title: PAR-1-dependent pp60src activation is dependent on protein kinase C and increased [Ca2+]: evidence that pp60src does not regulate PAR-1-dependent Ca2+ entry in human platelets.
abstract: BACKGROUND
  The role of the tyrosine kinase pp60src in PAR-1-dependent Ca2+ entry was investigated in human platelets. pp60src 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
  pp60src tyr416 phosphorylation was used to assess pp60src 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 pp60src 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 pp60src activation. Although Gö6976, 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 pp60src 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 pp60src 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.
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
authors.forename: M T
authors.initials: MT
authors.lastname: Harper
authors.forename: S O
authors.initials: SO
authors.lastname: Sage
mesh_terms: D000199:Actins / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002227:Carbazoles / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010742:Phospholipid Ethers / Q000494:pharmacology; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C412373:AG 1879; D000199:Actins; D002227:Carbazoles; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007211:Indoles; D010446:Peptide Fragments; D010742:Phospholipid Ethers; D011743:Pyrimidines; D044463:Receptor, PAR-1; C082835:thrombin receptor peptide (42-47); C081021:Go 6976; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C026659:edelfosine; D015638:Cytochalasin D; D004533:Egtazic Acid; D019284:Thapsigargin; D016392:Proto-Oncogene Proteins pp60(c-src); D011493:Protein Kinase C; D010738:Type C Phospholipases; D053499:Plasma Membrane Calcium-Transporting ATPases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium