Platelet ADP Receptors

血小板 ADP 受体

• 批准号: 7407378
• 负责人: Satya P. Kunapuli
• 金额: $ 44.98万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7407378
• 关键词: ADP Receptors; Adenylate Cyclase; Agonist; Alanine; Alpha Granule; Amino Acids; Aspirin; Atherosclerosis; Basic Amino Acids; Binding; Biochemical; Blood Cells; Blood Platelets; Charge; Combined Modality Therapy; Complement; Cytoplasmic Granules; Data; Drug Combinations; Event; Extracellular Domain; Fibrinogen; Foundations; Funding; GTP-Binding Proteins; Generations; Grant; Growth; Hemostatic function; Hydrophobicity; Inflammation; Label; Laboratories; Ligand Binding; MRS2179; Mammalian Cell; Mediating; Modeling; Molecular; Molecular Genetics; Mus; Mutate; Mutation; Nucleotides; Numbers; P2Y2 receptor; Personal Satisfaction; Phosphorylation; Physiological; Physiological Processes; Platelet Activation; Play; Process; Protein Isoforms; Protein Tyrosine Kinase; Receptor Activation; Relative (related person); Research Personnel; Role; Shapes; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Testing; Thromboembolism; Thrombosis; Thromboxane A2; Thrombus; Work; chemokine; clopidogrel; extracellular; in vivo; in vivo Model; inorganic phosphate; inward rectifier potassium channel; member; mutant; programs; purinoceptor P2Y1; receptor; response; size; src-Family Kinases; uridine triphosphate receptors

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.

描述(申请人提供)：ADP是一种重要的血小板激活激动剂，在止血和血栓形成中发挥重要作用。ADP使血小板改变形状，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对血小板的生理作用和细胞内反应已经被很好地描述，并且介导这些作用的受体已经被克隆。然而，ADP介导的这些生理过程的分子机制仍然不清楚。在之前的资助期间，我们已经证实了血小板上的三种ADP受体亚型，即P2Y1，P2Y12和P2X1，并阐明了血小板中的一些信号机制。在这个授权期，我们建议通过阐明ADP诱导血小板激活的分子机制来加强我们的理解，并评估P2Y受体在血栓形成中的相对贡献。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家族激酶参与了纤维蛋白原受体的激活，血栓素A2的产生，以及P2Y12受体激活下游的PKC-5亚型和Akt的磷酸化。我们建议通过药理学、生化和分子遗传学的方法来检验这一假说。我们推测，ADP中带负电荷的磷酸盐与P2Y12受体胞外区的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的结合。我们将使用嵌合受体和定点突变来验证这一假设。我们将通过定点突变将P2Y12受体中保守的碱性氨基酸残基转变为丙氨酸，在哺乳动物细胞中瞬时表达突变的受体，并检测其对标记核苷酸结合和ADP诱导的腺苷环化酶抑制的影响。此外，我们还将对我们实验室产生的一种具有结构性活性的P2Y12受体进行药理学鉴定。我们推测，在体内，P2Y1和P2Y12受体对血栓的生长和稳定性有协同作用。我们进一步建议通过使用P2Y1或P2Y12缺乏的小鼠以及使用拮抗剂和体内血栓形成和血栓栓塞症的模型来评估P2Y1和P2Y12受体在血栓形成中的相对贡献。