title: Regulation of protein kinase C by nitroarachidonic acid: impact on human platelet activation.
abstract: Platelet activation represents a key event in normal hemostasis as well as during platelet plug formation related to thrombosis. Nitro-fatty acids are novel endogenously produced signaling mediators exerting pluripotent anti-inflammatory actions in cells and tissues. We have recently shown that nitroarachidonic acid inhibits thromboxane synthesis during platelet activation by affecting prostaglandin endoperoxide H synthase (PGHS). Herein, we investigated the regulation of human platelet activation by NO(2)AA and describe a novel mechanism involving protein kinase C (PKC) inhibition. NO(2)AA-mediated antiplatelet effects were characterized using mass spectrometry, confocal microscopy, flow cytometry, western blot and aggregometry. Incubation of NO(2)AA with human platelets caused a significant reduction in platelet sensitivity to thrombin, ADP, arachidonic acid (AA), and phorbol ester (PMA). These effects were cGMP-independent and did not involve Ca(2+) store-dependent mobilization. In contrast, signaling downstream of conventional PKC activation, such as α-granule secretion and extracellular signal regulated kinase 2 activation was strongly inhibited by NO(2)AA. Immunofluorescence confocal microscopy confirmed NO(2)AA-mediated inhibition of PKCα translocation to the membrane. In summary, we demonstrate that NO(2)AA inhibits platelet activation through modulation of PKCα activity as a potential novel mechanism for platelet regulation in vivo.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125 C.P 11800, Montevideo, Uruguay.
authors.forename: L
authors.initials: L
authors.lastname: Bonilla
authors.forename: V B
authors.initials: VB
authors.lastname: O'Donnell
authors.forename: S R
authors.initials: SR
authors.lastname: Clark
authors.forename: H
authors.initials: H
authors.lastname: Rubbo
authors.forename: A
authors.initials: A
authors.lastname: Trostchansky
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport; D002417:Cattle; D006801:Humans; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*
chemical_list: D016718:Arachidonic Acid; D011493:Protein Kinase C