Isolation of bound thrombin consisting of thrombin and fibrin N-terminal fragment from clot lysate

It has been reported that thrombin is liberated from fibrin clots by the action of fibrinolytic enzymes. It has also been reported that the liberated thrombin complexes with fibrin fragment E or (DD)E, which are denoted as bound thrombin. However, bound thrombin has not been isolated from clot lysate, and the structural characteristics of isolated bound thrombin have not been specified. In this study, we attempted to isolate the bound thrombin from clot lysate and to clarify its structural features. Rabbit fibrinogen was clotted with bovine thrombin, and clot lysate was prepared with urokinase. The bound thrombin was isolated from clot lysate by serial chromatography using a Sepharose 4B column immobilizing an anti-bovine thrombin antibody and a Sepharose 4B column immobilizing an anti-rabbit fibrinogen antibody. SDS-PAGE under unreduced conditions demonstrated that there were two different protein bands in the isolated bound thrombin. On a C4 reverse-phase HPLC, the bound thrombin from clot lysate was resolved by 4 M urea into alpha-thrombin and a fibrin fragment, the N-terminal regions of which were identified as alpha-, beta- and gamma-chains. Thus, in the bound thrombin, thrombin molecule would bind to rabbit fibrin fragment consisting of N-terminal central domain.