Abstract
The substrate specificity of thrombin is regulated by binding of macromolecular substrates and effectors to exosites I and II. Exosites I and II have been reported to be extremely linked allosterically, such that binding of a ligand to one exosite results in near-total loss of affinity for ligands at the alternative exosite, whereas other studies support the independence of the interactions. An array of fluorescent thrombin derivatives and fluorescein-labeled hirudin(54-65) ([5F]Hir(54-65)(SO(3)(-))) were used as probes in quantitative equilibrium binding studies to resolve whether the affinities of the exosite I-specific ligands, Hir(54-65)(SO(3)(-)) and fibrinogen, and of the exosite II-specific ligands, prothrombin fragment 2 and a monoclonal antibody, were affected by alternate exosite occupation. Hir(54-65)(SO(3)(-)) and fibrinogen bound to exosite I with dissociation constants of 16-28 nm and 5-7 microm, respectively, which were changed < or =2-fold by fragment 2 binding. Native thrombin and four thrombin derivatives labeled with different probes bound fragment 2 and the antibody with dissociation constants of 3-12 microm and 1.8 nm, respectively, unaffected by Hir(54-65)(SO(3)(-)). The results support a ternary complex binding model in which exosites I and II can be occupied simultaneously. The thrombin catalytic site senses individual and simultaneous binding of exosite I and II ligands differently, resulting in unique active site environments for each thrombin complex. The results indicate significant, ligand-specific allosteric coupling between thrombin exosites I and II and catalytic site perturbations but insignificant inter-exosite thermodynamic linkage.
Highlights
The substrate specificity of thrombin is regulated by binding of macromolecular substrates and effectors to exosites I and II
An array of fluorescent thrombin derivatives and fluorescein-labeled hirudin54–65 ([5F]Hir54–65(SO3؊)) were used as probes in quantitative equilibrium binding studies to resolve whether the affinities of the exosite I-specific ligands, Hir54–65(SO3؊) and fibrinogen, and of the exosite II-specific ligands, prothrombin fragment 2 and a monoclonal antibody, were affected by alternate exosite occupation
This was an important goal because studies employing hirudin peptides or fragment 2 (F2) as probes of exosite involvement in other thrombin interactions could not be interpreted unambiguously, and it was uncertain whether the effects were due to competitive binding of alternate exosite I or II ligands or to extremely negative exosite linkage
Summary
Fibrinogen; ATA-FPR-CH2Cl, N␣[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl; ATA-FFR-CH2Cl, N␣-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl; T, human ␣-thrombin, T*, human ␣-thrombin fluorescently labeled at the active site; [5F]FPR-T, [5-(acetamido)fluorescein]-D-Phe-Pro-Arg-thrombin; [6F]FPR-T, [6-(acetacarboxyl-terminal hirudin sequence [5, 6], thrombomodulin [7], the thrombin receptor [8, 9], and an acidic sequence on the serpin, heparin cofactor II [10, 11]. Mido)fluorescein]-D-Phe-Pro-Arg-thrombin; [4ЈF]FPR-T, [4Ј-{[(acetyl)amino]methyl}fluorescein]-D-Phe-Pro-Arg-thrombin; [ANS]FPR-T, [2-[(4Ј-acetamido)anilino]naphthalene-6-sulfonic acid]-D-Phe-Pro-Argthrombin; [ANS]FFR-T, [2-[(4Ј-acetamido)anilino]naphthalene-6sulfonic acid]-D-Phe-Phe-Arg-thrombin; F2, human prothrombin fragment 2; mAb, human anti-exosite II monoclonal antibody; Hir, Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln; Hir54 – 65(SO3Ϫ), Tyr63-sulfated hirudin54 – 65; [5F]Hir54 – 65(SO3Ϫ), Hir54 – 65(SO3Ϫ) labeled at the amino terminus with 5-carboxy(fluorescein); H*, [5F]Hir54–65(SO3Ϫ); IAANS, 2-[(4Ј-iodoacetamido)-anilino]naphthalene6-sulfonic acid; AT, antithrombin. To understand the mechanism of exosite regulation of thrombin further, the present work was undertaken to resolve whether the affinities of the exosite I-specific ligands, Hir (SO3Ϫ) and fibrinogen, and of the exosite II-specific ligands, prothrombin fragment 2 and a monoclonal antibody, were affected by alternate exosite occupation This was an important goal because studies employing hirudin peptides or F2 as probes of exosite involvement in other thrombin interactions could not be interpreted unambiguously, and it was uncertain whether the effects were due to competitive binding of alternate exosite I or II ligands or to extremely negative exosite linkage. Changes in binding affinity of factors V and Va, thrombomodulin, fibrin, and high molecular weight heparin affected by model exositespecific ligands are likely to be due to competitive overlapping binding sites or additional interactions between the ligands themselves, but not to extreme inter-exosite allosteric linkage
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