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Abstract
Nitric oxide (NO)-mediated inhibition of platelet function occurs primarily through elevations in cGMP, although cGMP-independent mechanisms such as S-nitrosylation have been suggested as alternative NO-signaling pathways. In the present study we investigated the potential for S-nitrosylation to act as a NO-mediated cGMP-independent signaling mechanism in platelets. The NO-donor, S-nitrosoglutathione (GSNO), induced a concentration-dependent inhibition of platelet adhesion to immobilized collagen. In the presence of the soluble guanylyl cyclase inhibitor, ODQ, NO-mediated activation of the cGMP/protein kinase G signaling pathway was ablated. However, ODQ failed to completely abolish the inhibitory effect of NO on collagen-mediated adhesion, confirming that cGMP-independent signaling events contribute to the regulation of platelet adhesion by NO. Biotin-switch analysis of platelets demonstrated the presence of several S-nitrosylated proteins under basal conditions. Treatment of platelets with exogenous NO-donors, at concentrations that inhibited platelet adhesion, increased the number of S-nitrosylated bands and led to hyper-nitrosylation of basally S-nitrosylated proteins. The extent of S-nitrosylation in response to exogenous NO was unaffected by platelet activation. Importantly, platelet activation in the absence of exogenous NO failed to increase S-nitrosylation beyond basal levels, indicating that platelet-derived NO was unable to induce this type of protein modification. Our data demonstrate that S-nitrosylation of platelet proteins in response to exogenous NO may act as a potentially important cGMP-independent signaling mechanism for controlling platelet adhesion.
| Abbreviations | ||
| cGMP | = | cyclic guanosine monophosphate |
| DTPA-NONOate | = | dipropylenetriamine-NONOate |
| DTT | = | dithiothretol |
| ECM | = | extracellular matrix |
| GSH | = | glutathione |
| GSNO | = | S-nitrosoglutathione |
| H89 | = | N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide |
| HS | = | human serum |
| NO | = | nitric oxide |
| ODQ | = | 1H-CitationCitationCitationoxadiazolo[4,3-a]quinoxalin-1-one |
| sGC | = | soluble guanylyl cyclase |
| PKG | = | protein kinase G |
| WP | = | washed platelets |
| VASP | = | vasodilator stimulated phosphoprotein |
| Abbreviations | ||
| cGMP | = | cyclic guanosine monophosphate |
| DTPA-NONOate | = | dipropylenetriamine-NONOate |
| DTT | = | dithiothretol |
| ECM | = | extracellular matrix |
| GSH | = | glutathione |
| GSNO | = | S-nitrosoglutathione |
| H89 | = | N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide |
| HS | = | human serum |
| NO | = | nitric oxide |
| ODQ | = | 1H-CitationCitationCitationoxadiazolo[4,3-a]quinoxalin-1-one |
| sGC | = | soluble guanylyl cyclase |
| PKG | = | protein kinase G |
| WP | = | washed platelets |
| VASP | = | vasodilator stimulated phosphoprotein |