Non enzymatic upregulation of tissue factor expression by gamma-glutamyl transferase in human peripheral blood mononuclear cells.
BACKGROUND
Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar.


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 TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient.


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 <0.1 ng/mL practically 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 NFkB-driven, redox-sensitive transcriptional site of action.


CONCLUSIONS
GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation.
Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell'Area Critica, Università di Pisa, Pisa, Italy.
Valentina
V
Scalise
Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell'Area Critica, Università di Pisa, Pisa, Italy.
Cristina
C
Balia
Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell'Area Critica, Università di Pisa, Pisa, Italy.
Silvana
S
Cianchetti
Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell'Area Critica, Università di Pisa, Pisa, Italy.
Tommaso
T
Neri
Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell'Area Critica, Università di Pisa, Pisa, Italy.
Vittoria
V
Carnicelli
Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell'Area Critica, Università di Pisa, Pisa, Italy.
Riccardo
R
Zucchi
Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Università di Pisa, Pisa, Italy.
Maria
M
Franzini
Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Università di Pisa, Pisa, Italy.
Alessandro
A
Corti
Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Università di Pisa, Pisa, Italy.
Aldo
A
Paolicchi
Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell'Area Critica, Università di Pisa, Pisa, Italy.
Alessandro
A
Celi
Dipartimento di Patologia Chirurgica, Medica, Molecolare e dell'Area Critica, Università di Pisa, Pisa, Italy.
Roberto
0000-0001-7745-3890
R
Pedrinelli
Cytokines; Gamma-glutamyltransferase; NFkB; Oxidative Stress; Tissue Factor