Platelet activation mediated through membrane glycoproteins: involvement of tyrosine kinases.

Abstract

Fc gamma RII cross-linking and anti-CD9 mAbs included tyrosine phosphorylation of Fc gamma RII, Syk, and Lyn associated with Fc gamma RII in Fc gamma RII cross-linking but not in anti-CD9 mAb-induced platelet activation. We prepared various GST fusion proteins expressing one or two SH2 domains of Syk and evaluated the association between these GST fusion proteins with Fc gamma RII. Based on the results obtained from these experiments, we suggest that only one tyrosine residue in ITAM of Fc gamma RII is phosphorylated with anti-CD9 mAb and that both are phosphorylated with Fc gamma RII cross-linking. Platelet activation mediated by GPIb, the receptor for vWF, is also related with tyrosine phosphorylation. Botrocetin and vWF induced Syk activation. Shc 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 platelets were activated with vWF and botrocetin, an in vitro kinase assay revealed the transient association of a kinase activity with GPIb after platelet activation. Phosphoamino acid analysis of phosphorylated proteins in this assay demonstrated that only tyrosine residues but not serine or threonine were phosphorylated, suggesting that the kinase was indeed a tyrosine kinase.