While the involvement of signaling of the ADP P2Y1 and the thromboxane A2 receptors (TPR) in the pathogenesis of thrombotic diseases is well documented, there are no antagonists that are currently available for clinical use, and which target either of these pathways. This derives, in part, from lack of knowledge regarding receptor signaling and structure. The present application proposes experiments that address fundamental aspects of the structural biology and signaling of platelet TPR and P2Y1 receptors: 1) The biological significance of antagonism of P2Y1 ligand-binding region (i.e., the second extracellular loop;EL2 in the prevention of thrombotic diseases. Our hypothesis is that EL2 of P2Y1 plays an important role in platelet activation. To address this hypothesis, our experiments will evaluate the ability of a novel functional antibody targeting EL2 (abbreviated as EL2Ab) to block ADP-induced platelet activation. Subsequent studies we will investigate the effects of EL2Ab and an EL2 peptide-based "vaccine" on bleeding time, and thrombosis development. 2. The G-protein coupling domains of TPR. Our hypothesis is that the intracellular (IL) domains of TPR contain separate regions that confine coupling to a specific G-protein. In this regard, one of our most intriguing results is that a peptide mimicking the first IL of TPR (abbreviated Myr-IL1pep) blocked TPR-mediated aggregation but not shape change. This finding suggests that the IL1 domain of TPR participates in receptor coupling to Gq, but not G13. Similarly, the role of other IL regions in G-protein coupling will be determined by examining the effects of their corresponding peptides on TPR-triggered platelet aggregation, shape change and secretion. Also, the effects of any biologically-active IL peptides on bleeding time and thrombosis development will be investigated. Collectively, results obtained from these studies will provide fundamental information concerning TPR and P2Y1 receptor biology and structure, and may define new therapeutic targets and/or aid molecular modeling study predictions for organic derivatives/agents for treating thrombotic disease states.
Collectively, the experiments presented in this application- some of which are translational in nature- address fundamental questions concerning the platelet thromboxane A2 and P2Y1 receptor structure, signaling and function. Results obtained from these studies should provide knowledge and lay down the foundation for defining new and novel targets, as well as significantly aid in the molecular modeling study predictions for designing organic derivatives/agents for the therapeutic management of thromboembolic-based disease states.