Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II

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 (Arg(93) --> Ala, Arg(97) --> Ala, and Arg(101) --> Ala thrombin) is substituted for thrombin, Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha(1)-antitrypsin Met(358) --> 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.