Bound Thrombin from Crushed Clots Is Composed of α-Thrombin and the N-Terminal Regions of α- and γ-Chains of Fibrinogen

We aimed at clarifying the structural characteristics of the bound thrombin that is liberated by mechanical breakdown of fibrin clots. Fibrin clots were prepared with bovine thrombin and rabbit fibrinogen, and were crushed mechanically with a glass rod. The supernatant of the crushed clots was subjected to immunoaffinity chromatography to isolate the bound thrombin. Western blotting analysis revealed that the bound thrombin could be reacted with both antithrombin and antifibrinogen under unreduced conditions. SDS-PAGE under reduced conditions revealed that there were three bands, two of which were found to be the N-terminal fragments of the alpha- and gamma-chains of fibrinogen. The bound thrombin could be dissociated into three distinct fibrin fragments and bovine alpha-thrombin when denatured by 8 M urea. Thus, the bound thrombin liberated from crushed clots is a stable complex between bovine alpha-thrombin and fibrin fragments of the N-terminal regions of rabbit alpha- and gamma-chains.