Abstract
Background and AimsNitric 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.MethodsHuman washed platelet aggregation and adhesion assays, as well as flow cytometry for αIIbβ3 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).ResultsBAY 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 Ca2+ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca2+ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced αIIbβ3 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.ConclusionThe inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca2+ levels and αIIbβ3 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.
Highlights
Platelets are a specialized set of blood cells that are an integral component of hemostasis and thrombosis
Ligand binding to this integrin mediates platelet adhesion and aggregation, and triggers the ‘‘outside-in’’ signaling, resulting in platelet biochemical and morphological responses linked to cytoskeletal remodeling [1,2]
Soluble guanylyl cyclase is a heterodimeric complex consisting of two subunits, a and b, each of which contains three common domains, namely, the N-terminal haem-binding domain that mediates the nitric oxide (NO) sensitivity of the enzyme, the dimerization domain that exists in the middle of the structure for each subunit, and the C-terminal catalytic domain, which is the most highly conserved region between the subunits, and is responsible for the conversion of GTP to cyclic guanosine monophosphate (cGMP) [5]
Summary
Platelets are a specialized set of blood cells that are an integral component of hemostasis and thrombosis. Platelets are activated by adhesive proteins and soluble agonists through receptor-specific platelet activation pathways inducing the ‘‘inside-out’’ signaling process that lead to activation of the ligand binding function of integrin aIIbb3 [1,2]. Ligand binding to this integrin mediates platelet adhesion and aggregation, and triggers the ‘‘outside-in’’ signaling, resulting in platelet biochemical and morphological responses linked to cytoskeletal remodeling [1,2]. Endothelial cellderived nitric oxide (NO) exerts an inhibitory effect in the platelet function by activation of soluble guanylyl cyclase (sGC) –3,5-cyclic guanosine monophosphate (cGMP) signaling pathway, preventing adhesion and aggregation of platelets to the vascular wall [3,4]. The hypothesis that sGC oxidation potentiates the antiplatelet activities of BAY 60-2770 has been tested
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
