BLOOD PLATELET THROMBOXANE RECEPTORS

尽管TXA2和PGH2被认为参与了 止血和某些形式心血管疾病的发生， 关于TXA2/PGH2的具体机制，人们知之甚少。 受体的相互作用导致了血小板功能的调节。这 缺乏资料主要有两方面的考虑：1. 存在于血小板内的多种反馈机制；以及2. 纯化的信号转导组件，例如受体，不可用 蛋白质，相关的G蛋白等。从而在一个高度偶联的，阳性的 反馈系统，显然发生在TXA2/PGH2途径中， 一个地方的药物干预无疑会影响多个地方 其他地点的生化过程。反过来，这又显著地 使观测结果的解释复杂化。在此基础上 思考--研究TXA2/PGH2信号的最权威途径 转导将是识别、分离和表征原始的 参与这一途径的成分。一旦这一点完成，你就会发现 可以开始检查药理和生理调节 在没有外来影响的情况下重组组件。这个 本申请建议表征TXA2/PGH2信号 使用这种方法的转导途径。具体地说，研究是 设计目的：1.建立抗肽和单抗抗体库 它将被用来探测TXA2/PGH2受体及其相关的G- 亲和层析纯化TXA2/PGH2受体 程序；3.纯化、鉴定和鉴定受体相关G- 蛋白质；4.确定受体/G蛋白偶联结构域；4；鉴定 CAMP/cGMP介导的TXA2/PGH2受体/G- 蛋白质复合体；5.研究它们之间的关系 A受体/G蛋白复合体的磷酸化和偶联 重组系统；6.应用特定的光亲和配基技术 不可逆转地给血小板贴上标签。TXA2/PGH2受体，并鉴定 受体配体结合域；以及7.利用新合成的， 生物素标记的TXA2/P6H2受体拮抗剂(与 抗体)来标记和定义静息中的受体位置/分布 并激活了血小板。 这些研究代表了对人类的严谨和深入的描述 血小板TXA2/PGH2受体及其信号转导机制 特别参与了这条途径。这些研究的可行性是 目前纯化的受体/G-G的可用性大大增强了 蛋白质，高亲和力TXA2/PGH2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，它是 预计这些研究将对更多的 明确TXA2/PGH2介导的血小板的潜在机制 激活，进而，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.