Ecotin modulates thrombin activity through exosite-2 interactions

Int J Biochem Cell Biol. 2006;38(11):1893-900. doi: 10.1016/j.biocel.2006.05.001. Epub 2006 May 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Dose-Response Relationship, Drug
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism
  • Escherichia coli Proteins / pharmacology*
  • Humans
  • Periplasmic Proteins / chemistry
  • Periplasmic Proteins / metabolism
  • Periplasmic Proteins / pharmacology*
  • Platelet Aggregation / drug effects
  • Platelet Aggregation Inhibitors / pharmacology
  • Protein Binding / drug effects
  • Protein Conformation / drug effects
  • Serine Endopeptidases / metabolism
  • Serine Proteinase Inhibitors / chemistry
  • Serine Proteinase Inhibitors / metabolism
  • Serine Proteinase Inhibitors / pharmacology
  • Spectrometry, Fluorescence
  • Thrombin / metabolism*


  • Eco protein, E coli
  • Escherichia coli Proteins
  • Periplasmic Proteins
  • Platelet Aggregation Inhibitors
  • Serine Proteinase Inhibitors
  • Serine Endopeptidases
  • Thrombin