Platelet activation mediated through membrane glycoproteins: Involvement of tyrosine kinases

Pc gamma RII cross-linking and anti-CD9 mAbs incuded tyrosine phosphorylati on of Pc gamma RII, Syk, and Lyn associated with Pc gamma RII in Pc gamma R II cross-linking but not in anti-CD9 mAb-induced platelet activation. We pr epared various GST fusion proteins expressing one or two SH2 domains of Syk and evaluated the association between these GST fusion proteins with Fc ga mma RII. Based on the results obtained from these experiments, we suggest t hat only one tyrosine residue in ITAM of Pc gamma RII is phosphorylated wit h anti-CD9 mAb and that both are phosphorylated with Pc gamma RII cross-lin king. Platelet activation mediated by GPIb, the receptor for vWF, is also r elated with tyrosine phosphorylation. Botrocetin and vWF induced Syk activa tion. She was also rapidly and heavily tyrosine phosphorylated. Sre and Lyn , a 54-kDa tyrosine kinase, was associated with cytoskeletal proteins. When GPIb was immunoprecipitated with nonfunctional anti-GPIb mAbs after platel ets were activated with vWF and botrocetin, an in vitro kinase assay reveal ed the transient association of a kinase activity with GPIb after platelet activation. Phosphoamino acid analysis of phosphorylated proteins in this a ssay demonstrated that only tyrosine residues but not serine or threonine w ere phosphorylated, suggesting that the kinase was indeed a tyrosine kinase .