title: Activation of haem-oxidized soluble guanylyl cyclase with BAY 60-2770 in human platelets lead to overstimulation of the cyclic GMP signaling pathway.
abstract: OBJECTIVE
  Nitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested.
  
  
  METHODS
  Human washed platelet aggregation and adhesion assays, as well as flow cytometry for α(IIb)β(3) integrin activation and Western blot for α1 and β1 sGC subunits were performed. Intracellular calcium levels were monitored in platelets loaded with a fluorogenic calcium-binding dye (FluoForte).
  
  
  RESULTS
  BAY 60-2770 (0.001-10 µM) produced significant inhibition of collagen (2 µg/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 µM). In fibrinogen-coated plates, BAY 60-2770 significantly inhibited platelet adhesion, an effect potentiated by ODQ. BAY 60-2770 increased the cGMP levels and reduced the intracellular Ca(2+) levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca(2+) levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced α(IIb)β(3) activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 µM) were all prevented by ODQ. Incubation with ODQ (10 µM) significantly reduced the protein levels of α1 and β1 sGC subunits, which were prevented by BAY 60-2770.
  
  
  CONCLUSIONS
  The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca(2+) levels and α(IIb)β(3) activation are all potentiated in haem-oxidizing conditions. BAY 60-2770 prevents ODQ-induced decrease in sGC protein levels. BAY 60-2770 could be of therapeutic interest in cardiovascular diseases associated with thrombotic complications.
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), São Paulo, Brazil.
authors.forename: Camila B
authors.initials: CB
authors.lastname: Mendes-Silverio
authors.forename: Luiz O S
authors.initials: LO
authors.lastname: Leiria
authors.forename: Rafael P
authors.initials: RP
authors.lastname: Morganti
authors.forename: Gabriel F
authors.initials: GF
authors.lastname: Anhê
authors.forename: Sisi
authors.initials: S
authors.lastname: Marcondes
authors.forename: Fabíola Z
authors.initials: FZ
authors.lastname: Mónica
authors.forename: Gilberto
authors.initials: G
authors.lastname: De Nucci
authors.forename: Edson
authors.initials: E
authors.lastname: Antunes
mesh_terms: D001565:Benzoates / Q000494:pharmacology*; D001713:Biphenyl Compounds / Q000494:pharmacology*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000494:pharmacology*; D015539:Platelet Activation / Q000187:drug effects*; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000071756:Soluble Guanylyl Cyclase
chemical_list: C528797:4-(((4-carboxybutyl) (2- (5-fluoro-2-((4'-(trifluoromethyl) biphenyl-4-yl)methoxy)phenyl)ethyl) amino)methyl)benzoic acid; D001565:Benzoates; D001713:Biphenyl Compounds; D020536:Enzyme Activators; D006845:Hydrocarbons, Fluorinated; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D018160:Receptors, Cytoplasmic and Nuclear; D000242:Cyclic AMP; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP; D002118:Calcium