LIPID METABOLISM IN STIMULATED HUMAN PLATELETS

目前的建议旨在了解 静息和刺激人类血小板和 中性粒细胞调节游离花生四烯酸的水平。它一般都是 公认环氧合酶和脂氧合酶只能利用 以游离的未酯化的花生四烯酸为底物。这些酶 氧合花生四烯酸到不稳定的中间体，然后 转化为生物活性的化合物，如 促聚集的血小板衍生血栓素A2，参与 止血、血栓形成和动脉粥样硬化。监管 游离花生四烯酸水平也是形成的重要因素 其他具有生物活性的二十烷类化合物，因为a)内皮细胞 利用血小板衍生的花生四烯酸盐产生抗 聚集性前列环素，b)未受刺激的中性粒细胞代谢 12HETE(血小板脂氧合酶产物)转化为二羟基 二十烷类化合物，以及c)刺激的中性粒细胞产生趋化性 从花生四烯酸酯中释放出促炎症的白三烯B4 通过刺激的血小板。我计划调查：1) 含磷脂的花生四烯酸酯的合成(重点) 关于所涉及的磷脂的个别分子物种) 静息和刺激的血小板和中性粒细胞； 中性粒细胞在哪些基础上合成额外的磷脂 刺激，这是中性粒细胞可能需要的一个过程 吞噬作用；在这方面，血小板和中性粒细胞不同 它们在细胞刺激下合成磷脂的能力； 白蛋白对I.二十碳烷类化合物产生的影响 限制游离花生四烯酸的可利用性并通过增加 不稳定中间体的半衰期，以及II。(Re)血小板的合成 和中性粒细胞磷脂2)溶血磷脂酶活性(S) 血小板和中性粒细胞中，以及这些酶如何调节 溶血磷脂水平，溶血磷脂对细胞有溶解作用，但可以 用作形成新磷脂的底物；3) 有或没有特征性质膜和 中性粒细胞来源的内质网标志酶 细胞质是否明确建立质膜 和胞浆含有所有需要的酶设备 观察刺激细胞中的脂类代谢过程；4)鉴定 细胞上钙离子储存和释放的特定亚细胞位置 与肌醇磷脂代谢有关的激活，以及 肌醇磷脂代谢的调节。大多数技术适用于 这些研究(TLC、GLC、HPLC、放射化学程序和 超速离心法)在实验室中运行。 首席调查员。

The current proposal is aimed at an understanding of the mechanisms by which resting and stimulated human platelets and neutrophils regulate levels of free arachidonate. It Is generally accepted that cyclooxygenase and lipoxygenase can only utilize free, unesterified arachidonate as substrate. These enzymes oxygenate arachidonate to labile intermediates which can then be converted to biologically active compounds, such as the proaggregatory plateletderived thromboxane A2, involved in hemostasis, thrombosis and atherosclerosis. The regulation of levels of free arachidonate is also important for formation of other biologically active eicosanoids since a) endothelial cells utilize plateletderived arachidonate to generate anti- aggregatory prostacyclin, b) unstimulated neutrophils metabolize 12HETE (the platelet lipoxygenase product) to dihydroxy eicosanoids, and c) stimulated neutrophils produce chemotactic and proinflammatory leukotriene B4 from arachidonate liberated by stimulated platelets. I plan to investigate: 1) pathways of synthesis of arachidonate containing phospholipids (with emphasis on individual molecular species of the phospholipids involved) in resting and stimulated platelets and neutrophils; mechanisms by which neutrophils synthesize additional phospholipids upon stimulation, a process which may be necessary for neutrophil phagocytosis; in which respects platelets and neutrophils differ in their capability to synthesize phospholipids upon cell stimulation; the influence of albumin on i. production of eicosanoids, both by limiting availability of free arachidonate and by increasing the half life of labile intermediates, and ii. (re)synthesis of platelet and neutrophil phospholipids 2) the activity of lysophospholipase(s) in platelets and neutrophils, and how these enzymes may regulate levels of lysophospholipids, which are lytic to cells but can be utilized as substrate for formation of new phospholipids; 3) presence or absence of characteristic plasma membrane and endoplasmic reticulum marker enzymes in neutrophilderived cytoplasts to establish unequivocally whether plasma membrane and cytosol contain all the enzymatic equipment required for the lipid metabolic processes observed in stimulated cells; 4) identify specific subcellular sites of calcium storage and release upon cell activation as related to phosphoinositide metabolism, and the regulation of inositide metabolism. Most of the techniques for these studies (TLC, GLC, HPLC, radiochemical procedures and ultracentrifugation) are operational in the laboratory of the Principal Investigator.