Inhibition of human platelet aggregation by diazeniumdiolates: extent of inhibition correlates with nitric oxide load delivered.

Abstract

The profile of nitric oxide (NO) release from the diazeniumdiolate class of NO donors was evaluated using inhibition of platelet aggregation as a model. At 37 degrees C, the NO complexes (Z)-1-¿N-methyl-N-[6-(N-methylammoniohexyl)amino]¿-diazen-1- ium-1,2-diolate (dimethylhexanediamine complex), sodium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (diethylamine complex), (Z)-1-¿N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]amino diazen-1-ium-1,2-diolate (spermine complex), and (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (diethylenetriamine complex) have half-lives of 1, 2 and 39 min, and 20 h, respectively. All the diazeniumdiolates caused concentration-dependent inhibition of platelet aggregation; IC50 values (values for which the effect was half the maximum) were 26.0+/-24.1, 34.9+/-24.0 and 14.9+/-6.4 nM for dimethylhexanediamine complex, diethylamine complex and spermine complex, respectively, when pre-incubated with platelets for one half-life. Inhibition by all compounds was time-dependent. Pretreatment of platelets with spermine complex for 5 and 39 min resulted in IC50 values of 1.7+/-0.85 microM and 19.7+/-0.12 nM, whereas IC50 values for sodium nitroprusside were 27.3+/-1.25 nM and 25 nM (average, n = 2), at 5 and 39 min, respectively. Pre-incubation of each diazeniumdiolate at a concentration of 100 nM for 5 min at 37 degrees C, which resulted in the theoretical delivery of NO loads from 96.9% down to 0.3%, resulted in decreasingly efficacious inhibition of platelet aggregation. Linear regression analysis of the theoretical NO load delivered against the actual maximum inhibition (%) showed a strong correlation (r2 = 0.975). All four diazeniumdiolates caused concentration- and time-dependent inhibition of agonist-stimulated elevation of intra-platelet Ca2+ levels; IC50 values were, respectively, 8.7+/-1.49 nM and 11.5+/-1.36 nM for dimethylhexanediamine complex and diethylamine complex at their half-lives, and 176+/-16.9 nM and >100 microM, for spermine complex and diethylenetriamine complex at 2 min pre-incubation time. The respective nucleophiles not complexed with NO did not show anti-aggregatory properties or inhibition of agonist-induced elevation of intra-platelet Ca2+ levels. The inhibitory effects of all diazeniumdiolates tested were attenuated by 10 microM haemoglobin. These studies indicate that these compounds induce controlled, predictable release of NO at biological pH and temperature.