title: Ecotin modulates thrombin activity through exosite-2 interactions.
abstract: Ecotin is a Escherichia coli-derived protein that has been characterized as a potent inhibitor of serine-proteases. This protein is highly effective against several mammalian enzymes, which includes pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. In this work we showed that ecotin binds to human alpha-thrombin via its secondary binding site, and modulates thrombin catalytic activity. Formation of wild type ecotin-alpha-thrombin complex was observed by native PAGE and remarkably, gel filtration chromatography showed an unusual 2:1 ecotin:enzyme stoichiometry. Analysis of the protease inhibitor effects on thrombin biological activities showed that (i) it decreases the inhibition of thrombin by heparin/antithrombin complex (IC50=3.2 microM); (ii) it produces a two-fold increase in the thrombin-induced fibrinogen clotting; and (iii) it inhibits thrombin-induced platelet aggregation (IC50=4.5 microM). Allosteric changes on thrombin structure were then evaluated. Complex formation with ecotin caused a three-fold increase in the rate of thrombin inhibition by BPTI, suggesting a displacement of the enzyme's 60-loop. In addition, ecotin modulated the enzyme's catalytic site, as demonstrated by changes in the fluorescence emission of fluorescein-FPRCK-alpha-thrombin (EC50=3.5 microM). Finally, solid phase competition assays demonstrated that heparin and prothrombin fragment 2 prevents thrombin interaction with ecotin. Altogether, these observations strongly support an ecotin interaction with thrombin anion-binding exosite-2, resulting in modulation of its biological activities. At this point, ecotin might be useful as a new tool for studying thrombin allosteric modulation.
authors.affiliation: LaBioMol, Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Outeiro São João Batista, Niterói, RJ 24001-970, Brazil.
authors.forename: Helena C
authors.initials: HC
authors.lastname: Castro
authors.forename: Robson Q
authors.initials: RQ
authors.lastname: Monteiro
authors.forename: Mariane
authors.initials: M
authors.lastname: Assafim
authors.forename: Natália I V
authors.initials: NI
authors.lastname: Loureiro
authors.forename: Charles
authors.initials: C
authors.lastname: Craik
authors.forename: Russolina B
authors.initials: RB
authors.lastname: Zingali
mesh_terms: D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D033903:Periplasmic Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism*
chemical_list: C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D010975:Platelet Aggregation Inhibitors; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D013917:Thrombin