Genetic and pharmacological analyses of involvement of Src-family, Syk and Btk tyrosine kinases in platelet shape change -: Src-kinases mediate integrin αIIbβ3 inside-out signaling during shape change

Platelet shape change was found to be associated with an increase in protein tyrosine phosphorylation upon stimulation of thrombin-, ADP- and thromboxane Az-G-protein coupled receptors in human platelets and thromboxane A, receptors in mouse platelets. By using PP1 and PD173956, two structurally unrelated specific inhibitors of Src-family tyrosine kinases, and mouse platelets deficient in the Src-kinase Fyn or Lyn, we show that Src-family kinases cause the increase in protein tyrosine phosphorylation. We further detected that the non-Src tyrosine kinase Syk was activated during shape change in a manner dependent on Src-family kinaseactivation. The pharmacological experiments and the studies on Fyn-, Lyn- and Syk-deficient mouse platelets showed that neither Src-family kinases nor Syk are functionally involved in shape change. Also human platelets deficient of the tyrosine kinase Btk showed a normal shape change. Binding of PAC-1 that recognizes activated integrin alpha (IIb)beta (3) complexes on the platelet surface was enhanced during shape change and blocked by inhibition of Src-kinases. We conclude that the activation of Src-kinases and the subsequent Syk stimulation upon activation of G-protein coupled receptors are not involved in the cytoskeletal changes underlying shape change of human and mouse platelets, but that the stimulation of this evolutionary conserved pathway leads to integrin alpha (IIb)beta (3) exposure during shape change.