Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates

Cell Chem Biol. 2021 Jan 21;28(1):26-33.e8. doi: 10.1016/j.chembiol.2020.10.002. Epub 2020 Oct 22.


Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.

Keywords: Glossina morsitans; X-ray crystallography; anticoagulant; coagulation inhibitor; macromolecular recognition; post-translational modification; protein-protein interaction; three-dimensional structure; thrombin inhibitor; tyrosine sulfation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anticoagulants / chemical synthesis
  • Anticoagulants / chemistry
  • Anticoagulants / pharmacology*
  • Antithrombin Proteins / chemical synthesis
  • Antithrombin Proteins / chemistry
  • Antithrombin Proteins / pharmacology*
  • Cell Line
  • Humans
  • Insect Proteins / chemical synthesis
  • Insect Proteins / chemistry
  • Insect Proteins / pharmacology*
  • Molecular Structure
  • Thrombin / antagonists & inhibitors*
  • Thrombin / metabolism
  • Tsetse Flies
  • Tyrosine / analogs & derivatives*
  • Tyrosine / chemical synthesis
  • Tyrosine / chemistry
  • Tyrosine / pharmacology


  • Anticoagulants
  • Antithrombin Proteins
  • Insect Proteins
  • tsetse thrombin inhibitor
  • tyrosine O-sulfate
  • Tyrosine
  • Thrombin