Genetic and pharmacological analyses of involvement of Src-family, Syk and Btk tyrosine kinases in platelet shape change. Src-kinases mediate integrin α_IIbβ₃ inside-out signaling durin

Abstract

Summary: Platelet shape change was found to be associated with an increase in protein tyrosine phosphorylation upon stimulation of thrombin-, ADP-and thromboxane A₂ -G-protein coupled receptors in human platelets and thromboxane A₂ 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 ac-tivated integrin α_IIbβ₃ 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 α_IIbβ₃ exposure during shape change.