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

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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.