Characterization of proexosite I on prothrombin

Abstract

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.