Anophelin: Kinetics and mechanism of thrombin inhibition
Anophelin: Kinetics and mechanism of thrombin inhibition
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DOI：
10.1021/bi991231p
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发表时间：
1999-12-14
期刊：
影响因子：
2.9
通讯作者：
Ribeiro, JMC
中科院分区：
文献类型：
--
作者：
Francischetti, IMB;Valenzuela, JG;Ribeiro, JMC
按蚊蛋白是从白纹伊蚊唾液腺中分离出来的一种6.5 kDa的多肽，具有α-凝血酶抑制物的作用。本文对按蚊抑制α-凝血酶的作用进行了动力学分析和机理研究。Anophelin被确定为一种可逆的、缓慢的、紧密结合的α-凝血酶抑制物，表现为竞争性抑制。计算得到的解离速率常数(k(1))为2.11+/-0.06x10(8)M-1 S(-1)，解离速率常数(k(-1))为4.05+/-0.97x10(-4)S(-1)。在0.15和0.4M的氯化钠存在下，按蚊-α-凝血酶复合体的K-I分别增加了17.6倍和207倍，表明离子相互作用在按蚊-α-凝血酶复合体的形成中起重要作用。将α-凝血酶与与α-凝血酶阴离子结合外切酶1(TABE1)结合的C-末端水飞蓟素片段54-65孵育可减弱按蚊蛋白对α-凝血酶的抑制作用；按蚊蛋白还可阻断TABE1依赖的胰酶；介导α-凝血酶的蛋白分解。利用γ-凝血酶，一种阴离子结合位被破坏的α-凝血酶衍生物，Anophelin作为一种快速而经典的竞争性抑制小显色底物的伽玛-凝血酶水解酶(K-I=0.694+/-0.063 nm)。此外，用D-Phe-Pro-Arg-氯甲基酮(PPACK)处理酶可防止按蚊-伽马-凝血酶复合体的形成，PPACK是一种不可逆转地阻断凝血酶催化活性的试剂。结论是，Anophelin是一种有效的cr-凝血酶双重抑制物，因为它既与TABE1结合，又与催化部位结合，最佳结合取决于这两个结构域的可用性。最后，Anophelin抑制凝血结合的cr-凝血酶，IC50为45 nM，并在激活凝血的内在途径后，增加了体外爆炸性α-凝血酶产生之前的滞后期。由于其独特的一级序列，按蚊蛋白可能成为研究α-凝血酶结构和功能的一种新试剂。
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 or-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 sire of thrombin. It; is concluded that anophelin is a potent dual inhibitor of cr-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 cr-thrombin with an IC50 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.