Regulation of protein kinase C by nitroarachidonic acid: Impact on human platelet activation

• We evaluated the effect of nitrated arachidonic acid on human platelet activation.
• NO2AA regulates PKC, key protein for platelet activation.
• The anti-platelet effects were independent of cGMP or Ca2+ mobilization.
• Our observations provide a possible novel mechanism for platelet in vivo regulation.

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 NO2AA and describe a novel mechanism involving protein kinase C (PKC) inhibition. NO2AA-mediated antiplatelet effects were characterized using mass spectrometry, confocal microscopy, flow cytometry, western blot and aggregometry. Incubation of NO2AA 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 Ca2+ 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 NO2AA. Immunofluorescence confocal microscopy confirmed NO2AA-mediated inhibition of PKCα translocation to the membrane. In summary, we demonstrate that NO2AA inhibits platelet activation through modulation of PKCα activity as a potential novel mechanism for platelet regulation in vivo.