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

1 S-Nitrosothiols are nitric oxide (NO) donor drugs that have been shown to inhibit platelet aggregation in platelet rich plasma (PRP) in vitro and to inhibit platelet activation in vivo. The aim of this study was to compare the platelet effects of a novel S-nitrosated glyco-amino acid, RIG200, with an established S-nitrosothiol, S-nitrosoglutathione (GSNO) in PRP, and to investigate the effects of cell-free haemoglobin and red blood cells on S-nitrosothiol-mediated inhibition of platelet aggregation.

2 The effects of GSNO and RIG200 in collagen (2.5 mug ml(-1))-induced platelet aggregation in PRP and whole blood were investigated in vitro. Both compounds were found to be powerful inhibitors of aggregation in PRP, and RIG200 was significantly more potent (IC50=2.0 muM for GSNO and 0.8 muM for RIG200, P=0.04).

3 Neither compound inhibited aggregation in whole blood, even at concentrations of 100 muM. Red blood cell concentrations as low as 1% of the haematocrit, and cell-free haemoglobin (greater than or equal to2.5 muM), significantly reduced their inhibitory effects on platelets.

4 Experiments involving measurement of cyclic GMP levels, electrochemical detection of NO and electron paramagnetic resonance of haemoglobin in red blood cells, indicated that scavenging of NO generated from S-nitrosothiols by haemoglobin was responsible for the lack of effect of S-nitrosothiols on platelets in whole blood.

5 These studies suggest that scavenging of NO by haemoglobin in blood might limit the therapeutic application of S-nitrosothiols as anti-platelet agents.