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Anophelin: Kinetics and Mechanism of Thrombin Inhibition

凝血酶 化学 离解常数 水蛭素 立体化学 结合位点 动力学 非竞争性抑制 生物物理学 生物化学 血小板 受体 生物 物理 免疫学 量子力学
作者
Ivo M.B. Francischetti,Jesús G. Valenzuela,José M. C. Ribeiro
出处
期刊:Biochemistry [American Chemical Society]
卷期号:38 (50): 16678-16685 被引量:104
标识
DOI:10.1021/bi991231p
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 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.
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