Anophelin is a 6.5-kDa peptide isolated from the salivary gland of Anopheles albimanus that behaves as an α-thrombin inhibitor. In this paper, kinetic analyses and the study of mechanism of α-thrombin inhibition by anophelin were performed. Anophelin was determined to be a reversible, slow, tight-binding inhibitor of α-thrombin, displaying a competitive type of inhibition. The binding of anophelin to α-thrombin is stoichiometric with a dissociation constant (K(i)) of 5.87 ± 1.46 pM, a calculated association rate constant (k(i)) of 2.11 ± 0.06 x 108 M-1 s-1, and a dissociation rate constant (k-1) of 4.05 ± 0.97 X 10-4 s-1. In the presence of 0.15 and 0.4 M NaCl, a 17.6- and 207-fold increase in the K(i) of anophelin α- thrombin complex was observed, respectively, indicating that ionic interactions are important in anophelin-α-thrombin complex formation. Incubation of α-thrombin with C-terminal hirudin fragment 54-65 that binds to α-thrombin anion binding exosite 1 (TABE1) attenuates α-thrombin inhibition by anophelin; anophelin also blocks TABE1-dependent trypsin- mediated proteolysis of α-thrombin. Using γ-thrombin, an α-thrombin derivative where the anion binding exosite has been disrupted, anophelin behaves as a fast and classical competitive inhibitor of γ-thrombin hydrolysis of small chromogenic substrate (K(i) = 0.694 ± 0.063 nM). In addition, anophelin-γ-thrombin complex formation is prevented by treatment of the enzyme with D-Phe-Pro-Arg-chloromethyl ketone (PPACK), a reagent that irreversibly blocks the catalytic site of thrombin. It is concluded that anophelin is a potent dual inhibitor of α-thrombin because it binds both to TABE1 and to the catalytic site, optimal binding being dependent on the availability of both domains. Finally, anophelin inhibits clot-bound α- thrombin with an IC50 of 45 nM and increases the lag phase that precedes explosive in vitro α-thrombin generation after activation of intrinsic pathway of blood coagulation. Because of its unique primary sequence, anophelin may be used as a novel reagent to study the structure and function of α-thrombin.
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