PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease-activated receptor-1 but not protease-activated receptor-4 in human platelets

Background: Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca2+ signaling must be tightly controlled. Thrombin-induced Ca2+ signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca2+ signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. Objectives: To investigate the mechanisms by which PKC inhibitors enhance thrombin-induced Ca2+ signaling, and to determine the importance of this pathway in platelet activation. Methods: Cytosolic Ca2+ signaling was monitored in fura-2-loaded human platelets. Phosphatidylserine ( PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. Results: PKC inhibition by bisindolylmaleimide-I (BIM-I) enhanced alpha-thrombin-induced Ca2+ signaling in a concentration-dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or gamma-thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM-I had little effect on alpha-thrombin-induced signaling following treatment with the PAR1 antagonist, SCH-79797. BIM-I enhanced Ca2+ release from intracellular stores and Ca2+ entry, as assessed by Mn2+ quench. However, the plasma membrane Ca2+ ATPase inhibitor, 5(6)-carboxyeosin, did not prevent the effect of BIM-I. PKC inhibition strongly enhanced alpha-thrombin-induced PS exposure, which was reversed by blockade of PAR1. Conclusions: Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca2+ release and Ca2+ entry, which leads to reduced platelet PS exposure.