Characterization of proexosite I on prothrombin.

Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin. Quantitative affinity chromatography and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand, hirudin(54-65) ([5F]Hir(54-65) (SO(3)(-)), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir(54-65)(SO(3)(-)) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human prothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 micrometer and 25 +/- 2 nm, respectively, demonstrating a 130-fold increase in affinity for the active proteinase. The bovine proteins similarly showed a 150-fold higher affinity of [5F]Hir(54-65)(SO(3)(-)) for thrombin compared with prothrombin, despite a 2-5-fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr(63)-sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir(54-65)(SO(3)(-)) to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a approximately 100-fold increase in affinity when thrombin is formed.

Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation.

Regulatory exosite I of thrombin is present on prothrombin in a precursor state (proexosite I) that specifically binds the Tyr(63)-sulfated peptide, hirudin(54-65) (Hir(54-65)(SO(3)(-))) and the nonsulfated analog. The role of proexosite I in the mechanism of factor Va acceleration of prothrombin activation was investigated in kinetic studies of the effects of peptide binding. The initial rate of human prothrombin activation by factor Xa was inhibited by the peptides in the presence of factor Va but not in the absence of the cofactor. Factor Xa and factor Va did not bind the peptide with significant affinity compared with prothrombin. Maximum inhibition reduced the factor Va-accelerated rate to a level indistinguishable from the rate in the absence of the cofactor. The effect of Hir(54-65)(SO(3)(-)) on the kinetics of prothrombin activation obeyed a model in which binding of the peptide to proexosite I prevented productive prothrombin interactions with the factor Xa-factor Va complex. Comparison of human and bovine prothrombin as substrates demonstrated a similar correlation between peptide binding and inhibition of factor Va acceleration. Inhibition of prothrombin activation by hirudin peptides was opposed by assembly on phospholipid vesicles of the membrane-bound factor Xa-factor-Va-prothrombin complex. Factor Va interactions of human and bovine prothrombin activation are concluded to share a common mechanism in which proexosite I participates in productive interactions of prothrombin as the substrate of the factor Xa-factor Va complex, possibly by directly mediating productive prothrombin-factor Va binding.

Role of regulatory exosite I in binding of thrombin to human factor V, factor Va, factor Va subunits, and activation fragments.

The blood coagulation proteinase, thrombin, converts factor V into factor Va through a multistep activation pathway that is regulated by interactions with thrombin exosites. Thrombin exosite interactions with human factor V and its activation products were quantitatively characterized in equilibrium binding studies based on fluorescence changes of thrombin covalently labeled with 2-anilinonaphthalene-6-sulfonic acid (ANS) linked to the catalytic site histidine residue by Nalpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl ([ANS]FPR-thrombin). Exosite I was shown to play a predominant role in the binding of factor V and factor Va from the effect of the exosite I-specific ligand, hirudin54-65, on the interactions. Factor V and factor Va bound to exosite I of [ANS]FPR-thrombin with similar dissociation constants of 3.4 +/- 1.3 and 1.1 +/- 0.4 microM and fluorescence enhancements of 182 +/- 41 and 127 +/- 17%, respectively. Native thrombin and labeled thrombin bound with similar affinity to factor Va. Among factor V activation products, the factor Va heavy chain was shown to contain the site of exosite I binding, whereas exosite I-independent, lower affinity interactions were observed for activation fragments E and C1, and no detectable binding was observed for the factor Va light chain. The results support the conclusion that the factor V activation pathway is initiated by exosite I-mediated binding of thrombin to a site in the heavy chain region of factor V that facilitates the initial cleavage at Arg709 to generate the heavy chain of factor Va. The results further suggest that binding of thrombin through exosite I to factor V activation intermediates may regulate their conversion to factor Va and that similar binding of thrombin to the factor Va produced may reflect a mode of interaction involved in the regulation of prothrombin activation.

Demonstration of exosite I-dependent interactions of thrombin with human factor V and factor Va involving the factor Va heavy chain: analysis by affinity chromatography employing a novel method for active-site-selective immobilization of serine proteinases.

In an essential step of blood coagulation, factor V is proteolytically processed by thrombin to generate the activated protein cofactor, factor Va, and to release the activation fragments E and C1. For the identification and characterization of sites of thrombin binding to factor V and its activation products, a new method was developed for immobilizing thrombin and other serine proteinases specifically (>/=92%) through their active sites and used in affinity chromatography studies of the interactions. Interactions of factor V with exosite I of thrombin were shown to regulate the factor V activation pathway from the 93% +/- 12% inhibition of the rate of activation correlated with specific binding of hirudin54-65 to this exosite. Chromatography of factor V on active-site-immobilized thrombin showed only a weak interaction, while the factor Va heterodimer bound specifically and with apparently higher affinity, in an interaction that was prevented by hirudin54-65. The heavy chain of subunit-dissociated factor Va bound to immobilized thrombin, while the light-chain subunit and fragment E had no detectable affinity. These results demonstrate a previously undescribed, exosite I-dependent interaction of thrombin with factor Va that occurs through the factor Va heavy chain. They support the further conclusion that similar exosite I-dependent binding of thrombin to the heavy-chain region of factor V contributes to recognition of factor V as a specific thrombin substrate and thereby regulates proteolytic activation of the protein cofactor.