Blood Platelet Thromboxane Receptors

血小板血栓素受体

• 批准号: 7800286
• 负责人: GUY C LEBRETON
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7800286
• 关键词: Accounting; Address; Adenosine; Agonist; Binding Sites; Biology; Blood Platelets; Cardiovascular Diseases; Cell Line; Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease; Event; Goals; Human; In Vitro; Integrins; Investigation; Isoprostanes; Link; Measures; Mediating; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; Pathway interactions; Phosphorylation; Phosphorylation Site; Platelet Activation; Platelet aggregation; Prevention; Process; Receptor Signaling; Signal Pathway; Signal Transduction; Site; Thromboxane Receptor; Thrombus; base; defined contribution; in vivo; mutant; novel; prevent; public health relevance; receptor; receptor coupling; research study; rho; therapeutic development

DESCRIPTION (provided by applicant): The present application proposes experiments that address three fundamental aspects of TXA2 receptor (TPR) signaling in platelets: 1. The molecular and functional consequences of G13 phosphorylation. Our hypothesis is that PKA-mediated phosphorylation of G Switch Region 1 selectively regulates signaling through G13-coupled platelet receptors. This model predicts that G13 phosphorylation at Thr203 prevents interaction of activated G13 with its downstream effectors, and hence G13 signal transduction. To address this question, the initial experiments will employ both human platelets and a T203A mutant cell line to examine the mechanism by which G13 phosphorylation by cAMP modulates Rho A activation and inhibits platelet function. Subsequent experiments will develop a T203A mutant mouse that is insensitive to cAMP-mediated phosphorylation of G13. These studies will define the contribution of G13 phosphorylation to platelet signaling and functional activation both in vitro and in vivo. 2. The involvement of ADP-mediated Gi signaling in TPR-induced platelet aggregation. Our hypothesis is that TPR signaling is itself sufficient to cause integrin activation and platelet aggregation. We further hypothesize that the contribution of secreted ADP to TPR-mediated human platelet activation has been substantially overestimated. This hypothesis is based on our recent findings that adenosine- based P2Y12 receptor antagonists increase human platelet cAMP levels, and that this increased can account for the bulk of their inhibitory activity. The proposed experiments will define the underlying mechanism by which this elevation in cAMP occurs, e.g., through activation of a Gs-coupled receptor or through inhibition of Gi signaling. Subsequent experiments will employ human platelets and P2Y12 deficient mice to examine the contribution of P2Y12 signaling to the TPR-mediated platelet activation process. 3. The modulation of platelet function by isoprostanes. Our hypothesis is that isoprostanes signal in platelets through two opposing pathways: one linked to TPRs; and the other linked to a novel receptor. This hypothesis is derives from two recent results: a) there is a significant inhibitory component of isoprostane signaling that is only revealed when TPR signaling is blocked; and b) isoprostanes interact with platelets at two distinct binding sites. The planned studies will define the isoprostane inhibitory mechanism, and characterize the putative isoprostane receptor in human platelets. Separate experiments will develop a TPR mutant mouse (F194A) that effectively separates the two pathways of isoprostane signaling. Studies using these mice will measure the contributions of the inhibitory component of isoprostane signaling to both platelet function and thrombus formation in vivo. PUBLIC HEALTH RELEVANCE: Taken together, the experiments presented in this application address fundamental questions concerning human blood platelet signaling and function. They will define the molecular events associated with platelet activation/inhibition, and how such events modulate platelets, both in vitro and in vivo. As such these experiments will provide new and important information concerning fundamental aspects of platelet biology, which in turn will facilitate the development of therapeutic strategies for the control/prevention of thromboembolic disorders.

描述（由申请人提供）：本申请提出了解决血小板中TXA2受体（TPR）信号传导的三个基本方面的实验： 1.G13磷酸化的分子和功能后果。我们的假设是 PKA 介导的 G Switch Region 1 磷酸化选择性地调节通过 G13 偶联血小板受体的信号传导。该模型预测 G13 Thr203 处的磷酸化可阻止激活的 G13 与其下游效应子的相互作用，从而阻止 G13 信号转导。为了解决这个问题，最初的实验将使用人类血小板和 T203A 突变细胞系来检查 cAMP 磷酸化 G13 调节 Rho A 激活和抑制血小板功能的机制。后续实验将开发一种对 cAMP 介导的 G13 磷酸化不敏感的 T203A 突变小鼠。这些研究将明确 G13 磷酸化对体外和体内血小板信号传导和功能激活的贡献。 2. ADP介导的Gi信号传导参与TPR诱导的血小板聚集。我们的假设是 TPR 信号传导本身足以引起整合素激活和血小板聚集。我们进一步假设分泌的 ADP 对 TPR 介导的人血小板活化的贡献被大大高估了。这一假设基于我们最近的发现，即基于腺苷的 P2Y12 受体拮抗剂会增加人血小板 cAMP 水平，并且这种增加可以解释其抑制活性的大部分。拟议的实验将定义 cAMP 升高的潜在机制，例如通过 Gs 偶联受体的激活或通过 Gi 信号传导的抑制。后续实验将使用人类血小板和 P2Y12 缺陷小鼠来检查 P2Y12 信号传导对 TPR 介导的血小板激活过程的贡献。 3.异前列烷对血小板功能的调节。我们的假设是，异前列烷通过两种相反的途径在血小板中发出信号：一种与 TPR 相关；另一种与 TPR 相关。另一个与一种新的受体有关。这一假设源自最近的两个结果：a) 异前列烷信号传导存在显着的抑制成分，只有当 TPR 信号传导被阻断时才会显现出来； b) 异前列腺素在两个不同的结合位点与血小板相互作用。计划中的研究将确定异前列烷抑制机制，并表征人血小板中假定的异前列烷受体。单独的实验将开发一种 TPR 突变小鼠 (F194A)，它可以有效分离异前列腺素信号传导的两条途径。使用这些小鼠的研究将测量异前列烷信号传导的抑制成分对体内血小板功能和血栓形成的贡献。公共健康相关性：总而言之，本申请中提出的实验解决了有关人类血小板信号传导和功能的基本问题。他们将定义与血小板激活/抑制相关的分子事件，以及这些事件如何在体外和体内调节血小板。因此，这些实验将提供有关血小板生物学基本方面的新的重要信息，这反过来将有助于制定控制/预防血栓栓塞性疾病的治疗策略。