title: Activation of human platelets by 2-arachidonoylglycerol: role of PKC in NO/cGMP pathway modulation.
abstract: We demonstrated that the endocannabinoid 2-arachidonoylglycerol (2-AG) activated dose-dependently washed human platelets and increased intracellular calcium levels. Moreover 2-AG activated protein kinase C measured as p47pleckstrin phosphorylation. These parameters were prevented by the tromboxane A2 receptor antagonist SQ29548, by phospholipase C pathway (U73122) and protein kinase C (GF109203X) inhibitors. No effect on 2-AG-induced platelet activation and calcium elevation in the presence of inhibitors of fatty acid amide hydrolase or monoacylglycerol lipase was observed. In addition we have shown that 2-AG dose-dependently increased NO and cGMP levels. These effects were abolished by U73122, GF109203X, EGTA and the intracellular calcium chelator BAPTA/AM. Moreover, 2-AG enhanced eNOS activity through the phosphorylation of its positive regulatory residue ser1177 and by dephosphorylation of the negative one thr495. The eNOS ser1177 phosphorylation was inhibited by U73122 and GF109203X but it was unaffected by the PI3K/AKT pathway inhibitors LY294002 and MK2206. The dephosphorylation of thr495 was reversed by low concentrations of calyculin A. Taken together these data suggest that 2-AG behaves as a true platelet agonist stimulating PKC activation and calcium elevation. Likely 2-AG can modulate platelet activation by increasing NO levels through eNOS activation.
authors.affiliation: Department of Experimental Medicine, Biochemistry section, Genoa University, Genoa, Italy.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Signorello
authors.forename: Enrica
authors.initials: E
authors.lastname: Giacobbe
authors.forename: Alessia
authors.initials: A
authors.lastname: Segantin
authors.forename: Luciana
authors.initials: L
authors.lastname: Avigliano
authors.forename: Fabiola
authors.initials: F
authors.lastname: Sinigaglia
authors.forename: Mauro
authors.initials: M
authors.lastname: Maccarrone
authors.forename: Giuliana
authors.initials: G
authors.lastname: Leoncini
mesh_terms: D001095:Arachidonic Acids / Q000378:metabolism* / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D063385:Cannabinoid Receptor Modulators / Q000378:metabolism* / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D063388:Endocannabinoids; D004789:Enzyme Activation / Q000502:physiology; D005989:Glycerides / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D005989:Glycerides; D009569:Nitric Oxide; C094503:glyceryl 2-arachidonate; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C; D006152:Cyclic GMP