NITRIC-OXIDE RELEASED FROM ACTIVATED PLATELETS INHIBITS PLATELET RECRUITMENT

Vessel injury and thrombus formation are the cause of most ischemic co ronary syndromes and, in this setting, activated platelets stimulate p latelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets, To further define the capacity of platelets to produce nitric oxide (NO) , as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet a ggregometer, thereby permitting simultaneous measurement of platelet a ggregation and NO production, Treatment of platelets with the NO synth ase inhibitor L-N-G-nitroarginine methyl ester (L-NAME), reduced NO pr oduction by 92+/-8% in response to 5 mu M ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% in crease in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second rec ruitable population of platelets added to stimulated platelets at the peak of NO production, To study platelet recruitment accurately, we de veloped an assay that monitors two platelet populations simultaneously . Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP, At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11 % increase in the number of P selectin-positive platelets in the nonbi otinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylat ed population, the number of P selectin positive biotinylated platelet s increased by 180+/-32% as compared to biotinylated platelets added t o the control, In summary, stimulated platelets produce NO that modest ly inhibits platelet activation but markedly inhibits additional plate let recruitment. These data suggest that platelet-derived NO may regul ate platelet recruitment to a growing thrombus.