THE PI EFFECT AND PLATELET ALPHA-ADRENERGIC STIMULATION

已有研究表明，磷脂酰肌醇(PI)的水解性 门控功能在一种机制中的门控功能，通过这种功能，受体在 细胞表面诱导钙离子的动员。人类的血小板已经被 显示含有被激活的PI专一性磷脂酶C(PI-PLC) 通过使血小板暴露于凝血酶。它们还含有α-肾上腺素能 受刺激后导致钙跨血浆转运的受体 膜，促进PI的合成，抑制腺苷环化酶 活动。我们建议确定1)水解物是否 肌醇磷脂存在于肾上腺素激活的血小板中。 各种条件，以及是否有控制PI的情况 这种血小板中的代谢与猪的钙门控作用是一致的 周转，2)胆固醇增加/消耗或治疗的影响 PI-PLC对血小板膜腺苷环化酶活性的影响 血小板中是否存在多聚磷脂酰肌醇特异性PLC 膜，4)其他磷脂和脂肪酸对膜的影响 可溶性PI-PLC的活性，以及5)不同浓度的 肾上腺素或胶原的腺苷环化酶抑制剂SQ22536，ADP， 和小剂量凝血酶诱导的血小板磷脂的水解性。为 在大多数这些研究中，我们将使用脂类分解技术 我们所熟悉的，包括薄层色谱、高效液相色谱、气相色谱。血小板将会是 富含或不含胆固醇的高胆固醇或低胆固醇 用现在使用的方法分离磷脂微囊及其膜 通常在这个实验室里，它产生的制剂是腺苷 环化酶对肾上腺素和PGD2有反应。PI的营业额将是 用放射性同位素标记和质量法进行评估 决定。阐明α-肾上腺素能的作用 刺激，特别是作为其他激动剂和AS的协同队列 一个已经被证明特别容易被 胆固醇，应该提高我们对控制事件的理解 正常和病理状态下的血小板聚集，如血栓形成。

It has been suggested that the hydrolysis of phosphatidylinositol (PI) has a gating function in a mechanism whereby the activation of receptors at cell surfaces induces the mobilization of Ca++. Human platelets have been shown to contain a PI-specific phospholipase C (PI-PLC) which is activated by exposure of platelets to thrombin. They also contain Alpha-adrenergic receptors which, upon stimulation, lead to Ca++ transport across the plasma membrane, enhanced synthesis of PI, and depressed adenylate cyclase activity. We propose to determine 1) whether hydrolysis of phosphoinositides occurs in platelets activated by epinephrine under a variety of conditions, and whether the circumstances controlling PI metabolism in such platelets are consistent with a Ca++-gating role for PI turnover, 2) the effects of cholesterol enrichment/depletion or treatment with PI-PLC upon the activity of platelet membrane adenylate cyclase, 3) whether a polyphosphoinositide-specific PLC is present in platelet membranes, 4) the effects of other phospholipids and fatty acid on the activity of soluble PI-PLC, and 5) the extent of potentiation by epinephrine or the adenylate cyclase inhibitor SQ22536 of collagen, ADP, and low-dose thrombin-induced hydrolysis of platelet phospholipid. For most of these studies, we will employ techniques of lipid resolution with which we are familiar, including TLC, HPLC, and GC. Platelets will be loaded with or depleted of cholesterol using cholesterol-rich or -poor phospholipid vesicles, and their membranes isolated by a method now used routinely in this laboratory, which yields preparations whose adenylate cyclase is responsive to epinephrine and PGD2. Turnover of PI will be assessed with the aid of both radioisotopic labeling and mass determinations. The clarification of the role of Alpha-adrenergic stimulation, particularly as a synergistic cohort for other agonists and as an event already shown to be especially susceptible to modification by cholesterol, should enhance our understanding of the events controlling platelet aggregation in normal and pathological states such as thrombosis.