Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.
Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor.
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由于蛋白质二聚化，活性位点抑制因子 VIIa 的功能活性大幅增强：深入了解组织因子组装/分解的机制。
DOI：
10.1021/bi050007z
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Nelsestuen,GaryL
中科院分区：
文献类型：
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作者：
Stone,MatthewD;Harvey,StephenB;Martinez,MichaelB;Bach,RonaldR;Nelsestuen,GaryL
活性位点抑制的凝血因子VIIa（fVIIai）与组织因子（TF）结合，组织因子是一种在损伤时暴露并启动凝血级联的细胞表面受体。FVIIai阻断因子VII的相应酶（fVIIa）或酶原（fVII）形式的结合并抑制凝血。尽管几项研究表明，fVIIai可能具有上级体内抗凝作用，但使用fVIIai的挑战是生产成本。本研究报告了通过其活性位点交联的fVIIai的二聚体形式的性质。二聚野生型fVIIai在阻断fVIIa与TF结合方面比单体fVIIai有效至少75倍。具有更高膜亲和性的突变体fVIIai的二聚体是1600倍更有效。任何形式的fVIIai的抗凝与肝素等药物有很大不同，并显示出延迟的作用模式。凝血在最初几分钟内正常进行，随着平衡结合的建立，抑制作用增加。这表明，在碰撞依赖性反应中，fVIIa（i）与TF的结合使抑制剂和酶对TF具有同等的接近性。组装不受二聚体的较高亲和力和较慢解离的影响。因此，抗凝治疗被延迟，直到反应达到平衡。不同解离实验的性质表明，从TF的fVIIai的解离发生由两个步骤的机制。第一步是分离TF−fVIIa（i），同时两种蛋白质仍与膜结合，第二步是从膜上解离fVIIa（i）。这些结果表明fVIIai的新作用，使其与阻断凝血级联的后续步骤的大多数抗凝剂区分开来。
Active site-inhibited blood clotting factor VIIa (fVIIai) binds to tissue factor (TF), a cell surface receptor that is exposed upon injury and initiates the blood clotting cascade. FVIIai blocks binding of the corresponding enzyme (fVIIa) or zymogen (fVII) forms of factor VII and inhibits coagulation. Although several studies have suggested that fVIIai may have superior anticoagulation effects in vivo, a challenge for use of fVIIai is cost of production. This study reports the properties of dimeric forms of fVIIai that are cross-linked through their active sites. Dimeric wild-type fVIIai was at least 75-fold more effective than monomeric fVIIai in blocking fVIIa association with TF. The dimer of a mutant fVIIai with higher membrane affinity was 1600-fold more effective. Anticoagulation by any form of fVIIai differed substantially from agents such as heparin and showed a delayed mode of action. Coagulation proceeded normally for the first minutes, and inhibition increased as equilibrium binding was established. It is suggested that association of fVIIa(i) with TF in a collision-dependent reaction gives equal access of inhibitor and enzyme to TF. Assembly was not influenced by the higher affinity and slower dissociation of the dimer. As a result, anticoagulation was delayed until the reaction reached equilibrium. Properties of different dissociation experiments suggested that dissociation of fVIIai from TF occurred by a two-step mechanism. The first step was separation of TF−fVIIa(i) while both proteins remained bound to the membrane, and the second step was dissociation of the fVIIa(i) from the membrane. These results suggest novel actions of fVIIai that distinguish it from most of the anticoagulants that block later steps of the coagulation cascade.