Inhibition of nitric oxide biosynthesis promotes P-selectin expression in platelets: Role of protein kinase C

Abstract

Inhibition of NO synthesis promotes P-selectin expression on endothelial cells; however, the precise mechanism is unclear. Because NO has been shown to inhibit protein kinase C (PKC) activity, we examined the hypothesis that the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) stimulates P-selectin expression on platelets via PKC activation. Ten-minute incubation with either phorbol 12-myristate 13-acetate (PMA), thrombin, or L- NAME significantly increased P-selectin expression on platelets (as assessed by flow-cytometric analysis) and PKC activity of platelet membranes. Increased P-selectin expression induced by either PMA, thrombin, or L-NAME was significantly attenuated by the selective PKC inhibitor UCN-01 (7- hydroxystaurosporine). Furthermore, L-NAME induced P selectin expression was significantly attenuated by either L-arginine, 8-bromo-cGMP, or sodium nitroprusside (SNP). Interestingly, L-NAME further potentiated P-selectin upregulation by thrombin. L-NAME, thrombin, and PMA also significantly increased polymorphonuclear leukocyte adherence to the coronary artery endothelium, an effect that was significantly attenuated by the anti P- selectin monoclonal antibody PBL3 or by UCN-01, L-arginine, 8-bromo-cGMP or SNP but not by D-arginine or the nonblocking anti-P-selectin monoclonal antibody NBPL6. These results indicate that inhibition of NO synthesis induces rapid P-selectin expression, which appears to be at least partially mediated by PKC activation in platelets. Similar effects and mechanisms of L- NAME on P-selectin function were also observed in endothelial cells, another site of P-selectin expression. Thus, PKC activation may play an important role in cell to-cell interaction when NO production is compromised.