Background: A highly consistent association links circulating Gamma-Glutamyl-Transferase (GGT) to incident acute coronary events to which Tissue Factor (TF), the principal initiator of the coagulation process, is known to contribute actively. GGT and TF are also coexpressed by plaques harvested from atherosclerotic patients, thus making it conceivable a direct role of the former on TF expression and activation. The aim of our study was to assess the direct effect of GGT on TF antigen (ag) , TF mRNA and TF pro-coagulant activity (PCA) in human peripheral blood mononuclear cells. All experiments we run using an enzymatically inactive human recombinant (hr) GGT to exclude confounding from its enzymatic activity. Methods: Human peripheral blood mononuclear cells were obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and incubated with hrGGT (0.5ng/μl) either alone or with anti-hrGGT, a specific polyclonal antibodies (2,5μg/ml), BAY- 11-7082 (10-5M) a selective NF-κB inhibitor, and N-acetylcysteine (10-3M) an antioxidant. TFag expression was assessed by ELISA, TF mRNA by real-time PCR and PCA by a 1-stage clotting assay. Results: hrGGT increased TFag expression (from 85±59 to 536±317 pg/mL,n=13, p<0.001) and stimulated PCA (from 0.008±0.007 to 0.37±0.3 arbitrary units, n=14, p<0.001) and TF mRNA (from 0.006±0.002 to 0.048±0.04 fold-increase,n=9,p< 0.001). hrGGT-induced TFag and PCA was inhibited by anti-hrGGT antibodies (PCA: from 0.70±0.56 to 0.27±0.34 arbitrary units, n=8, p<0.01,-64%; TFag: from 489±394 to 193±65pg/mL, n=6, p<0.01) as well as by pre-treatment with BAY-11-7082 (PCA: from 0.21±0.17 to 0.08±0.11 arbitrary units, n=7, p<0.01, -70%; TFag: from 410±255 to 146± 94 ρg/mL, n=7, p<0.01) and N-acetylcysteine (PCA: from 0.386±0.17 to 0.08±0.13 arbitrary units, n=7,p<0.01). Conclusion: These data represent the first demonstration of a direct effect of GGT on TF expression independent of its own enzymatic activity, a behavior consistent with a cytokine-like mechanism acting through NF-κB stimulation. This mechanism might contribute to promote acute thrombotic events, a possibility in need, however, of further evaluation.
GAMMA-GLUTAMYLTRANSFERASE STIMULATES TISSUE FACTOR EXPRESSION INDEPENDENT OF ITS ENZYMATIC ACTIVITY IN HUMAN MONONUCLEAR CELLS
SCALISE, VALENTINA;BALIA, CRISTINA;CIANCHETTI, SILVANA;FRANZINI, MARIA;CARNICELLI, VITTORIA;PETRINI, SILVIA;NERI, TOMMASO;ZUCCHI, RICCARDO;CELI, ALESSANDRO;CORTI, ALESSANDRO;
2015-01-01
Abstract
Background: A highly consistent association links circulating Gamma-Glutamyl-Transferase (GGT) to incident acute coronary events to which Tissue Factor (TF), the principal initiator of the coagulation process, is known to contribute actively. GGT and TF are also coexpressed by plaques harvested from atherosclerotic patients, thus making it conceivable a direct role of the former on TF expression and activation. The aim of our study was to assess the direct effect of GGT on TF antigen (ag) , TF mRNA and TF pro-coagulant activity (PCA) in human peripheral blood mononuclear cells. All experiments we run using an enzymatically inactive human recombinant (hr) GGT to exclude confounding from its enzymatic activity. Methods: Human peripheral blood mononuclear cells were obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and incubated with hrGGT (0.5ng/μl) either alone or with anti-hrGGT, a specific polyclonal antibodies (2,5μg/ml), BAY- 11-7082 (10-5M) a selective NF-κB inhibitor, and N-acetylcysteine (10-3M) an antioxidant. TFag expression was assessed by ELISA, TF mRNA by real-time PCR and PCA by a 1-stage clotting assay. Results: hrGGT increased TFag expression (from 85±59 to 536±317 pg/mL,n=13, p<0.001) and stimulated PCA (from 0.008±0.007 to 0.37±0.3 arbitrary units, n=14, p<0.001) and TF mRNA (from 0.006±0.002 to 0.048±0.04 fold-increase,n=9,p< 0.001). hrGGT-induced TFag and PCA was inhibited by anti-hrGGT antibodies (PCA: from 0.70±0.56 to 0.27±0.34 arbitrary units, n=8, p<0.01,-64%; TFag: from 489±394 to 193±65pg/mL, n=6, p<0.01) as well as by pre-treatment with BAY-11-7082 (PCA: from 0.21±0.17 to 0.08±0.11 arbitrary units, n=7, p<0.01, -70%; TFag: from 410±255 to 146± 94 ρg/mL, n=7, p<0.01) and N-acetylcysteine (PCA: from 0.386±0.17 to 0.08±0.13 arbitrary units, n=7,p<0.01). Conclusion: These data represent the first demonstration of a direct effect of GGT on TF expression independent of its own enzymatic activity, a behavior consistent with a cytokine-like mechanism acting through NF-κB stimulation. This mechanism might contribute to promote acute thrombotic events, a possibility in need, however, of further evaluation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
