MECHANISM OF PLATELET ACTIVATION BY MAJOR BASIC PROTEIN

一般认为，在血管外血小板活化中， 肺可能有助于许多肺部疾病的发病机制， 炎症性疾病，包括哮喘。 虽然激活 血小板与哮喘有关，但对血小板的作用知之甚少。 可能的激动剂负责其激活。 我们最近 主要碱性蛋白（MBP），一种嗜酸性粒细胞颗粒蛋白， 当嗜酸性粒细胞被激活时， 激动剂 这些研究还表明，激活机制 不同于其他强血小板激动剂。 它是漫长的- 这项建议的长期目标是阐明MBP 激活人体血小板 初步研究将集中在 证实了初步研究表明MBP不会激活 磷脂酶C（PLC），第一个酶激活的最强 激动剂 这将涉及甘油二酯和肌醇的定量- 1，4，5-三磷酸，两个第二信使，由酶促产生， PLC的作用和PLC抑制剂U- 73，122，对MBP介导的血小板活化。 其次，研究测量 使用45 Ca的钙内流和使用钙内流抑制剂的其他方法 将进行测试的假设，缓慢上升， 在MBP活化的血小板中观察到的细胞内钙是由于 细胞外流入而不是从细胞内储存释放。 尽管缺乏显著水平的第二信使 通常与蛋白激酶的激活有关，正常的 MBP中普列克底物蛋白和肌球蛋白轻链的磷酸化- 刺激的血小板清楚地表明蛋白激酶被激活 在MBP。 不饱和脂肪酸在活化血管内皮细胞中的作用 蛋白激酶C同工酶将使用放射性示踪剂 在MBP过程中形成的脂肪酸的技术和质量测量- 介导的血小板活化。 蛋白酪氨酸激酶的作用将 也被调查。 蛋白质酪氨酸磷酸化的诱导 将通过使用抗磷酸酪氨酸的免疫印迹来测量 抗体的 最后，蛋白酪氨酸的功能意义 将通过检查蛋白质的作用来研究磷酸化 酪氨酸激酶抑制剂，染料木素，对信号转导的各个步骤 转导

It is generally accepted that activation of extravascular platelets in the lungs may contribute to the pathogenesis of many pulmonary inflammatory diseases including asthma. Although the activation of platelets has been implicated in asthma, little is known about the possible agonists responsible for their activation. We have recently reported that major basic protein (MBP), an eosinophil granule protein that is secreted when eosinophils are activated, is a potent platelet agonist. These studies also indicated that the mechanism of activation differed from that of other strong platelet agonists. It is the long- term goal of this proposal to elucidate the novel mechanism by which MBP activates human platelets. Initial studies will concentrate on confirming preliminary studies indicating that MBP does not activate phospholipase C (PLC), the first enzyme activated by most strong agonists. This will involve quantitation of diacylglycerol and inositol- 1,4,5-trisphosphate, the two second messengers, produced by the enzymatic action of PLC and examination of the effect of the PLC inhibitor, U- 73,122, on MBP-mediated platelet activation. Next, studies measuring calcium influx with 45Ca and others using inhibitors of calcium influx will be performed to test the hypothesis that the slow rise in intracellular calcium seen in MBP activated platelets is due to extracellular influx rather than release from intracellular stores. Despite the absence of significant levels of the second messengers normally associated with the activation of protein kinases, the normal phosphorylation of both pleckstrin and myosin light chain in MBP- stimulated platelets clearly indicates that protein kinases are activated by MBP. The role of unsaturated fatty acids in the activation of an isoenzyme of protein kinase C will be examined using both radiotracer techniques and mass measurements of the fatty acids formed during MBP- mediated platelet activation. The role of protein tyrosine kinases will also be investigated. The induction of protein tyrosine phosphorylation will be measured by an immunoblotting with an anti-phosphotyrosine antibody. Finally, the functional significance of protein tyrosine phosphorylation will be studied by examining the effects of the protein tyrosine kinase inhibitor, genistein, on the various steps in signal transduction.