PAR-1-dependent pp60src activation is dependent on protein kinase C and increased [Ca2+]i:: evidence that pp60src does not regulate PAR-1-dependent Ca2+ entry in human platelets

Background: The role of the tyrosine kinase pp60(src) in PAR-1-dependent Ca2+ entry was investigated in human platelets. pp60(src) plays a role in thapsigargin (TG)-evoked store-operated Ca2+ entry (SOCE), which is thought to be a major component of thrombin-evoked Ca2+ entry. Methods: pp60(src) tyr(416) phosphorylation was used to assess pp60(src) activation. Fura-2-loaded platelets were used to monitor intracellular Ca2+ concentration ([Ca2+](i)). Results: Activation of PAR-1 with the specific agonist SFLLRN increased pp60(src) activation within 10 s. This required phospholipase C (PLC) activity, Ca2+ release and a rise in intracellular Ca2+. PP2, an inhibitor of Src-family tyrosine kinases, inhibited SFLLRN-evoked Ca2+ entry, but also inhibited Ca2+ release and the extrusion of Ca2+ by the plasma membrane Ca2+ ATPase. Actin polymerization and conventional protein kinase C (cPKC) activity were required for TG- and SFLLRN-evoked pp60(src) activation. Although Go6976, an inhibitor of cPKCs, inhibited TG-evoked SOCE, it had little effect on SFLLRN- or thrombin-evoked Ca2+ entry. Conclusions: These data indicate that stimulation of PAR-1 leads to activation of pp60(src) in human platelets, through PLC and cPKC activation, Ca2+ release and actin polymerization. However, as PKC and actin polymerization are not needed for SFLLRN-evoked Ca2+ entry, we suggest that pp60(src) is also not required. The apparent inhibition of SFLLRN-evoked Ca2+ entry by PP2 is likely to be secondary to reduced Ca2+ release. These data argue against a contribution of this SOCE pathway to PAR-1-dependent Ca2+ entry.