Anophelin: kinetics and mechanism of thrombin inhibition.

摘要: 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 study mechanism inhibition by anophelin were performed. was determined to be reversible, slow, tight-binding inhibitor alpha-thrombin, displaying competitive type inhibition. The binding stoichiometric with dissociation constant (K(i)) 5.87 +/- 1.46 pM, calculated association rate (k(1)) 2.11 0.06 x 10(8) M(-1) s(-1), (k(-1)) 4.05 0.97 10(-4) s(-1). presence 0.15 0.4 M NaCl, 17.6- 207-fold increase in K(i) anophelin-alpha-thrombin complex observed, respectively, indicating ionic interactions are important formation. Incubation C-terminal hirudin fragment 54-65 binds anion exosite 1 (TABE1) attenuates anophelin; also blocks TABE1-dependent trypsin-mediated proteolysis alpha-thrombin. Using gamma-thrombin, derivative where has been disrupted, fast classical gamma-thrombin hydrolysis small chromogenic substrate (K(i) = 0. 694 0.063 nM). addition, anophelin-gamma-thrombin formation prevented treatment enzyme D-Phe-Pro-Arg-chloromethyl ketone (PPACK), reagent irreversibly catalytic site thrombin. It concluded potent dual because it both TABE1 site, optimal being dependent on availability domains. Finally, inhibits clot-bound IC(50) 45 nM increases lag phase precedes explosive vitro generation after activation intrinsic pathway blood coagulation. Because its unique primary sequence, may used novel structure function