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, hirudin54–65 ([5F]Hir54–65(SO3−), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir54–65(SO3−) 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 μm 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]Hir54–65(SO3−) for thrombin compared with prothrombin, despite a 2–5-fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr63-sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir54–65(SO3−) to human and bovine prothrombin and thrombin, exhibiting similar, 40–70-fold higher affinities for the proteinases, although nonsulfated Hir54–65 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 ∼100-fold increase in affinity when thrombin is formed.