Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 60-2770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway

Background and Aims: 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 antiplatelet activities of BAY 60-2770 has been tested. Methods: Human washed platelet aggregation and adhesion assays, as well as flow cytometry for alpha(IIb)beta(3) integrin activation and Western blot for alpha 1 and beta 1 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 mu M) produced significant inhibition of collagen (2 mu g/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 mM). 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 Ca2+ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 mM) inhibited platelet aggregation and Ca2+ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen-and thrombin-induced alpha(IIb)beta(3) activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 mu M) were all prevented by ODQ. Incubation with ODQ (10 mu M) significantly reduced the protein levels of alpha 1 and beta 1 sGC subunits, which were prevented by BAY 60-2770. Conclusion: The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca2+ levels and alpha(IIb)beta(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. Mendes-Silverio CB, Leiria LOS, Morganti RP, Anhe GF, Marcondes S, et al. (2012) Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 602770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway. PLoS ONE 7(11): e47223. doi:10.1371/journal.pone.004722