Becker D L, Fredenburgh J C, Stafford A R, Weitz J I
Department of Medicine, McMaster University and Hamilton Civic Hospitals Research Centre, Hamilton, Ontario L8V 1C3, Canada.
J Biol Chem. 1999 Mar 5;274(10):6226-33. doi: 10.1074/jbc.274.10.6226.
Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.
当纤维蛋白与凝血酶的外位点1结合且与纤维蛋白结合的肝素与外位点2结合时，会形成三元凝血酶 - 肝素 - 纤维蛋白复合物，该复合物会使抗凝血酶和肝素辅因子II对肝素催化的凝血酶抑制速率分别降低58倍和247倍。肝素辅因子II的降低幅度更大，这反映了其在抑制过程中需要接近外位点1。抗凝血酶和肝素辅因子II的抑制作用受到保护需要外位点1和2都被连接，因为当用外位点1变体（γ-凝血酶和凝血酶Quick 1）或外位点2变体（Arg93→Ala、Arg97→Ala和Arg101→Ala凝血酶）替代凝血酶时，观察到的保护作用最小。同样，在可溶性纤维蛋白和肝素存在的情况下，肝素非依赖性抑制剂α1-抗胰蛋白酶Met358→Arg对凝血酶的抑制速率降低不到2倍。相比之下，凝血酶受到共价抗凝血酶 - 肝素复合物的抑制保护，这表明当三元复合物形成时，肝素接近凝血酶外位点2受到阻碍。这些结果揭示了凝血酶外位点1和2在三元复合物组装以及随后保护凝血酶免受肝素催化抑制剂抑制方面的重要性。
