Investigation of the anticoagulant mechanisms of a covalent antithrombin-heparin complex

Recently, we developed a covalent antithrombin-heparin complex (ATH) as a p ossible treatment for respiratory distress syndrome. ATH reacted rapidly wi th thrombin and efficiently catalyzed the inhibition of either thrombin or factor Xa by exogenous antithrombin, In order to investigate mechanisms for the conjugate's unusual anticoagulant properties, changes in fluorescence due to covalent linkage or addition of exogenous antithrombin were studied in relation to reaction with thrombin derivatives or factor Xa. The emissio n spectrum of ATH was similar to that of antithrombin plus heparin mixtures . ATH quickly inhibited thrombin or factor Xa activities, as measured by a fluorogenic substrate. Fluorescein-labeled heparin was displaced from eithe r thrombin or active site blocked thrombin by ATH, indicating that thrombin must bind to the conjugate's heparin moiety. Interaction of thrombin with ATH's heparin component was confirmed by a slow reaction rate of conjugate with a thrombin mutant that has weak heparin binding. Total intrinsic fluor escence increased when exogenous antithrombin was added to ATH, indicating that the catalytic mechanism may occur through a second inhibitor binding s ite. Thus, ATH reacts directly with thrombin through a bridge mechanism and probably catalyzes the reaction of thrombin with antithrombin by a second binding sequence on its heparin chain.