• thrombosis
• gamma-glutamyltransferase
• diabetes
• cardiovascular disease
• tissue factor

Background: Gamma-glutamyltransferase (GGT), a member of the structural superfamily of the N-terminal nucleophilic hydrolases expressed by a wide number of cell types including circulating monocytes, hydrolyzes extracellular glutathione (GSH) to provide cysteine for its intracellular re-synthesis. Along with its pivotal role in that antioxidant biological process, however, GGT also generates reactive oxygen species (ROS) and activates NFkB, a redox-sensitive transcription factor key in Tissue Factor (TF) gene expression, a major regulator of haemostasis and thrombosis. Therefore, deductive reasoning makes it plausible to hypothesize a cross-talk between GGT and TF, a possibility consistent with the highly consistent epidemiological association of circulating GGT levels with acute coronary events, that coexisting diabetes increases exponentially, a pathological process in which TF contribute actively. An assumption, corroborated by recent reports of enzymatically active protein in human atheromatous plaques, requires, however, to document a mechanistic link between GGT and TF for which at the moment no evidence is available.
Aim: To evaluate the effect of GGT on TF expression in peripheral blood mononuclear cells (PBMCs), and the potential intracellular mechanism involved, exposed to normal glucose (NG) and high glucose (HG) concentration, the diabetic hallmark.
Methods: Experiments were run with either equine, enzymatically active GGT or human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert because of missing post translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen (ELISA) and TF mRNA (real-time PCR) were assessed in unpooled PBMCs suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient and exposed to NG and HG concentrations. Specificity of the effect GGT-induced was tested by an anti-hrGGT polyclonal antibody. The role of NFκB was indirectly assessed by using BAY-11-7082, a validated NFκB inhibitor, as well as N-acetyl-L-cysteine (NAC), an antioxidant with inhibitory effect on NFκB .
Results: Equine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin (LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination (LAL method) of LPS concentrations <0.1ng/ml devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl- L-cysteine, an antioxidant, was consistent with a NFκB-driven, redox-sensitive transcriptional site of action. High doses of glucose, up to 50mM concentration, potentiated the effect of hrGGT on TF expression in a concentration-dependent manner, both PCA, antigen and mRNA expression.
Conclusions: GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behavior mediated by NFκB activation that might perhaps promote acute thrombotic events. HG concentrations amplifies that effect, potentially contributing to atherothrombotic risk conferred by higher GGT levels in diabetic patients.