ROLE OF C-KINASE IN PLATELET ARACHIDONATE RELEASE

C-激酶在血小板花生四烯酸释放中的作用

• 批准号: 3471187
• 负责人: STEPHEN P HALENDA
• 金额: $ 8.31万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3471187
• 关键词: G protein; arachidonate; autoradiography; bacterial toxins; biological information processing; biological signal transduction; calcium; calcium flux; cell free system; eicosanoid metabolism; enzyme mechanism; enzyme substrate; gel electrophoresis; hemostasis; human subject; phospholipase A2; phospholipase inhibitor; phosphorylation; platelets; protein kinase C; radiotracer; terpene saponin; thrombin; thrombosis; thromboxanes; tritium

Aside from its crucial role in the normal hemostatic process, the blood platelet is firmly implicated in the pathogenesis of thrombosis and other vascular diseases; thus it is important to understand the cellular mechanisms which regulate platelet function. Formation of the key platelet activator thromboxane A2 during platelet aggregation is limited by the availability of its chemical precursor, arachidonic acid. This project seeks to clarify the poorly understood mechanism by which arachidonic acid mobilization in human platelets is governed. The roles of three major intracellular signals, Ca2+, the Ca2+/phospholipid-dependent protein kinase (C-kinase), and guanine nucleotide-binding proteins (N-proteins) in the cellular mechanism of arachidonic acid release, will be studied in human platelets in experiments involving intact cells, permeabilized platelets, and platelet-derived subcellular preparations. Previous studies in intact platelets which suggest an involvement of C- kinase in Ca2+-dependent arachidonic acid release will be extended by examining the direct effect of purified C-kinase on the activity of phospholipase A2, the major lipolytic enzyme responsible for arachidonic acid liberation in stimulated platelets. Identification of the C-kinase substrate which mediates activation of phospholipase A2 will be sought. The possible involvement of endogenous phospholipase A2 inhibitors (lipocortins) and their regulation by C-kinase will be studied. And finally, the role of N- proteins will be investigated by using guanine nucleotides and pertussis toxin in permeabilized platelets and studying the effect of purified N-protein subunits on platelet phospholipase A2. Possible interactions among the different signal transducers (Ca2+, C-Kinase, N-proteins) for control of arachidonic acid release will be examined. The goals of this project and future studies are: (1) to identify the cellular factors which control the liberation of arachidonic acid, the precursor for prostaglandins, thromboxanes, and other biologically important substances; and (2) more broadly, to define the intra- and intercellular signalling pathways that regulate platelet aggregation and secretion, as these events are centrally involved in hemostasis and thrombosis.

除了在正常止血过程中的关键作用外， 血小板是坚定地牵连在发病机制 血栓形成和其他血管疾病;因此， 了解调节血小板的细胞机制 功能 关键血小板激活剂血栓素的形成 A2在血小板聚集过程中受到其可用性的限制。 化学前体，花生四烯酸。 本项目谋求 阐明了花生四烯酸 控制人血小板中的酸动员。 三种主要的细胞内信号，Ca ~（2+）， Ca 2 +/磷脂依赖性蛋白激酶（C-激酶），和 细胞内的鸟嘌呤核苷酸结合蛋白（N-蛋白） 花生四烯酸释放的机制，将在人体内进行研究 在涉及完整细胞的实验中， 血小板和血小板衍生的亚细胞制剂。 先前 对完整血小板的研究表明， Ca 2+依赖花生四烯酸释放中的激酶， 通过检查纯化的C-激酶对 主要脂解酶磷脂酶A2的活性 负责刺激血小板中花生四烯酸的释放。 鉴定介导活化的C-激酶底物 磷脂酶A2的活性。 可能参与的 内源性磷脂酶A2抑制剂（脂皮质素）及其 将研究C-激酶的调节。 最后，N的作用- 蛋白质将通过使用鸟嘌呤核苷酸和 透化血小板中的百日咳毒素及其作用的研究 血小板磷脂酶A2上纯化的N蛋白亚基。 不同信号转换器之间可能的相互作用 (Ca2+，C-激酶，N-蛋白）用于控制花生四烯酸 释放将被审查。 本研究的目标和未来的研究是：（1）识别 控制花生四烯酸释放的细胞因子 酸，前列腺素、血栓素和其他 生物学上重要的物质;（2）更广泛地说，定义 细胞内和细胞间的信号通路， 血小板聚集和分泌，因为这些事件集中在 参与止血和血栓形成。