Investigation of the existence and biological role of l‐arginine/nitric oxide pathway in human platelets by spin‐trapping/EPR studies

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Abstract
The aim of the present study was to apply spin trapping/EPR spectroscopy to investigate the existence and biological role of the l‐arginine/nitric oxide pathway in human platelet aggregation. Three different spin traps were used: two nitroso, 3,5‐dibromo‐4‐nitrosobenzenesulfonate (DBNBS) and 2‐methyl‐2‐nitrosopropane (MNP), and a nitrone, 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO). The effect of spin‐trap concentration on the collagen‐induced human platelet aggregation was compared to the anti‐aggregatory effect caused by l‐arginine. The results show that the nitroso spin traps (DBNBS and MNP) are more effective than l‐arginine in preventing platelet aggregation. DMPO has virtually no effect on the collagen‐induced aggregation except at a high concentration (300 mM). Furthermore, activation of platelets with a low concentration of collagen (17 μg/ml) and in the presence of DBNBS or MNP yields several EPR‐detectable spin adducts. Some of the observed spin adducts do not correspond to those originating from the interaction of a free radical, nitric oxide (NO˙) gas, with the spin traps [Arroyo, C. M. & Kohno, M. (1991) Free Radical Res. Commun. 14, 145–155]. Only one adduct of DBNBS, with a relative intensity of 0.1, observed in the washed‐platelet experiment and in the presence of superoxide dismutase, is similar to the EPR spectrum obtained following a reaction of pure NO˙ gas with DBNBS. This suggests that the EPR spectrum of the DBNBS adduct consisting of a triplet may originate from the production of NO˙ by these cells. Additional DBNBS and MNP spin adducts were generated during platelet activation in the presence of Ca2+ and of a cytosol‐depleted l‐arginine preparation from washed platelets to which l‐arginine was subsequently added. The formation of these DBNBS and MNP spin adducts were inhibited by Nω‐methyl‐l‐arginine (MeArg, 100 μM), suggesting that these originated from a product of NO synthase. Furthermore, the formation of DBNBS and MNP spin adducts in platelet suspensions was enhanced by the presence of superoxide dismutase; however, their formation was prevented by the endothelial‐derived relaxing factor (EDRF) inhibitors methylene blue and hemoglobin. The results from the MeArg and EDRF inhibitor experiments support the existence of the l‐arginine NO pathway in platelets. In addition, the prevention of spin‐adduct formation by EDRF inhibitors, suggests that the mechanisms of EDRF formation and the l‐arginine NO pathway in endothelial cells and platelets are similar. The potent platelet anti‐aggregatory effect shown by DBNBS and MNP, in addition to the results obtained from experiments involving MeArg and EDRF inhibitors, raise doubts with respect to the true identity of EDRF which has previously been suggested to be identical to NO˙ originating from l‐arginine. The results suggest that EDRF may be a nitroxylated compound, a hydroxyguanidinium cation radical, with strong nucleophilic properties and with pharmacological properties similar to NO˙. It is possible, following an electron reduction, that such a hydroxyguanidinium cation radical could yield NO˙.