MECHANISM OF PLATELET ACTIVATION BY MAJOR BASIC PROTEIN

人们普遍认为，血管外血小板的激活在 肺可能参与了许多肺疾病的发病机制。 包括哮喘在内的炎症性疾病。尽管激活了 血小板与哮喘有牵连，对此我们知之甚少 可能的激动剂负责它们的激活。我们最近做了 报道了主要碱性蛋白(MBP)，一种嗜酸性粒细胞颗粒蛋白 当嗜酸性粒细胞被激活时分泌的，是一种有效的血小板 激动剂。这些研究还表明，激活的机制 与其他强效血小板激动剂不同。它是长长的- 本提案的长期目标是阐明MBP的新机制 激活人类的血小板。初步研究将集中在 确认初步研究表明MBP不会被激活 磷脂酶C(PLC)，最强激活的第一种酶 激动剂。这将涉及双甘油和肌醇的定量- 1，4，5-三磷酸，两个第二信使，由酶 PLC的作用及PLC抑制剂U-2的作用效果检测 73,122，对MBP介导的血小板活化的影响。接下来，研究测量 用45Ca和其他使用钙内流抑制剂的钙内流 将被执行，以检验假设，即 MBP激活的血小板中出现的细胞内钙是由于 细胞外流入，而不是从细胞内储存中释放。 尽管没有显著水平的第二信使 正常情况下与蛋白激酶的激活有关，正常的 MBP中pleckstrin和myosin轻链的磷酸化- 受刺激的血小板清楚地表明蛋白激酶被激活 被MBP。不饱和脂肪酸在激活血管紧张素转换酶中的作用 将使用两种放射性示踪剂来检测蛋白激酶C同工酶 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 将通过用抗磷酸酪氨酸进行免疫印迹来测量 抗体。最后，蛋白质酪氨酸的功能意义 将通过检测蛋白质的影响来研究磷酸化。 酪氨酸激酶抑制剂金雀异黄素在信号传导中的各个步骤 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.