Blood Platelet Thromboxane Receptors

描述(由申请人提供)：本申请提出的实验涉及血小板中血栓素A2受体(TPR)信号的三个基本方面：1.G13磷酸化的分子和功能后果。我们的假设是，PKA介导的G切换区1的磷酸化通过G13偶联的血小板受体选择性地调节信号。该模型预测，Thr203处的G13磷酸化阻止了激活的G13与其下游效应器的相互作用，从而阻止了G13信号转导。为了解决这个问题，最初的实验将使用人血小板和T203A突变细胞系来研究G13被cAMP磷酸化调控Rho A激活和抑制血小板功能的机制。随后的实验将培育出对cAMP介导的G13磷酸化不敏感的T203A突变小鼠。这些研究将确定G13磷酸化在体外和体内对血小板信号和功能激活的贡献。2.ADP介导的GI信号在TPR诱导的血小板聚集中的作用。我们的假设是，TPR信号本身足以引起整合素激活和血小板聚集。我们进一步假设，分泌型ADP对TPR介导的人血小板活化的贡献被大大高估了。这一假说是基于我们最近的发现，即基于腺苷的P2Y12受体拮抗剂增加了人血小板cAMP水平，并且这种增加可以解释其大部分抑制活性。拟议的实验将确定cAMP升高发生的潜在机制，例如，通过激活Gs偶联受体或通过抑制GI信号。随后的实验将使用人类血小板和P2Y12缺陷小鼠来检查P2Y12信号在TPR介导的血小板激活过程中的作用。3.异前列腺素对血小板功能的调节作用。我们的假设是，异前列腺素通过两条相反的途径在血小板上传递信号：一条与TPR相关；另一条与一种新的受体相关。这一假说源于最近的两个结果：a)异前列腺素信号有一个重要的抑制成分，只有当TPR信号被阻断时才被揭示；b)异前列腺素与血小板在两个不同的结合部位相互作用。计划中的研究将确定异前列腺素抑制机制，并表征人类血小板中假定的异前列腺素受体。单独的实验将开发出一种TPR突变小鼠(F194A)，它可以有效地分离两条异前列腺素信号通路。利用这些小鼠进行的研究将测量异前列腺素信号的抑制成分对体内血小板功能和血栓形成的贡献。

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.