title: Anophelin: kinetics and mechanism of thrombin inhibition.
abstract: Anophelin is a 6.5-kDa peptide isolated from the salivary gland of Anopheles albimanus that behaves as an alpha-thrombin inhibitor. In this paper, kinetic analyses and the study of mechanism of alpha-thrombin inhibition by anophelin were performed. Anophelin was determined to be a reversible, slow, tight-binding inhibitor of alpha-thrombin, displaying a competitive type of inhibition. The binding of anophelin to alpha-thrombin is stoichiometric with a dissociation constant (K(i)) of 5.87 +/- 1.46 pM, a calculated association rate constant (k(1)) of 2.11 +/- 0.06 x 10(8) 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-alpha-thrombin complex was observed, respectively, indicating that ionic interactions are important in anophelin-alpha-thrombin complex formation. Incubation of alpha-thrombin with C-terminal hirudin fragment 54-65 that binds to alpha-thrombin anion binding exosite 1 (TABE1) attenuates alpha-thrombin inhibition by anophelin; anophelin also blocks TABE1-dependent trypsin-mediated proteolysis of alpha-thrombin. Using gamma-thrombin, an alpha-thrombin derivative where the anion binding exosite has been disrupted, anophelin behaves as a fast and classical competitive inhibitor of gamma-thrombin hydrolysis of small chromogenic substrate (K(i) = 0. 694 +/- 0.063 nM). In addition, anophelin-gamma-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 alpha-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 alpha-thrombin with an IC(50) of 45 nM and increases the lag phase that precedes explosive in vitro alpha-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 alpha-thrombin.
authors.affiliation: Medical Entomology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0425, USA.
authors.forename: I M
authors.initials: IM
authors.lastname: Francischetti
authors.forename: J G
authors.initials: JG
authors.lastname: Valenzuela
authors.forename: J M
authors.initials: JM
authors.lastname: Ribeiro
mesh_terms: D000818:Animals; D000852:Anopheles; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D006629:Hirudins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
chemical_list: D006629:Hirudins; D019476:Insect Proteins; D012471:Salivary Proteins and Peptides; C121275:anophelin protein, Anopheles albimanus; D013917:Thrombin