ACTIVATION-INDUCED EXPOSURE OF THE THROMBIN ANION-BINDING EXOSITE - INTERACTIONS OF RECOMBINANT MUTANT PROTHROMBINS WITH THROMBOMODULIN ANDA THROMBIN EXOSITE-SPECIFIC ANTIBODY

The activation of serine protease zymogens involves conformational cha nges that increase the affinity of substrate binding and the activity of the catalytic center. The activation of prothrombin is particularly complex and requires several cleavages in the proenzyme region in add ition to the conserved activation cleavage after Arg320. To understand how these cleavages lead to the exposure of the thrombin anion-bindin g exosite, a major macromolecular recognition site, interactions of re combinant human prothrombin derivatives with thrombomodulin, and an ex osite-specific antibody were studied by competition binding and immuno precipitation. By either method, the anion-binding exosite is not func tional on prethrombin 2, which is cleaved after Arg271 and lacks fragm ent 1.2, nor on meizothrombin, which is cleaved only after Arg320. In contrast, the exosite is fully exposed on meizothrombin des-Fl, which is cleaved after both Arg320 and Arg155 and therefore lacks amino-term inal fragment 1 (Fl). Thus, two events are required to create the exos ite. First, cleavage after Arg320 causes conformational changes that a re much more extensive than those accompanying the activation of tryps inogen. Second, removal of amino-terminal Fl is necessary, perhaps to relieve steric hindrance. These results indicate that the F1 fragment regulates access to the thrombin exosite. The properties of meizothrom bin des-Fl suggest that this prothrombin derivative could have a biolo gical function.