title: Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin.
abstract: Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin(54-65) labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir(54-65)(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir(54-65)(SO3-)). [NBD]Hir(54-65)(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir(54-65)(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir(54-65)(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin.F1.2 complex bound [5F]Hir(54-65)(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.
authors.affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Anderson
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000614:Aminocaproates; D001665:Binding Sites; D004792:Enzyme Precursors / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism / Q000502:physiology*; D011498:Protein Precursors / Q000502:physiology*; D011516:Prothrombin / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism*
chemical_list: D000614:Aminocaproates; D004792:Enzyme Precursors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006629:Hirudins; D010446:Peptide Fragments; D011498:Protein Precursors; C091596:hirudin (54-65); C045879:prothrombin fragment 1.2; D015943:Factor Va; C029733:prothrombin fragment 1; C045132:4-carboxyfluorescein; C072800:7-nitrobenzoxadiazole-6-aminohexanoic acid; D011516:Prothrombin; C012007:prethrombins; D013917:Thrombin; C026812:meizothrombin; D009327:4-Chloro-7-nitrobenzofurazan