title: PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease-activated receptor-1 but not protease-activated receptor-4 in human platelets.
abstract: BACKGROUND
  Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca(2+) signaling must be tightly controlled. Thrombin-induced Ca(2+) signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca(2+) signal, although the mechanisms by which this occurs and its physiological relevance are still unclear.
  
  
  OBJECTIVE
  To investigate the mechanisms by which PKC inhibitors enhance thrombin-induced Ca(2+) signaling, and to determine the importance of this pathway in platelet activation.
  
  
  METHODS
  Cytosolic Ca(2+) 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 Ca(2+) 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 Ca(2+) release from intracellular stores and Ca(2+) entry, as assessed by Mn(2+) quench. However, the plasma membrane Ca(2+) 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 Ca(2+) release and Ca(2+) entry, which leads to reduced platelet PS exposure.
authors.affiliation: School of Physiology and Pharmacology, University of Bristol, Bristol, UK. m.harper@bris.ac.uk
authors.forename: M T
authors.initials: MT
authors.lastname: Harper
authors.forename: A W
authors.initials: AW
authors.lastname: Poole
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D020013:Calcium Signaling / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D044463:Receptor, PAR-1 / Q000187:drug effects* / Q000378:metabolism; D018179:Receptors, Thrombin / Q000187:drug effects* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors
chemical_list: D017304:Annexin A5; D007211:Indoles; D008301:Maleimides; D009842:Oligopeptides; D010446:Peptide Fragments; D010718:Phosphatidylserines; D047428:Protein Kinase Inhibitors; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C455901:alanyl-tyrosyl-prolyl-glycyl-lysyl-phenylalanine; C082835:thrombin receptor peptide (42-47); D011493:Protein Kinase C; D013917:Thrombin; D053499:Plasma Membrane Calcium-Transporting ATPases; C112908:protease-activated receptor 4; C070515:bisindolylmaleimide I