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.

尽管只有 32 个残基，采采蝇凝血酶抑制剂 (TTI) 是所描述的最有效的抗凝剂之一，对凝血酶具有亚皮摩尔抑制活性。出乎意料的是，从果蝇中分离的 TTI 比合成和重组变体的活性高 2000 倍，重 180 Da。我们预测了酪氨酸O的存在TTI 的 -硫酸盐翻译后修饰，促使我们研究修饰对抗凝活性的影响。化学合成和功能分析的组合用于揭示硫酸化显着提高了 TTI 对凝血酶的抑制活性。使用 X 射线晶体学，我们显示 TTI 的 N 端硫酸化片段结合凝血酶的基本外位点 II，建立类似于生理底物的相互作用，而 C 端片段消除了凝血酶的催化活性。这种非规范的抑制模式，加上其效力和小尺寸，使 TTI 成为设计新型抗血栓药物的有吸引力的支架。