓素A2的生成、PKC-5亚型的磷酸化和P2Y12受体激活下游的Akt。我们建议通过药理学、生物化学和分子遗传学的方法来检验这一假设。我们假设ADP中带负电荷的磷酸盐与P2Y12受体胞外结构域中一些带正电荷的碱性氨基酸残基之间的电荷相互作用参与了配体结合。我们将使用嵌合受体和定点诱变来验证这一假设。我们将通过位点定向诱变将P2Y12受体中保守的碱性氨基酸残基改变为丙氨酸，在哺乳动物细胞中短暂表达突变受体，并确定其对标记核苷酸结合和adp诱导的腺苷酸环化酶抑制的影响。此外，我们将在药理学上表征我们实验室产生的组成活性P2Y12受体。我们假设P2Y1和P2Y12受体协同促进血栓在体内的生长和稳定。我们进一步提出评估P2Y1和P2Y12受体对血栓形成的相对贡献，使用P2Y1或P2Y12缺陷小鼠，用拮抗剂和体内血栓形成和血栓栓塞模型进行治疗。

DESCRIPTION (provided by applicant): ADP is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. ADP causes platelets to change their shape, to aggregate, to release contents of granules, and to produce thromboxane A2, another potent agonist for platelets. The physiological effects and intracellular responses of ADP on platelets have been well characterized and the receptor mediating these effects has been cloned. However the molecular mechanism of these ADP-mediated physiological processes remains obscure. During the previous grant period, we have demonstrated 3 ADP receptor subtypes, P2Y1, P2Y12, and P2X1, on platelets and elucidated a number of signaling mechanisms in platelets. In this grant period, we propose to enhance our understanding by elucidating the molecular mechanisms of ADP-induced platelet activation and evaluate the relative contribution of P2Y receptors to thrombus formation. We hypothesize that different tyrosine kinases are activated downstream of different P2Y receptor subtypes. Our studies in the previous grant period led to the conclusions that Src family kinases contribute to fibrinogen receptor activation, thromboxane A2 generation, and phosphorylation of PKC-5 isoform and Akt downstream of the P2Y12 receptor activation. We propose to test this hypothesis by pharmacological, biochemical, and molecular genetic approaches. We hypothesize that charge interactions between negatively charged phosphates in ADP and some of the positively charged basic amino acid residues in the extracellular domains of the P2Y12 receptor are involved in ligand binding. We will test this hypothesis using chimeric receptors and site-directed mutagenesis. We will change the conserved basic amino acid residues in the P2Y12 receptor to alanine by site-directed mutagenesis, express the mutant receptors transiently in mammalian cells, and determine the effect on binding of labeled nucleotides and ADP-induced inhibition of adenylyl cyclase. In addition, we will pharmacologically characterize a constitutively active P2Y12 receptor generated in our lab. We hypothesize that the P2Y1 and P2Y12 receptors synergistically contribute to the thrombus growth and stability in vivo. We further propose to evaluate the relative contribution of the P2Y1 and the P2Y12 receptors to thrombus formation, using P2Y1 or P2Y12 deficient mice and treatment with antagonists and models of in vivo thrombosis and thromboembolism.