Anticoagulant Decapeptide\nInteracts with Thrombin\nat the Active Site and Exosite‑I

Thrombin can be used as a target for its inhibitors to\nprevent\nblood coagulation. A novel peptide (TKLTEEEKNR, PfCN) identified from\nαS2-casein (fragments 211–220) with high anticoagulant\nactivity was screened and prepared. The activated partial thromboplastin\ntime, prothrombin time, and thrombin time, at the concentration of\n4 mM, prolonged about 19, 2.5 and 5.5 s, respectively. At the same\nconcentration, the fibrinogen clotting time prolonged from 25.5 ±\n0.7 to 38.3 ± 1.3 s. The thrombin inhibitory efficiency in vitro\n(IC50 value of 29.27 mM) and antithrombosis effect in vivo\nwere determined.\nThe secondary structure of thrombin, which was influenced by PfCN,\nindicates that PfCN can bind to thrombin. Isothermal titration calorimetry\nand the chromogenic substrate test showed that PfCN belongs to the\nbivalent thrombin inhibitor like bivalirudin. Although the effect\nwas not as good as bivalirudin, in the animal experiment, bleeding\noccurred in the bivalirudin group but not in the PfCN group. Moreover,\nmolecular docking illustrates the mechanism for the antithrombin activity\nof PfCN. These results indicated that PfCN could be used as an effective\nthrombin inhibitor with broad potential for the prevention of thrombotic\nacute pulmonary embolism and other thrombotic events.