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

使用蛋白质组学和细胞渗透性试剂分析血小板分析中的信号转导机制

基本信息

批准号：
16570163
负责人：
MOROI Masaaki
金额：
$ 2.37万
依托单位：
Kurume University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16570163/
关键词：
Platelet tyrosine phosphorylation Rap 1 GPVI

项目摘要

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.

1.血小板中Rap-1活性的分析我们建立了一种定量检测活化的Rap-1的方法，并测定了当血小板被激活时，血小板中活化的Rap-1的量。我们发现Rap1的激活与整合素的激活无关，而是与血小板释放反应有关，尤其是当血小板通过GPVI被激活时。我们还指出，Rap 1的激活是由PI 3-激酶依赖的酪氨酸磷酸化介导的。2.寻找Rap1相互作用蛋白我们现在正在筛选与Rap1在血小板中相互作用的蛋白质。从大肠杆菌中制备了GST融合的活性Rap1(Rap1V12)和失活Rap1(Rap1N17)，并与谷胱甘肽-琼脂糖偶联制得。将这些凝胶与血小板裂解液反应，经双向凝胶电泳法分析结合蛋白。由于细胞骨架蛋白被这些组分严重污染，我们目前还无法鉴定与Rap 1相互作用的特异性蛋白。3.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，但仍难以显示其特异性。4.分析GPVI依赖的激活机制众所周知，胶原与GPVI结合并通过酪氨酸磷酸化激活血小板。然而，我们发现，在某些条件下，酪氨酸磷酸化和整合素激活没有很好的相关性。我们现在正在分析磷酸化非依赖性激活的机制。