Blood Platelet Thromboxane Receptors

描述（由申请人提供）：本申请提出了解决血小板中TXA 2受体（TPR）信号传导的三个基本方面的实验：G13磷酸化的分子和功能后果。我们的假设是PKA介导的G开关区1的磷酸化通过G13偶联血小板受体选择性地调节信号传导。该模型预测在Thr 203处的G13磷酸化阻止活化的G13与其下游效应物的相互作用，并因此阻止G13信号转导。为了解决这个问题，最初的实验将采用人血小板和T203 A突变细胞系来检查cAMP调节Rho A活化和抑制血小板功能的G13磷酸化的机制。随后的实验将开发T203 A突变小鼠，其对cAMP介导的G13磷酸化不敏感。这些研究将确定G13磷酸化对体外和体内血小板信号传导和功能活化的贡献。2. ADP介导的Gi信号通路参与TPR诱导的血小板聚集。我们的假设是TPR信号本身足以引起整合素活化和血小板聚集。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。该假设基于我们最近的发现，即基于腺苷的P2 Y12受体拮抗剂增加人血小板cAMP水平，并且这种增加可以解释其抑制活性的大部分。所提出的实验将定义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信号传导。随后的实验将使用人血小板和P2 Y12缺陷小鼠来检查P2 Y12信号传导对TPR介导的血小板活化过程的贡献。3.异前列腺素对血小板功能的调节。我们的假设是，异前列烷通过两个相反的途径在血小板中传递信号：一个与TPR相关;另一个与新受体相关。这一假设来源于两个最近的结果：a）存在异前列烷信号传导的显著抑制组分，其仅在TPR信号传导被阻断时才被揭示;和B）异前列烷在两个不同的结合位点与血小板相互作用。计划的研究将确定异前列烷的抑制机制，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（F194 A），其有效地分离了异前列烷信号传导的两种途径。使用这些小鼠的研究将测量异前列烷信号传导的抑制组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.