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.
Highlights
The results indicated that NBD-labeled hirudin54–65 reported differences in the environments of proexosite I on the Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) zymogen forms and the activated enzymes
Binding of [NBD]Hir54–65(SO3Ϫ) to Pre 1 resulted in a 12 Ϯ 1% quench of the fluorescence instead of the enhancement seen with Pro, indicating a significant effect of the removal of fragment 1 (F1) on the change in probe environment. [NBD]Hir54–65(SO3Ϫ) bound to Pre 1 with a 7-fold increased affinity in comparison to Pro, confirming the 6-fold increase in affinity observed for [5F]Hir54–65(SO3Ϫ) [28]
The pathway of exosite I expression on human Pro activation intermediates and products and the role of F1 in exosite expression were characterized in quantitative equilibrium binding studies for the first time
Summary
Pre 1, prethrombin 1; Pre 2, prethrombin 2; Pro, prothrombin; F1, fragment 1; F2, fragment 2; F1.2, fragment 1.2; Gla, ␥-carboxyglutamic acid; MzT(-F1), meizothrombin des-fragment 1; MzT, meizothrombin; T, thrombin; FPR-CH2Cl, D-Phe-ProArg-CH2Cl; Hir54–65(SO3Ϫ), Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-GluTyr(SO3Ϫ)-Leu-Gln; [5F]Hir54–65(SO3Ϫ), Hir54–65(SO3Ϫ) labeled at the amino terminus with 5-carboxy(fluorescein); [NBD]Hir54–65(SO3Ϫ), Hir54–65(SO3Ϫ) labeled at the amino terminus with succinimidyl 6-(N(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate; Mes, 4-morpholineethanesulfonic acid. The results characterize a new role of F1 in the expression of exosites I and II, which is disengaged on folding of the proteinase “activation domain” [29] into the catalytically active form, simultaneous with activation of exosite I These observations suggest that changes in (pro)exosite I interactions mediated by the F1 domain and differential expression of exosite I on the Pro activation intermediates may regulate factor Va-Pro interactions that control the activation pathway
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
