Non enzymatic upregulation of tissue factor expression by gamma-glutamyl transferase in human peripheral blood mononuclear cellsReport as inadecuate




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Thrombosis Journal

, 14:45

First Online: 04 November 2016Received: 11 August 2016Accepted: 26 October 2016

Abstract

BackgroundBesides maintaining intracellular glutathione stores, gamma-glutamyltransferaseGGT 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.

MethodsExperiments 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 antigenELISA and TFmRNAreal-time PCR were assessed in unpooled human peripheral blood mononuclear cellPBMC suspensions obtained from healthy donors through discontinuous Ficoll-Hystopaque density gradient.

ResultsEquine 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. EndotoxinLPS contamination of GGT preparations was excluded by heat inactivation studies and direct determinationLAL 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.

ConclusionsGGT 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.

KeywordsGamma-glutamyltransferase Tissue Factor Cytokines Oxidative Stress NFkB AbbreviationsagAntigen

AUArbitrary Units

CaCl2Calcium Chloride

DNADeoxyribonucleic acid

ELISAEnzyme-linked immunosorbent assay

GAPDHGlyceraldehyde-3-phosphate dehydrogenase

GGTGamma-glutamyltransferase

GSHGlutathione

HrHuman recombinant

IgImmunoglobulin

LALLimulus Amebocyte Lysate

LPSLipopolysaccharide

MTTDimethyl thiazolyl diphenyl tetrazolium

NACN-acetyl-L-cysteine

NADPNicotinamide adenine dinucleotide phosphate

NFkBNuclear factor kappa-light-chain-enhancer of activated B cells

PBMCPeripheral Blood Mononuclear Cell

PCAProcoagulant activity

PCRPolymerase Chain Reaction

RNADeoxyribonucleic acid

ROSReactive Oxygen Species

RPMIRoswell Park Memorial Institute

SDStandard deviation

TFTissue Factor

TNFTumour Necrosis Factor

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Author: Valentina Scalise - Cristina Balia - Silvana Cianchetti - Tommaso Neri - Vittoria Carnicelli - Riccardo Zucchi - Maria Fran

Source: https://link.springer.com/article/10.1186/s12959-016-0119-8



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