1 The effect of copper on the activity of the S-nitrosothiol compounds S-nitrosocysteine (cysNO) and S-nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sulphonate (BCS), and human washed platelets as target cells.2 Chelation of trace copper with BCS (10 gLM) in washed platelet suspensions reduced the inhibition of thrombin-induced platelet aggregation by GSNO; however, BCS had no significant effect on the anti-aggregatory action of cysNO. BCS inhibited cyclic GMP generation in response to both cysNO and GSNO. 3 The effect of BCS was rapid (within 30 s), and could be abolished by increasing the platelet concentration to 500 x 109 1 -'. 4 In BCS-treated platelet suspensions, the addition of Cu2" ions (0.37-2.37 fiM) led to a restoration of both guanylate cyclase activation and platelet aggregation inhibition by GSNO. 5 The anti-aggregatory activity of GSNO was reduced in a concentration-dependent manner by the copper (I)-specific chelators BCS and neocuproine, and to a smaller extent by desferal.No effect was observed with the copper (II) specific chelator, cuprizone, the iron-specific chelator, bathophenanthroline sulphonate, or the broader-specificity copper chelator, D-penicillamine. 6 In both BCS-treated and -untreated platelet suspensions, cys NO was more potent than GSNO as a stimulator of guanylate cyclase. In BCS-treated platelet suspensions there was no significant difference between the anti-aggregatory potency of cysNO and GSNO; however, in untreated suspensions, GSNO was significantly more potent than cysNO. Thus, when copper was available, GSNO produced a greater inhibition of aggregation than cysNO, despite being a less potent activator of guanylate cyclase. 7 The breakdown of cysNO and GSNO was measured spectrophotometrically by decrease in absorbance at 334 nm. In Tyrode buffer, cysNO (10 !LM) broke down at a rate of 3.3 tAM min-'. BCS (10 tLM) reduced this to 0.5 ZlM min-'. GSNO, however, was stable, showing no fall in absorbance over a period of 7 min even in the absence of BCS. 8 We conclude that copper is required for the activity of both cysNO and GSNO, although its influence on anti-aggregatory activity is only evident with GSNO. The stimulatory effect of copper is unlikely to be explained solely by catalysis of S-nitrosothiol breakdown. The enhancement by copper of th anti-aggregatory activity of GSNO, relative to cysNO, suggests that copper may be required for biological activity of GSNO which is independent of guanylate cyclase stimulation.

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.

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1 We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were ®ve s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two nonnitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP). 2 Using 10 mM of each donor compound, mean+s.e.mean rate of NO release ranged from 0.04+0.001 nmol min 71 (for SNP) to 3.15+0.29 nmol min 71 (for cysNO); cyclic GMP accumulation ranged from 0.43+0.05 pmol per 10 8 platelets (for SNP) to 2.67+0.31 pmol per 10 8 platelets (for cysNO), and inhibition of platelet aggregation ranged from 40+6.4% (for SNP) to 90+3.8% (for SNAC). 3 There was a signi®cant positive correlation between the rate of NO release and the ability of the di erent NO donors to stimulate intra-platelet cyclic GMP accumulation (r=0.83; P=0.02). However, no signi®cant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the di erent NO donors (r=70.17), nor was there a signi®cant correlation between cyclic GMP accumulation and inhibition of aggregation by the di erent NO donor compounds (r=0.34). 4 Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P50.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P50.01 vs DEANO, and P50.001 vs 8-bromo cyclic GMP). 5 The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all signi®cantly diminished by the copper (I) (Cu + ) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no e ect on either the rate of NO release, or the anti-platelet action of the non-nitrosothiol compound DEANO. 6 Cyclic GMP accumulation in response to GSNO (10 79 ± 10 75 M) was undetectable following treatment of platelets with ODQ (100 mM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP-independent component in its anti-platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu + ions were required for the cyclic GMP-independent pathway to operate. 7 The cyclic GMP-independent action of GSNO, observed in ODQ-treated platelets, could not be explained by an increase in intra-platelet cyclic AMP. 8 The impermeable thiol modifying agent p-chloromercuriphenylsulphonic acid (...

We have examined the integrity of J774 cell nitric oxide (NO) production and glutathione maintenance, whilst NADPH supply was compromised by inhibition of the pentose pathway with 6-aminonicotinamide. In resting cells 6-phosphogluconate accumulation began after 4 h and glutathione depletion after 24 h of 6-aminonicotinamide treatment. Cellular activation by lipopolysaccharide/interferon-V V decreased glutathione by V50% and synchronous 6-aminonicotinamide treatment exacerbated this to 31.2% of control (P 6 6 0.05). In activated cells xy 3 P production was inhibited by 60% with 6-aminonicotinamide (P 6 6 0.01), and superoxide production by 50% (P 6 6 0.01) in zymosan-activated cells. NADPH production via the pentose pathway is therefore important to sustain macrophage NO production whilst maintaining protective levels of glutathione.z 1998 Federation of European Biochemical Societies.

S-nitrosothiol compounds are important mediators of NO signalling and can give rise to various redox derivatives of NO: nitrosonium cation (NO+), nitroxyl anion (NO-) and NO* radical. Several enzymes and transporters have been implicated in the intracellular delivery of NO from S-nitrosothiols. In the present study we have investigated the role of GPx (glutathione peroxidase), the L-AT (L-amino acid transporter) system and PDI (protein disulfide-isomerase) in the delivery of NO redox derivatives into human platelets. Washed human platelets were treated with inhibitors of GPx, L-AT and PDI prior to exposure to donors of NO redox derivatives (S-nitrosoglutathione, Angeli's salt and diethylamine NONOate). Rapid delivery of NO-related signalling into platelets was monitored by cGMP accumulation and DAF-FM (4-amino-5-methylamino-2'7'-difluorofluorescein) fluorescence. All NO redox donors produced both a cGMP response and DAF-FM fluorescence in target platelets. NO delivery was blocked by inhibition of PDI in a dose-dependent manner. In contrast, inhibition of GPx and L-AT had only a minimal effect on NO-related signalling.PDI activity is therefore required for the rapid delivery into platelets of NO-related signals from donors of all NO redox derivatives. GPx and the L-AT system appeared to be unimportant in rapid NO signalling by the compounds used in the present study. This does not, however, exclude a possible role during exposure of cells to other S-nitrosothiol compounds, such as S-nitrosocysteine. These results further highlight the importance of PDI in mediating the action of a wide range of NO-related signals.

Haemodialysis patients have an exceptionally high incidence of death from cardiovascular causes, related in part to abnormalities of lipids and platelets. Eskimos, however, have a low incidence of myocardial infarction and have a high dietary intake offish, rich in ω-3 polyunsaturated fatty acids. We have, therefore, studied the effect of a fish oil MaxEPA, containing 3.6 g of the ω-3 polyunsaturated fatty acid eicosapentaenoic acid on lipids and platelet function in haemodialysis patients. Following 8 weeks of therapy there was a 35% fall in triglycerides, a 10% rise of high-density lipoprotein (HDL) cholesterol, a 36% rise of HDL₂ cholesterol fraction and a 54% rise of the HDL₂:HDL₃ cholesterol ratio. The platelet aggregation to adenosine diphosphate and collagen was significantly reduced. The activated whole-blood clotting time was prolonged from 141 to 153 s, and 69% of patients showed a reduction of factor VIII related antigen which is usually elevated in haemodialysis patients and is thought to be a marker of endothelial damage. The blood pressure fell from 147/82 to 124/74. We have thus shown that a dietary supplement of eicosapentaenoic acid produces potentially beneficial effects on lipids, platelets, and blood pressure and may help to protect against atheroma and thus cardiovascular mortality in high-risk haemodialysis patients.

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