LIPID METABOLISM IN STIMULATED HUMAN PLATELETS

目前的建议旨在了解 静息和刺激的人血小板和 中性粒细胞调节游离花生四烯酸的水平。 一般 认为环氧合酶和脂氧合酶只能利用 游离的未酯化的花生四烯酸酯作为底物。 这些酶 花生四烯酸转化为不稳定的中间体， 转化为生物活性化合物，例如 促血小板聚集衍生血栓素A2，参与 止血、血栓形成和动脉粥样硬化。 调控 游离花生四烯酸的水平对于形成 其他具有生物活性的类二十烷酸，因为a）内皮细胞 利用血小板衍生的花生四烯酸产生抗- 聚集性前列环素，B）未受刺激的中性粒细胞代谢 12 HETE（血小板脂氧合酶产物）转化为二羟基 类花生酸，和c）刺激的中性粒细胞产生趋化性 和花生四烯酸释放的促炎性白三烯B4 通过刺激血小板。 我计划研究：1） 含花生四烯酸酯磷脂的合成（重点 所涉及的磷脂的单个分子种类）， 静息和刺激的血小板和中性粒细胞; 中性粒细胞合成额外的磷脂 刺激，一个可能是中性粒细胞 吞噬作用;在这方面血小板和中性粒细胞不同， 它们在细胞刺激时合成磷脂的能力; 白蛋白对i. 类花生酸的生产， 限制游离花生四烯酸的供应， 不稳定中间体的半衰期，和ii. 血小板（再）合成 2）溶血磷脂酶的活性 以及这些酶如何调节 溶血磷脂的水平，这是溶解细胞，但可以 用作形成新磷脂的底物; 3） 存在或不存在特征性质膜， 中性粒细胞来源的内质网标记酶 细胞质以明确地确定质膜是否 和胞质溶胶含有所有酶所需的设备， 在刺激的细胞中观察到的脂质代谢过程; 4）鉴定 细胞内钙储存和释放的特定亚细胞位点 与磷酸肌醇代谢相关的活化， 肌醇代谢的调节。 大多数的技术， 这些研究（TLC、GLC、HPLC、放射化学方法和 超离心）在实验室中运行， 首席研究员。

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.