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.