Thrombin Aptamer HD1 Inhibits Prothrombin Activation by Binding Proexosite 1 on Prothrombin.
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abstract
AbstractFunctional maturation of exosites 1 and 2 during prothrombin activation endows thrombin with its physiological activities. Thrombin exosite 1 binds ligands such as fibrinogen, the thrombin receptor, and hirudin, and orients them to the active site. In contrast, thrombin exosite 2 binds ligands such as heparin, platelet glycoprotein Iba, and the chondroitin sulfate moiety of thrombomodulin molecules; ligands that do not directly interact with the active site. Neither exosite is fully functional in prothrombin. Exosite 2 only becomes functional when fragment 2 is released. In contrast, there is evidence for existence of a proexosite 1 in prothrombin which contributes to prothrombin activation by prothrombinase. This domain in prothrombin lacks fibrin-binding capacity, but has been implicated in the interaction with factor Va (fVa), a necessary step for enhanced prothrombin activation by prothrombinase. In the present study, we used fluorescent (F) HD1 and HD22, DNA aptamers that bind to exosites 1 and 2, respectively, to study the exosites in prothrombin, prothrombin intermediates and thrombin. F-HD22 does not bind to prothrombin and only binds prothrombin intermediates lacking fragment 2. In contrast, F-HD1 binds to prothrombin and all of the prothrombin intermediates with Kd values ranging from 80 to 25 nM for prothrombin and thrombin, respectively. These findings are different from what has been reported with the hirudin fragment, Hir54–65, which exhibited a 60-fold lower affinity for prothrombin than for thrombin. To better explore the involvement of proexosite 1 in the activation of prothrombin by prothrombinase, we examined the effect of HD1 on prothrombin activation by intact prothrombinase, or by various components of the prothrombinase complex. HD1 inhibits prothrombin activation by prothrombinase with an IC50 of 140 nM. Omitting fVa from the prothrombinase complex abolishes the inhibitory effect of HD1, supporting the concept that the inhibitory effect of HD1 reflects disruption of the interaction between prothrombin and fVa. In similar experiments, Hir54–65 inhibits prothrombin activation by prothrombinase with an IC50 of 225 nM, in agreement with previous studies. These data show that, like hirugen, HD1 binds to the fVa-binding domain on proexosite 1 of prothrombin. In order to refine the location of HD1 and Hir54–65 binding sites on thrombin and prothrombin, competitive binding experiments were performed. Hir54–65 displaces 80% of F-HD1 from thrombin and 70% from prothrombin. In reverse titrations, HD1 displaces 50% of F-Hir54–65 from thrombin, but does not displace F-Hir54–65 from prothrombin. Taken together, these data suggest that the HD1 binding domain is distinct, at least in part, from the Hir54–65 binding domain on prothrombin, an observation supported by examination of the crystal structures of complexes of HD1 or Hir54–65 with thrombin. In conclusion, our data demonstrate that the prsuggests that the HD1 binding site on prothrombin more closely coincides with the fVa-binding site than does the hirugen-binding site. These findings provide new avenues for investigations into the oexosite 1 domain on prothrombin is fully capacitated for binding HD1. This role of exosite 1, not only in thrombin, but also in prothrombin.
