Role of a copper (I)‐dependent enzyme in the anti‐platelet action of S‐nitrosoglutathione

Abstract

1 S-nitrosoglutathione (GSNO) is a potent and selective anti-platelet agent, despite the fact that its spontaneous rate of release of nitric oxide (NO) is very slow. Our aim was to investigate the mechanism of the anti-aggregatory action of GSNO. 2 The biological action of GSNO could be mediated by NO released from S-nitrosocystylglycine, following enzymatic cleavage of GSNO by y-glutamyl transpeptidase. The anti-aggregatory potency of GSNO was not, however, altered by treatment of target platelets with the y-glutamyl transpeptidase inhibitor acivicin (1 mM). y-Glutamyl transpeptidase is not, therefore, involved in mediating the action of GSNO. 3 The rate of breakdown of S-nitrosoalbumin was increased from 0.19 + 0.086 nmol min' to 1.52+0.24 nmol min-' (mean+s.e.mean) in the presence of cysteine (P<0.05, n=4). Inhibition of platelet aggregation by S-nitrosoalbumin was also significantly increased by cysteine (P <0.05, n = 4), suggesting that the biological activity of S-nitrosoalbumin is mediated by exchange of NO from the protein carrier to form the unstable compound cysNO. Breakdown of GSNO showed a non-significant acceleration in the presence of cysteine, from 0.56+0.22 to 1.77+0.27 nmol min-' (mean+s.e.mean) (P = 0.064, n = 4), and its ability to inhibit platelet aggregation was not enhanced by cysteine. This indicates that the anti-platelet action of GSNO is not dependent upon transnitrosation to form cysNO. 4 Platelets pretreated with the copper (I)-specific chelator bathocuproine disulphonic acid (BCS), then resuspended in BCS-free buffer, showed resistance to the inhibitory effect of GSNO. These findings suggest that BCS impedes the action of GSNO by binding to structures on the platelet, rather than by chelating free copper in solution. 5 Release of NO from GSNO was catalysed enzymatically by ultrasonicated platelet suspensions. This enzyme had an apparent Km for GSNO of 12.4+2.64 gIM and a Vmax of 0.21 +0.03 nmol min-' per 108 platelets (mean + s.e.mean, n = 5). It was inhibited by BCS, but not by the iron chelator bathophenathroline disulphonic acid, nor by acivicin. 6 We conclude that the stable S-nitrosothiol compound GSNO may exert its anti-platelet action via enzymatic, rather than spontaneous release of NO. This is mediated by a copper-dependent mechanism. The potency and platelet-selectivity of GSNO may result from targeted NO release at the platelet surface.

Inhibition of human platelet aggregation by a novel S‐nitrosothiol is abolished by haemoglobin and red blood cells in vitro: implications for anti‐thrombotic therapy

“…The amount of NO generated from both GSNO and RIG200 was greater in PRP than in PPP, indicative of a platelet-dependent mechanism that can accelerate S-nitrosothiol decomposition, as previously proposed (Radomski et al, 1992;Gordge et al, 1996).…”

Chelation of Cellular Cu(I) Raised the Degree of Glyoxalase I Inactivation in Human Endothelial Cells upon Exposure to S-Nitrosoglutathione through Stabilization of S-Nitrosothiols.

“…52) They also demonstrated GSNO lyase activity in several lines of cells. This activity was highest in fibroblasts and was slightly modified by metal chelators, such as BCS.…”

Thiols in Nitric Oxide Synthase-Containing Nocardia sp. Strain NRRL 5646

“…One anomeric proton signal for D-glucosamine (5.12 ppm; J H-1 ϭ 3.7 Hz) was consistent with an ␣-glycosidic linkage. By 13 C-NMR, all four methyl groups were observed between 8 and 25 ppm, and the spectrum also showed one anomeric carbon signal at 101.9 ppm for D-glucosamine. Figure 2 showed that Nocardia sp.…”