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

• Published in: Journal of thrombosis and haemostasis 2011-08, Vol.9 (8), p.1599-1607
• Main Authors: HARPER, M. T., POOLE, A. W.
• Format: Article
• Language: English
• Subjects: Annexin A5 - metabolism; Annexin V; bisindolylmaleimide; Blood Platelets - drug effects; Blood Platelets - enzymology; Ca super(2+)-transporting ATPase; Calcium; Calcium (intracellular); calcium entry; Calcium influx; Calcium Signaling - drug effects; Calcium signalling; Data processing; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Indoles - pharmacology; Maleimides - pharmacology; Oligopeptides - pharmacology; Peptide Fragments - pharmacology; phosphatidylserine; Phosphatidylserines - metabolism; plasma membrane calcium ATPase; Plasma Membrane Calcium-Transporting ATPases - metabolism; Plasma membranes; Platelets; Protein kinase C; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Protein Kinase Inhibitors - pharmacology; protein kinases; Proteinase-activated receptor 1; Receptor, PAR-1 - drug effects; Receptor, PAR-1 - metabolism; Receptors, Thrombin - drug effects; Receptors, Thrombin - metabolism; Signal transduction; thrombin; Thrombin - metabolism; Thrombosis; Time Factors
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/j.1538-7836.2011.04393.x

To cite this article: Harper MT, Poole AW. PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets. J Thromb Haemost 2011; 9: 1599–607. Summary.  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 α‐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 γ‐thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM‐I had little effect on α‐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 α‐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.