Inhibition of Nitric Oxide Biosynthesis Promotes P-selectin Expression in Platelets

Abstract

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 PB1.3 or by UCN-01, l -arginine, 8-bromo-cGMP or SNP but not by d -arginine or the nonblocking anti–P-selectin monoclonal antibody NBP1.6. 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.