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

1 Nitric oxide (NO) is a potent inhibitor of platelet activation, that inhibits the agonist‐induced increase in cytosolic Ca 2+ concentration through both cGMP‐dependent and independent pathways. However, the NO‐related (NO x ) 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 Ca 2+ 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  μ M ). All three NO x 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 n M ) from the NO x 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 NO x donors. 5 Analysis of Fura‐2 loaded WP revealed that only DEA/NO inhibited Ca 2+ 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 Ca 2+ trafficking by the NO x donors. 6 Taken together, our data suggest that extracellular NO (>40 n M ) 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.British Journal of Pharmacology (2005) 144 , 849–859. doi: 10.1038/sj.bjp.0706110