Analysis of Signal Transducing Mechanism in Platelet-Analysis using Proteomics and Cell-permeable Reagents

1.血小板中Rap - 1活化的分析我们开发了一种定量活化Rap - 1的方法，并在血小板活化时测量血小板中Rap - 1的活化量。我们发现Rap 1的激活与其他研究表明的整合素激活无关，而是与血小板释放反应有关，特别是当血小板通过GPVI激活时。我们还发现rap1的激活是由PI 3激酶依赖性酪氨酸磷酸化介导的。(Thromb.Res。出版社)2。寻找Rap - 1相互作用蛋白我们现在正在筛选血小板中与Rap - 1相互作用的蛋白。从大肠杆菌中制备gst融合活性Rap 1（Rap 1V12）或非活性Rap 1(Rap 1n17)，并与谷胱甘肽- sepharose偶联。这些凝胶与血小板裂解液反应，结合蛋白经二维凝胶电泳质谱分析。由于细胞骨架蛋白被这些组分严重污染，我们还不能确定特异性的Rap - 1相互作用蛋白。将蛋白质或多肽引入血小板我们正在寻找将蛋白质或多肽引入血小板的方法。我们尝试了几种方法，发现与脂肪酸结合的多肽是最好的方法，但仍难以显示特异性。GPVI依赖性激活机制分析胶原蛋白与GPVI结合，通过酪氨酸磷酸化激活血小板。然而，我们发现酪氨酸磷酸化和整合素激活在一定条件下并没有很好的相关性。我们现在正在分析磷酸化非依赖性激活的机制。

1.Analysis of Rap 1 activation in plateletsWe developed a method quantitating the activated Rap 1 and measured the amount of the activated Rap 1 in platelets when platelets are activated. We found that the Rap 1 activation is not related to integrin activation as indicated by other studies but rather related to platelet release reaction, especially when platelets were activated through GPVI. We also indicated that Rap 1 activation is mediated by PI 3-kinase-dependent tyrosine phosphorylation. (Thromb.Res.In press)2.Searching the Rap 1-interacting proteinWe are now screening the protein which interact with Rap 1 in platelets. GST-fused active Rap 1(Rap 1V12) or inactive Rap 1 (Rap 1 N17) was prepared from E.coli and conjugated with glutathione-Sepharose. These gels were reacted with platelet lysate and the bound proteins were analyzed by mass spectroscopy after 2D gel electorphoresis. Since cytoskeletal proteins were heavily contaminated to these fractions, we could not identify the specific Rap 1-interacting proteins yet.3.Introduction of proteins or peptides into plateletsWe are searching the method which introduce proteins or peptides into platelets. We tried several methods and found that peptides conjugated with fatty acid would be a best method but still it is difficult to show the specificity.4.Analyzing the mechanism of GPVI-dependent activationIt is well recognized that collagen binds to GPVI and activates platelets through tyrosine-phosphorylation. However, we found that tyrosine phosphorylation and integrin activation are not well related under certain conditions. We are now analyzing the mechanis of phosphorylation-independent activation.