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HUMAN PLATELET ACTIVATION AND PHOSPHOLIPID METABOLISM

人体血小板活化和磷脂代谢

基本信息

批准号：
3354903
负责人：
SUSAN E RITTENHOUSE
金额：
$ 37.86万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-01 至 1992-06-30
项目状态：
已结题

项目摘要

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.

血小板的活化对于止血过程很重要，因为血小板因此成为形成的完整参与者病变定向聚集体，血管收缩和血管收缩的分泌者有丝分裂物质和产生促凝剂的表面蛋白质因素。现在人们普遍认识到，水解定量的少量磷脂，磷酸肌醇（PI），通过磷酸肌醇酶 C (PIC) 是主要的初始事件之一将细胞表面定向激动剂与完整的细胞反应结合起来。描述了另一种激动剂诱导的血小板 PI 代谢变化最近，PI 种类被 3-激酶 (PI-3K) 磷酸化。这在有核细胞中，反应似乎是促有丝分裂所必需的回应。由此产生的 PI 的功能尚不清楚，尤其是在无核血小板中。由于PI-3K产品较差 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，例如磷脂酰胆碱 (PtdCho)，作为血栓素 A2 前体的储备，花生四烯酸 (C20:4)，通过活化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，无定形细胞质前体聚合成有组织的细胞骨架和糖蛋白改变它们与膜细胞骨架的连接。我们打算探索如何 PIC 和 PI-3K 催化的 PI 代谢变化以及 C20:4 的动员与血小板的改变相协调细胞骨架装置，时间上和功能上。附加四个可激活的血小板成分：酪氨酸激酶、硫醇蛋白酶将研究钙蛋白酶、G 蛋白和整合素 GPIIb/IIIa 关于 PIC、PI-3K 和 PIA 法规。监控技术通过放射性同位素和质量分析联合进行血小板 PIC 和 PI-3K 首创了薄层色谱（TLC）和高效液相色谱（HPLC），并在本实验室中常用。我们还展示了以下方面的专业知识通过 TLC 和 HPLC 测量 C20:4 动员和 PLA 活化。这些部分技术将应用于 Triton 不溶性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。两种PI-3K产品的效果凝溶胶蛋白和profilin功能（调节肌动蛋白聚合）将被测量。酪氨酸激酶、G 蛋白和钙蛋白酶的激活将通过免疫印迹技术及其功能进行监测，以及 GPIIb/IIIa 的值将受到适当抑制剂的干扰，并且激动剂并与 PIC、PI-3K 和 PLA 活性的变化进行比较。