Investigation of the existence and biological role of L-arginine/nitric oxide pathway in human platelets by spin-trapping/EPR studies.
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 micrograms/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 N omega-methyl-L-arginine (MeArg, 100 microM), 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Prónai
L
L
Department of Physiology, Tokai University School of Medicine, Isehara, Japan.
Ichimori
K
K
Nozaki
H
H
Nakazawa
H
H
Okino
H
H
Carmichael
A J
AJ
Arroyo
C M
CM
D001120:Arginine / Q000097:blood* / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D003094:Collagen / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006454:Hemoglobins / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007536:Isomerism; D008751:Methylene Blue / Q000494:pharmacology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000097:blood*; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D013113:Spin Labels
D006454:Hemoglobins; D010975:Platelet Aggregation Inhibitors; D013113:Spin Labels; D009569:Nitric Oxide; D003094:Collagen; D001120:Arginine; D008751:Methylene Blue