Abstract
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.
Highlights
Ϳ100-fold increase in affinity on thrombin formation [6]
Kinetic studies described here of the effect of Tyr63-sulfated hirudin54–65 (Hir54–65(SO3Ϫ)) and the nonsulfated peptide (Hir54–65) as probes of proexosite I demonstrated that acceleration of prothrombin activation by factor Va was selectively inhibited, with no significant effect of the peptides on prothrombin activation catalyzed by factor Xa alone
Affinity Chromatography of Factor Xa and Factor Va on Hir54–65(SO3Ϫ)-Agarose—To assess the specificity of hirudin peptide binding for prothrombin, the possibility of interactions of the peptide with factor Xa or factor Va was first tested by affinity chromatography on Hir54–65(SO3Ϫ)-agarose
Summary
Protein and Peptide Purification and Characterization—Human prothrombin, thrombin, and hirudin peptides were obtained and characterized as described in the preceding paper [6]. The first term is in Michaelis-Menten form and represents a hyperbolic increase in the factor Va-catalyzed reaction rate with increasing prothrombin concentration, characterized by an apparent Km (Km,app) and an apparent catalytic rate constant for prothrombin activation by the factor Xa-factor Va complex (kVa) Both of these parameters are functions of factor Va concentration, as shown by Equations 4 and 5. The rate of thrombin formation as a function of prothrombin concentration in the absence and presence of fixed levels of Hir54–65(SO3Ϫ) and factor Va was analyzed by fitting of Equations 6 – 8 with Km,app, kVa, ku, and the dissociation constant for hirudin peptide binding to prothrombin (KD) as the fitted constants.
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