Genetic and pharmacological analyses of involvement of Src-family, Syk andBtk tyrosine kinases in platelet shape change - Src-kinases mediate integrin alpha(IIb)beta(3) inside-out signaling during shape change

Platelet shape change was found to be associated with an increase in protei n tyrosine phosphorylation upon stimulation of thrombin-, ADP- and thrombox ane Az-G-protein coupled receptors in human platelets and thromboxane A, re ceptors in mouse platelets. By using PP1 and PD173956, two structurally unr elated specific inhibitors of Src-family tyrosine kinases, and mouse platel ets deficient in the Src-kinase Fyn or Lyn, we show that Src-family kinases cause the increase in protein tyrosine phosphorylation. We further detecte d that the non-Src tyrosine kinase Syk was activated during shape change in a manner dependent on Src-family kinaseactivation. The pharmacological exp eriments and the studies on Fyn-, Lyn- and Syk-deficient mouse platelets sh owed that neither Src-family kinases nor Syk are functionally involved in s hape change. Also human platelets deficient of the tyrosine kinase Btk show ed a normal shape change. Binding of PAC-1 that recognizes activated integr in alpha (IIb)beta (3) complexes on the platelet surface was enhanced durin g shape change and blocked by inhibition of Src-kinases. We conclude that t he activation of Src-kinases and the subsequent Syk stimulation upon activa tion of G-protein coupled receptors are not involved in the cytoskeletal ch anges underlying shape change of human and mouse platelets, but that the st imulation of this evolutionary conserved pathway leads to integrin alpha (I Ib)beta (3) exposure during shape change.