Platelet ADP Receptors

描述（由申请方提供）：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导致血小板改变其形状，聚集，释放颗粒内容物，并产生血栓素A2，另一种有效的血小板激动剂。ADP对血小板的生理效应和细胞内反应已被很好地表征，并且介导这些效应的受体已被克隆。然而，这些ADP介导的生理过程的分子机制仍然不清楚。在上一个研究期间，我们已经证明了血小板上的3种ADP受体亚型，P2 Y1，P2 Y12和P2 X1，并阐明了血小板中的一些信号转导机制。在此资助期间，我们建议通过阐明ADP诱导的血小板活化的分子机制来增强我们的理解，并评估P2 Y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P2 Y受体亚型下游被激活。我们在上一个资助期的研究得出的结论是，Src家族激酶有助于纤维蛋白原受体活化，血栓烷A2的产生，以及PKC-5亚型和P2 Y12受体活化下游Akt的磷酸化。我们建议通过药理学、生物化学和分子遗传学方法来验证这一假设。我们推测，ADP中带负电荷的磷酸盐和P2 Y12受体胞外结构域中带正电荷的碱性氨基酸残基之间的电荷相互作用参与配体结合。我们将使用嵌合受体和定点突变来检验这一假设。我们将通过定点突变将P2 Y12受体中保守的碱性氨基酸残基改变为丙氨酸，在哺乳动物细胞中瞬时表达突变受体，并确定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，我们还将对我们实验室中产生的组成型活性P2 Y12受体进行初步表征。我们假设P2 Y1和P2 Y12受体协同促进体内血栓的生长和稳定。我们进一步建议使用P2 Y1或P2 Y12缺陷小鼠和用拮抗剂治疗以及体内血栓形成和血栓栓塞模型来评估P2 Y1和P2 Y12受体对血栓形成的相对贡献。

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.