HUMAN PLATELET ACTIVATION AND PHOSPHOLIPID METABOLISM

血小板的激活对止血过程很重要， 由于血小板因此成为形成过程中的完全参与者 病变导向的聚集体，血管收缩和血管收缩的分泌物 有丝分裂物质和产生促凝剂的表面 蛋白质因素。现在已经很好地认识到，一种 磷脂的少量种类，磷脂酰肌醇(PI)，通过 磷脂酶C(PIC)是主要的起始事件之一 将细胞表面导向的激动剂与完整的细胞反应偶联。 另一种激动剂诱导的血小板PI代谢变化被描述 最近的研究是通过3-激酶(PI-3K)对PI物种进行磷酸化。这 有核细胞中的反应似乎是有丝分裂所必需的。 回应。所得到的PI的功能尚不清楚， 尤其是在无核的血小板中。因为PI-3K产品很差 PIC的底物，它们可能直接起作用，而不是作为 信号的前兆。主要磷脂，如磷脂酰胆碱 (PtdCho)，作为血栓素A2前体的储备， 花生四烯酸(C20：4)，通过激活的血小板释放 磷脂酶A(PLA)。所有这些变化似乎都是由 G蛋白。在血小板活化过程中，无定形胞浆 前体聚合成有组织的细胞骨架和糖蛋白 将它们的连接改为膜细胞骨架。我们打算探索如何 PIC和PI-3K催化的PI的代谢变化 C20：4的动员与血小板的变化是相协调的 细胞骨架装置，时间上和功能上。另外四个 可激活的血小板成分：酪氨酸激酶、硫醇蛋白水解酶 CaPain、G蛋白(S)和整合素GPIIb/IIIa将被研究 关于PIC、PI-3K和PIA的调节。监控技术 放射性同位素和质谱法联合检测血小板PIC和PI-3K 薄层层析(TLC)和高效液相色谱(HPLC)是最早的，并在 在这个实验室里很常见。我们还在以下方面展示了专业知识 用薄层层析法和高效液相色谱法测定C20：4的动员和聚乳酸的活化。这些 部分技术将应用于不溶于Triton的细胞骨架和 膜骨架制剂以及免疫沉淀 含磷酸酪氨酸的蛋白质。两个PI-3K产品的效果 明胶蛋白和轮廓蛋白的功能(调节肌动蛋白聚合)将 被测量。酪氨酸激酶、G蛋白和钙蛋白的激活将 通过免疫印迹技术及其功能进行监测，以及 GPIIb/IIIa，将被适当的抑制剂和 并与PIC、PI-3K和PLA活性的变化进行比较。

The activation of the platelet is important for the hemostatic process, since the platelet thereby becomes a full participant in the formation of lesion-directed aggregates, a secretor of vasoconstricting and mitogenic substances, and a surface for the generation of pro-coagulant protein factors. It is now well recognized that the hydrolysis of a quantitatively minor species of phospholipid, phosphoinositide (PI), by phosphoinositidase C (PIC) is one of the major initial events that couples a cell surface-directed agonist to a full cellular response. Another agonist-induced change in platelet PI metabolism described recently is phosphorylation of PI species by a 3-kinase (PI-3K). This reaction, in nucleated cells, appears to be required for mitogenic responses. The function of the resulting PI's is as yet unknown, especially in the anucleate platelet. Since PI-3K products are poor substrates for PIC, they probably function directly, rather than as precursors for signals. Major phospholipid, such as phosphatidylcholine (PtdCho), serves as a reserve for the thromboxane A2 precursor, arachidonic acid (C20:4), which is liberated in activated platelets via phospholipase A (PLA). All of these changes appear to be controlled by G-proteins. In the course of platelet activation, amorphous cytoplasmic precursors polymerize into organized cytoskeleton, and glycoproteins change their linkage to membrane cytoskeleton. We intend to explore how the metabolic changes in PI's catalyzed by PIC and PI-3K and the mobilization of C20:4 are coordinated with alterations in the platelet cytoskeletal apparatus, temporally and functionally. Four additional activatable platelet components: tyrosine kinase, the thiol protease calpain, G-protein(s), and the integrin GPIIb/IIIa, will be investigated with respect to PIC, PI-3K and PIA regulation. Techniques for monitoring platelet PIC and PI-3K by radioisotopic and mass analysis in conjunction with thin layer chromatography (TLC) and HPLC were pioneered, and are in common use in, this lab. We also have demonstrated expertise in measuring C20:4 mobilization and PLA activation by TLC and HPLC. These techniques, in part, will be applied to triton-insoluble cytoskeletal and membrane skeletal preparations as well as to immunoprecipitated phosphotyrosine-containing protein. The effects of two PI-3K products on gelsolin and profilin function (modulating actin polymerization) will be measured. Activation of tyrosine kinase, G-protein, and calpain will be monitored by immunoblotting techniques and their function, as well as that of GPIIb/IIIa, will be perturbed with appropriate inhibitors and agonists and compared with alterations in PIC, PI-3K, and PLA activities.