title: 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, 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.
authors.affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Anderson
authors.forename: A
authors.initials: A
authors.lastname: Nesset
authors.forename: K R
authors.initials: KR
authors.lastname: Dharmawardana
authors.forename: P E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002417:Cattle; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D006801:Humans; D011516:Prothrombin / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
chemical_list: D013431:Sulfates; D011516:Prothrombin; D013917:Thrombin