BLOOD PLATELET THROMBOXANE RECEPTORS

虽然TXA 2和PGH 2被认为参与了 止血和某些形式的心血管疾病的发生， 关于TXA 2/PGH 2的具体机制知之甚少， 受体相互作用导致血小板功能的调节。这 缺乏信息主要是出于两个考虑：1。的 存在于血小板内的多个反馈机制;以及2.的 纯化的信号转导组分的不可用性，例如，受体 蛋白质，相关的G蛋白等。因此，在一个高度偶联的，积极的 反馈系统，如明显发生在TXA 2/PGH 2通路中， 在一个部位的药物干预无疑会影响多个部位， 其他地点的生化过程。这反过来又意味着 使观察结果的解释复杂化。基于此 考虑，研究TXA 2/PGH 2信号的最确定方法 转导将是识别，分离和表征主要的 参与这一进程的组件。一旦完成， 可以开始检查药理学和生理学调节， 在不存在外来影响的情况下重构组分。的 本申请提出表征TXA 2/PGH 2信号 使用这种方法的转导途径。具体而言，研究 设计为：1.构建抗肽和单克隆抗体库 其将用于探测TXA 2/PGH 2受体及其相关的G- 蛋白质; 2.亲和层析法纯化TXA 2/PGH 2受体 程序; 3.纯化、鉴定和表征受体相关G- 蛋白质; 4.定义受体/G蛋白偶联结构域; 4;鉴定 cAMP/cGMP介导的TXA 2/PGH 2受体磷酸化位点/G- 蛋白质复合物; 5.调查这些之间的关系 受体/G蛋白复合物的磷酸化和偶联， 重组系统; 6.应用特定的光亲和配体技术， 不可逆标记血小板。TXA 2/PGH 2受体，并确定 受体配体结合结构域;和7.利用新合成的， 生物素化TXA 2/P6 H2受体拮抗剂（与 抗体）来标记和定义静息细胞中的受体位置/分布 和活化的血小板。 这些研究代表了对人类 血小板TXA_2/PGH_2受体及其信号转导机制 特别是在这条道路上。这些研究的可行性是 由于目前纯化的受体/G- 蛋白质类，高亲和力TXA 2/PGH 2类似物受体/G蛋白特异性 抗体和大量的初步数据。在此基础上 预计这些研究将在更多领域具有重要价值。 明确定义TXA 2/PGH 2介导的血小板聚集的潜在机制 激活，反过来，新的治疗方法来治疗 血栓栓塞性疾病

Although TXA2 and PGH2 are believed to be involved in the process of hemostasis and the genesis of certain forms of cardiovascular disease, little is known concerning the specific mechanisms by which TXA2/PGH2- receptor interaction leads to the modulation of platelet function. This lack of information is primarily due to two considerations: 1. the multiple feedback mechanisms which exist within platelets; and 2. the unavailability of purified signal transduction components, e.g., receptor protein, associated G-proteins, etc. Thus in a highly-coupled, positive feedback system, as apparently occurs in the TXA2/PGH2 pathway, pharmacological intervention at one site will undoubtedly effect multiple biochemical processes at additional sites. This, in turn, significantly complicates interpretation of the observed results. Based on this consideration, the most definitive approach to studying TXA2/PGH2 signal transduction would be to identify, isolate and characterize the primary components involved in this pathway. Once this has been accomplished one can begin to examine pharmacological and physiological modulation the reconstituted components in the absence of extraneous influences. The present application proposes to characterize the TXA2/PGH2 signal transduction pathway using this approach. Specifically, studies are designed to: 1. raise antipeptide and monoclonal antibodies libraries which will be used to probe TXA2/PGH2 receptors and their associated G- proteins; 2. purify TXA2/PGH2 receptors by affinity chromatography procedures; 3. purify, identify and characterize receptor-associated G- proteins; 4. define receptor/G-protein coupling domains; 4; identify cAMP/cGMP-mediated phosphorylation sites on the TXA2/PGH2 receptor/G- protein complex; 5. investigate the relationship between such phosphorylation and coupling of the receptor/G-protein complex in a reconstituted system; 6. apply specific photoaffinity ligand techniques to irreversibly label platelets. TXA2/PGH2 receptors, and identify the receptor ligand binding domains; and 7. utilize a newly synthesized, biotinylated TXA2/P6H2 receptor antagonist (in conjunction with antibodies) to label and define receptor location/distribution in resting and activated platelets. These studies represent a rigorous and in-depth characterization of human platelet TXA2/PGH2 receptors and the signal transduction mechanisms specifically involved in this pathway. The feasibility of these studies is great strengthened by the current availability of purified receptor/G- proteins, high affinity TXA2/PGH2 analogs receptor/G-protein-specific antibodies and substantial preliminary data. On this basis, it is anticipated that these studies will be of significant value in more clearly defining the underlying mechanisms of TXA2/PGH2-mediated platelet activation and, in turn, novel therapeutic approaches to the treatment of thromboembolic disorders.