title: Nitric oxide released from activated platelets inhibits platelet recruitment.
abstract: Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet 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 aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM 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% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed 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 nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.
authors.affiliation: Whitaker Cardiovascular Institute and Evans Memorial Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Freedman
authors.forename: J
authors.initials: J
authors.lastname: Loscalzo
authors.forename: M R
authors.initials: MR
authors.lastname: Barnard
authors.forename: C
authors.initials: C
authors.lastname: Alpert
authors.forename: J F
authors.initials: JF
authors.lastname: Keaney
authors.forename: A D
authors.initials: AD
authors.lastname: Michelson
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000328:Adult; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008839:Microelectrodes; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D012701:Serotonin / Q000378:metabolism; D013927:Thrombosis / Q000378:metabolism
chemical_list: D019007:P-Selectin; D009569:Nitric Oxide; D012701:Serotonin; D000244:Adenosine Diphosphate; D001710:Biotin; D019001:Nitric Oxide Synthase; D006152:Cyclic GMP; D019331:NG-Nitroarginine Methyl Ester