Nitrovasodilators and cGMP inhibit human platelet activation.
Endothelial relaxing factor has been identified as nitric oxide, formed from L-arginine by the soluble enzyme nitric oxide synthase. Nitric oxide inhibits platelet aggregation and adhesion by stimulating a soluble guanylate cyclase and increasing the intracellular concentration of cyclic GMP. Nitrovasodilators, such as sodium nitroprusside, release the active moiety, nitric oxide. In the present study, we have investigated the effect of sodium nitroprusside and of a permeable cGMP derivative on the aggregation and ATP secretion of human platelets stimulated with the protein kinase C activators 1-oleoyl-2-acetylglycerol or 4 beta-phorbol-12- myristate-13-acetate. Human platelets were treated with lysine acetylsalicylate, washed and resuspended in Tyrode-buffered solution. ATP secretion was evaluated by luciferin-luciferase luminescence. Nitroprusside (4-40 microM) or 8-Br-cGMP (0.1-2.4 mM) inhibited both platelet aggregation and ATP secretion evoked by 1-oleoyl-2-acetylglycerol (40 microM) or 4 beta-phorbol-12-myristate-13- acetate (4 nM) in a dose-dependent manner, in the presence of the selective inhibitor of cGMP phosphodiesterase, M&B 22948 (5 microM). The inhibitory effect of nitroprusside was reversed by hemoglobin, known to bind and inactivate nitric oxide. To study the calcium-dependent pathway, we treated platelets with the ionophore ionomycin. The ensuing aggregation and ATP secretion were rapid and were dependent on agonist concentration. Nitroprusside (4-40 microM) inhibited the aggregation evoked by ionomycin (0.4 microM) as well as ATP release, in a dose-dependent manner. We conclude that cGMP is able to inhibit both the protein kinase C-dependent and the calcium-dependent pathways leading to platelet activation.
Doni
M G
MG
Institute of Human Physiology, Faculty of Medicine and Surgery, University of Padova, Italy.
Alexandre
A
A
Padoin
E
E
Bertoncello
S
S
Deana
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology*; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004075:Diglycerides; D010975:Platelet Aggregation Inhibitors; D009599:Nitroprusside; C016276:8-bromocyclic GMP; D015759:Ionomycin; C038237:1-oleoyl-2-acetylglycerol; D000255:Adenosine Triphosphate; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate