Activation-induced exposure of the thrombin anion-binding exosite. Interactions of recombinant mutant prothrombins with thrombomodulin and a thrombin exosite-specific antibody.
The activation of serine protease zymogens involves conformational changes 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 addition to the conserved activation cleavage after Arg320. To understand how these cleavages lead to the exposure of the thrombin anion-binding exosite, a major macromolecular recognition site, interactions of recombinant human prothrombin derivatives with thrombomodulin, and an exosite-specific antibody were studied by competition binding and immunoprecipitation. By either method, the anion-binding exosite is not functional on prethrombin 2, which is cleaved after Arg271 and lacks fragment 1.2, nor on meizothrombin, which is cleaved only after Arg320. In contrast, the exosite is fully exposed on meizothrombin des-F1, which is cleaved after both Arg320 and Arg155 and therefore lacks amino-terminal fragment 1 (F1). Thus, two events are required to create the exosite. First, cleavage after Arg320 causes conformational changes that are much more extensive than those accompanying the activation of trypsinogen. Second, removal of amino-terminal F1 is necessary, perhaps to relieve steric hindrance. These results indicate that the F1 fragment regulates access to the thrombin exosite. The properties of meizothrombin des-F1 suggest that this prothrombin derivative could have a biological function.
Wu
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Q
Department of Medicine, Jewish Hospital of St. Louis, Washington University School of Medicine, Missouri 63110.
Picard
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V
Aiach
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Sadler
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JE
D000595:Amino Acid Sequence; D000906:Antibodies / Q000494:pharmacology*; D001323:Autoantibodies / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D001666:Binding Sites, Antibody; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011516:Prothrombin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D015183:Restriction Mapping; D013917:Thrombin / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism*
D000906:Antibodies; D001323:Autoantibodies; D011994:Recombinant Proteins; D018180:Thrombomodulin; D011516:Prothrombin; D013917:Thrombin