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.
Anticoagulants/pharmacology , Antithrombin Proteins/pharmacology , Insect Proteins/pharmacology , Thrombin/antagonists & inhibitors , Tyrosine/analogs & derivatives , Animals , Anticoagulants/chemical synthesis , Anticoagulants/chemistry , Antithrombin Proteins/chemical synthesis , Antithrombin Proteins/chemistry , Cell Line , Humans , Insect Proteins/chemical synthesis , Insect Proteins/chemistry , Molecular Structure , Thrombin/metabolism , Tsetse Flies , Tyrosine/chemical synthesis , Tyrosine/chemistry , Tyrosine/pharmacology
The RGD sequence was used to design potent hirudin isoform 3 mimetic peptides with both antithrombin activity and antiplatelet aggregation activity. The RGD and proline were inserted between the catalytic active binding domain (D-Phe-Pro-Arg-Pro) on the N-terminus and the anion-binding exosite binding domain (QGDFEPIPEDAYDE) on the Cterminus. Thrombin titration assay and ATP-induced platelet aggregation test revealed that the peptide with the linker RGDWP or RGDGP possessed potent antithrombin and antiplatelet activities, while other peptides without the Pro residue in the linker only showed antithrombin activity. Similar results were obtained in the RGD-containing hirulog-1 variants. Our study indicates that the inserted Pro residue facilitates the exposure of RGD and the binding of the peptide to glycoprotein IIb/IIIa (GPIIb/IIIa). The strategy of combining the RGD sequence and the Pro residue may be used for future designs of bifunctional antithrombotic agents.
Antithrombin Proteins/chemical synthesis , Antithrombins/chemical synthesis , Hirudins/chemistry , Peptide Fragments/chemistry , Platelet Aggregation Inhibitors/chemical synthesis , Animals , Antithrombins/chemistry , Catalytic Domain , Hirudins/chemical synthesis , Integrin beta3/metabolism , Male , Oligopeptides , Peptide Fragments/chemical synthesis , Platelet Membrane Glycoprotein IIb/metabolism , Protein Isoforms/chemical synthesis , Protein Isoforms/chemistry , Rats , Rats, Sprague-Dawley , Recombinant Proteins/chemical synthesis , Recombinant Proteins/chemistry , Whole Blood Coagulation Time
