Deciphering the platelet tyrosine phosphoproteome by SH2-domain profiling

Platelet signaling is predominantly governed by serine/threonine kinases, however increasing evidence is accumulating that tyrosine kinases play a significant role in signal transduction and functional regulation of platelets. Using 15 recombinant SH2-domains as highly specific probes in far-Western blot analyses, we endeavored to achieve a comprehensive characterization of the state of platelet tyrosine phosphorylation in dependency on the stimulant and the stimulated pathway. These data represent a global description of changes in tyrosine phosphorylation and demonstrate, that several platelet proteins are differentially phosphorylated on tyrosine residues depending on the stimulant, time and dose providing typical, pathway-related phosphorylation signatures. Finally, identification of phosphoproteins was achieved by SH2-domain pull down experiments in combination with mass spectrometry enabling us to decipher in detail phosphotyrosine-dependent signal transduction networks in platelets. Combination of both results leads us to a more precise understanding in tyrosine phosphorylation after platelet stimulation and also permits a quantitative description of identified phosphorylation events.