A potential role for extracellular nitric oxide generation in cGMP-independent inhibition of human platelet aggregation: biochemical and pharmacological considerations

Abstract

1. Nitric oxide (NO) is a potent inhibitor of platelet activation, that inhibits the agonist-induced increase in cytosolic Ca2+ concentration through both cGMP-dependent and independent pathways. However, the NO-related (NOx) species responsible for cGMP-independent signalling in platelets is unclear. We tested the hypothesis that extracellular NO, but not NO+ or peroxynitrite, generated in the extracellular compartment is responsible for cGMP-independent inhibition of platelet activation via inhibition of Ca2+ signalling. 2. Concentration-response curves for diethylamine diazeniumdiolate (DEA/NO; a spontaneous NO generator), S-nitroso-N-valerylpenicillamine (SNVP; an S-nitrosothiol) and 3-morpholinosydnonomine (SIN-1; a peroxynitrite generator) were generated in platelet-rich plasma (PRP) and washed platelets (WP) in the presence and absence of a supramaximal concentration of the soluble guanylate cyclase inhibitor, ODQ (20 microM). All three NOx donors displayed cGMP-independent inhibition of platelet aggregation in PRP, but only DEA/NO exhibited cGMP-independent inhibition of aggregation in WP. 3. Analysis of NO generation using an isolated NO-electrode revealed that cGMP-independent effects coincided with the generation of substantial levels of extracellular NO (>40 nM) from the NOx donors. 4. Reconstitution of WP with plasma factors indicated that the copper-containing plasma protein, caeruloplasmin (CP), catalysed the release of NO from SNVP, while Cu/Zn superoxide dismutase (SOD) unmasked NO generated from SIN-1. The increased generation of extracellular NO correlated with a switch to cGMP-independent effects with both NOx donors. 5. Analysis of Fura-2 loaded WP revealed that only DEA/NO inhibited Ca2+ signalling in platelets via a cGMP-independent mechanism. However, preincubation of SNVP and SIN-1 with CP and SOD, respectively, induced cGMP-independent inhibition of intraplatelet Ca2+ trafficking by the NOx donors. 6. Taken together, our data suggest that extracellular NO (>40 nM) is required for cGMP-independent inhibition of platelet activation. Plasma constituents may play an important pharmacological role in activating cGMP-independent signalling by S-nitrosothiols or peroxynitrite generators.

Figure 1

Generation of NO from DEA/NO, SNVP and SIN-1 in PRP and WP. Platelets were equilibrated at 37°C before the addition of DEA/NO (3 μM), SNVP (100 μM) or SIN-1 (100 μM) to PRP (a) or WP (b). Experiments involving the addition of SNVP (100 μM) and SIN-1 (100 μM) to WP are also shown on a smaller scale (inset). Data shown are the mean, with the s.e.m. indicated for every 60th (1-min) time point (n=4–5).

Figure 2

Inhibition of platelet aggregation by DEA/NO, SNVP and SIN-1 in PRP and WP in the presence and absence of ODQ. Platelets were equilibrated to 37°C before treatment with DEA/NO (a), SNVP (b) or SIN-1 (c) in PRP (i) or WP (ii). Platelet aggregation was then stimulated with U46619 (8 μM) 1-min after the addition of the NO_x donor. In experiments involving ODQ, platelets were preincubated with ODQ (20 μM) for 15-min before NO_x donor, followed by U46619 1-min later. ODQ did not affect DEA/NO and SNVP-mediated inhibition of aggregation in PRP (P>0.05), while it inhibited SIN-1-mediated inhibition of platelet aggregation in PRP (P<0.05). ODQ also inhibited DEA/NO-mediated inhibition of platelet aggregation in WP, and completely abolished SNVP and SIN-1-induced inhibition of platelet aggregation in WP. Data are expressed as the mean±s.e.m. (n=6). A representative trace of DEA/NO (30 nM–3 μM) in PRP (control) is included (iii).

Figure 3

Effect of plasma factors on the generation of NO from SNVP and SIN-1 in WP. Platelets were equilibrated to 37°C before the addition of SNVP (100 μM; a) or SIN-1 (100 μM; b). In experiments involving SNVP, WP were preincubated with CP (0.4 g l⁻¹) for 1-min prior to the addition of SNVP. In SIN-1 experiments, WP were preincubated with SOD (500 U ml⁻¹), Asc (100 μM) or HSA (4%) for 1-min before the addition of SIN-1. Data shown are the mean, with the s.e.m. indicated for every 60th (1-min) time point (n=4).

Figure 4

Effect of plasma factors on cGMP-independent inhibition of platelet aggregation by SNVP and SIN-1 in WP. Platelets equilibrated to 37°C were preincubated with ODQ (20 μM) for 14-min before the addition of CP (0.4 g l⁻¹) 1-min prior to the addition of SNVP (a). SOD (500 U ml⁻¹) was also added to ODQ-treated WP 1-min before the addition of SIN-1 (b). After incubation with SNVP or SIN-1 for 1-min, U46619 (8 μM) was added to induce aggregation. Previous data are added as a comparison. Data are expressed as the mean±s.e.m. (n=6).

Figure 5

Effect of DEA/NO, SNVP and SIN-1 on Ca²⁺signalling in Fura-2 labelled WP. Platelets loaded with Fura-2 were equilibrated to 37°C before the addition of DEA/NO (10 μM – a(i)), SNVP (100 μM – a(ii)) or SIN-1 (100 μM – a(iii)). In experiments involving ODQ, WP were preincubated with ODQ (20 μM) for 15-min before the addition of NO_x donor. In other experiments, ODQ-treated WP were reconstituted with CP (0.4 g l⁻¹) or SOD (500 U ml⁻¹) before the addition of SNVP or SIN-1 respectively. Representative traces are included (i–iii) alongside summary data obtained by measuring the AUC (b). ODQ did not affect DEA/NO-mediated inhibition of Ca²⁺signalling (P>0.05); however, it significantly attenuated the inhibitory action of SNVP and SIN-1 on Ca²⁺signalling (P<0.001). The effect of ODQ was significantly reversed by the addition of CP and SOD to SNVP and SIN-1, respectively (P<0.001). Summary data are expressed as the mean±s.e.m. (n=4–6).

Figure 6

Effect of ODQ incubation on NO generation by DEA/NO, SNVP and SIN-1 in PRP. Platelets were equilibrated to 37°C before the addition of DEA/NO (3 μM – a), SNVP (100 μM – b) or SIN-1 (100 μM – c). In experiments involving ODQ, PRP was treated with ODQ (20 μM) for 15-min before the addition of DEA/NO, SNVP or SIN-1. Data shown are the mean, with the s.e.m. indicated for every 60th (1-min) time point (n=4).