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.
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.
|Qian, Feng; Le Breton, Guy C; Chen, Jia et al. (2012) Role for the guanine nucleotide exchange factor phosphatidylinositol-3,4,5-trisphosphate-dependent rac exchanger 1 in platelet secretion and aggregation. Arterioscler Thromb Vasc Biol 32:768-77|
|Xie, An; Yan, Jun; Yue, Lan et al. (2011) 2-Aminoethyl methylphosphonate, a potent and rapidly acting antagonist of GABA(A)-ýý1 receptors. Mol Pharmacol 80:965-78|
|Gussin, Hélène A; Khasawneh, Fadi T; Xie, An et al. (2011) Subunit-specific polyclonal antibody targeting human ?1 GABA(C) receptor. Exp Eye Res 93:59-64|
|Huang, Jin-Sheng; Dong, Lanlan; Kozasa, Tohru et al. (2007) Signaling through G(alpha)13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion. J Biol Chem 282:10210-22|
|Huang, Jin-Sheng; Dong, Lanlan; Le Breton, Guy C (2006) Mass-dependent signaling between G protein coupled receptors. Cell Signal 18:564-76|
|Manganello, Jeanne M; Huang, Jin-Sheng; Kozasa, Tohru et al. (2003) Protein kinase A-mediated phosphorylation of the Galpha13 switch I region alters the Galphabetagamma13-G protein-coupled receptor complex and inhibits Rho activation. J Biol Chem 278:124-30|
|Turek, Joseph W; Halmos, Therese; Sullivan, Nora L et al. (2002) Mapping of a ligand-binding site for the human thromboxane A2 receptor protein. J Biol Chem 277:16791-7|
|Manganello, J M; Djellas, Y; Borg, C et al. (1999) Cyclic AMP-dependent phosphorylation of thromboxane A(2) receptor-associated Galpha(13). J Biol Chem 274:28003-10|
|Djellas, Y; Manganello, J M; Antonakis, K et al. (1999) Identification of Galpha13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors. J Biol Chem 274:14325-30|
|Halmos, T; Turek, J W; Le Breton, G C et al. (1999) Synthesis and biological characterization of sqbazide, a novel biotinylated photoaffinity probe for the study of the human platelet thromboxane A2 receptor. Bioorg Med Chem Lett 9:2963-8|
Showing the most recent 10 out of 30 publications