Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin
Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin
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DOI：
10.1074/jbc.m306916200
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发表时间：
2003-11-07
影响因子：
4.8
通讯作者：
Bock, PE
中科院分区：
文献类型：
--
作者：
Anderson, PJ;Bock, PE
凝血因子Xa激活凝血酶原（Pro）产生凝血酶催化位点和外切位点I和II。在平衡结合研究中，使用用6-（N-（7-硝基苯-2-氧杂-1，3-二唑-4-基）氨基）己酸酯（[NBD]Hir（54 - 65）（SO 3-））或5-（羧基）荧光素（[5 F]Hir（54 -65）（SO 3-））标记的水蛭素（54 - 65），表征了片段1（F1）在Pro活化期间外位点I表达途径中的作用。[NBD]Hir（54-65）（SO 3-）区分了Pro、凝血酶前体1（Pre 1）和凝血酶前体2（Pre 2）上的外位点I环境，但与[5 F]Hir（54-65）（SO 3-）具有相同的亲和力结合。Pro向Pre 1的转化导致对肽的亲和力增加7倍。相反，片段1.2（F1.2）使Pre 2对[5 F]Hir（54-65）（SO 3-）的亲和力降低3倍。与凝血酶相比，这与F1.2对Pre 2的亲和力增加16倍相关，表明F1对F1.2结合的增强作用。活性中间体，meizothrobin，表现出50- 220倍的增加外位点的亲和力。游离凝血酶和凝血酶.F1.2复合物以不可区分的亲和力结合[5 F]Hir（54-65）（SO 3-），表明F1对肽结合的影响在催化活性和外位点I表达后消除。结果表明F1在调节配体对外位点I的亲和力中具有新的酶原特异性作用。这可能反映了F1和Pre 2结构域之间的直接相互作用，在酶原激活结构域折叠后丢失。F1对（前）外位点I的影响和（前）外位点I在因子Va依赖性底物识别中的作用表明，Pro活化途径可能受（前）外位点I与因子Va相互作用的调节。
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.