BLOOD PLATELET THROMBOXANE RECEPTORS

阿司匹林被广泛用于预防冠状动脉或脑血管血栓形成的理由是它能阻止TXA2的合成。因此，TXA2信号转导在导致某些闭塞性血管疾病的事件的后遗症中起着关键作用。然而，尽管TXA2具有明显的生物学重要性，但其信号转导机制仍存在重大问题。在目前的应用中，我们将提出实验，这些实验将解决TXA2介导的信号转导的三个基本方面。具体目的I.将研究血小板七跨膜受体之间的信号转导。这一目的源于我们的假设，即G蛋白作为血小板受体池内的通讯载体。我们认为这是一项重要的发现，因为它为血小板(或其他细胞)中独立的受体信号通路的整合提供了分子机制。关于它在不同的血小板/内皮细胞受体之间的存在，它对配体结合亲和力的影响，以及它的分子作用机制，这一信号过程现在将得到进一步的定义。具体目的2.研究血小板血栓素A_2受体配体结合域(S)。这个目的是为了验证我们的假设，即TXA2受体蛋白的配体结合域(S)位于蛋白的胞外区。最近，我们成功地开发了一类新型的生物素化光亲和受体探针，目前正在应用这些探针来研究受体结合口袋。为了达到这一特定目的，还将采取两种互补的方法：a.受体的定点突变；b.针对该受体的位点特异性抗体。人们相信，从每种方法获得的联合信息将提供比单独使用光标记、突变或抗体靶向获得的更确定的信息。最后，特殊目的3.将研究cAMP介导的TXA2受体相关G-α13的磷酸化。这一目标将检验我们的假设，即通过G-α13传递的TXA2受体信号与血小板内非依赖于IP3的钙离子流相偶联；G-α13的磷酸化与TXA2信号对cAMP水平的特殊敏感性有关。最近，我们已经证明G-α13亚基与内源性血小板TXA2受体有关，并且这个α-亚基被cAMP磷酸化。现拟通过实验研究G-α13的功能(即钙离子通量)，其在血小板(表面或内膜)中的定位，以及其磷酸化对TXA2受体-G-α13复合体稳定性和G-蛋白异源三聚体复合体稳定性的生化影响。综上所述，从实验中获得的结果应该为通过TXA2受体途径进行信号转导提供新的和重要的信息。这些信息反过来将对开发更具体和有效的药理学方法来控制TXA2介导的血栓栓塞症有重大好处。

The rationale for the widespread use of aspirin in preventing coronary or cerebrovascular thrombosis is based on its ability to block the synthesis of TXA2. Thus, TXA2 signal transduction plays a critical role in the sequelae of events leading to certain occlusive vascular disorders. However, in spite of the clear biological importance of TXA2, significant questions remain concerning its signaling mechanisms. In the present application, we propose experiments which will address three fundamental aspects of TXA2-mediated signal transduction. Specific Aim I. will Investigate Signaling Between Platelet Seven-Transmembrane Receptors. This aim derives from our hypothesis that G-proteins serve as communication vectors within the platelet receptor pool. We believe this is a significant finding because it provides a molecular mechanism for the integration of the separate receptor signaling pathways in platelets (or other cells). This signaling process will now be further defined relative to its existence between different platelet/endothelial receptors, its effects on ligand binding affinities, and its molecular mechanism of action. Specific Aim 2. will Investigate the Platelet TXA2 Receptor Ligand Binding Domain(s). This aim is directed at testing our hypothesis that the ligand binding domain(s) of the TXA2 receptor protein resides in the extracellular region of the protein. Recently, we have succeeded in developing a new class of biotinylated photoaffinity receptor probes, and are currently applying these probes to investigate the receptor binding pocket. Two complementary approaches to this Specific Aim will also be taken: a. site- directed mutagenesis of the receptor; and b. site-specific antibodies against the receptor. It is believed that the combined information obtained from each approach will provide more definitive information than could be obtained by the utilization of either photolabeling, mutagenesis or antibody targeting alone. Finally, Specific Aim 3. will Investigate cAMP-Mediated Phosphorylation of TXA2 receptor-associated G-alpha13. This aim will test our hypothesis that TXA2 receptor signaling through G- alpha13 is coupled to IP3-independent Ca2+ fluxes in platelets; and that phosphorylation of G-alpha13 is associated with the peculiar sensitivity of TXA2 signaling to cAMP levels. Recently, we have demonstrated that the G-alpha13 subunit is associated with endogenous platelet TXA2 receptors, and that this alpha-subunit is phosphorylated by cAMP. Experiments are now proposed to study the function of G-alpha13 (i.e. Ca2+ fluxes), its localization in platelets (surface or internal membrane), and the biochemical consequences of its phosphorylation on the stability of the TXA2 receptor-G-alpha13 complex and the stability of the G-protein heterotrimer complex. In summary, the results obtained from the proposed experiments should provide new and important information regarding signal transduction through the TXA2 receptor pathway. This information will, in turn, be of significant benefit to the development of more specific and effective pharmacological approaches to the control of TXA2-mediated thromboembolism.