Role of a copper (I)-dependent enzyme in the anti-platelet action of S-nitrosoglutathione.
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 gamma-glutamyl transpeptidase. The anti-aggregatory potency of GSNO was not, however, altered by treatment of target platelets with the gamma-glutamyl transpeptidase inhibitor acivicin (1 mM). gamma-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-1 to 1.52 +/- 0.24 nmol min-1 (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-1 (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 K(m) for GSNO of 12.4 +/- 2.64 microM and a Vmax of 0.21 +/- 0.03 nmol min-1 per 10(8) 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.
Institute of Urology and Nephrology, University College London.
M P
MP
Gordge
J S
JS
Hothersall
G H
GH
Neild
A A
AA
Dutra
D000704:Analysis of Variance; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002417:Cattle; D003300:Copper / Q000097:blood; D003545:Cysteine / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology*; D010618:Phenanthrolines / Q000494:pharmacology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D026422:S-Nitrosoglutathione; D012710:Serum Albumin, Bovine / Q000037:antagonists & inhibitors / Q000378:metabolism*
D009603:Nitroso Compounds; D010618:Phenanthrolines; D010975:Platelet Aggregation Inhibitors; C091665:S-nitrosoalbumin; C030575:bathocuproine disulfonic acid-Dowex 1 X-4 resin; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D003300:Copper; D005978:Glutathione; D003545:Cysteine