Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis PROPERTIES OF KD1-L17R VARIANT
Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis PROPERTIES OF KD1-L17R VARIANT
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DOI：
10.1074/jbc.m110.191163
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发表时间：
2011-02-01
影响因子：
4.8
通讯作者：
Bajaj, S. Paul
中科院分区：
文献类型：
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作者：
Bajaj, Madhu S.;Ogueli, Godwin I.;Bajaj, S. Paul
组织因子途径抑制剂-2（TFPI-2）抑制因子XIa、血浆激肽释放酶和因子VIIa/组织因子;因此，已提出将其用作抗凝剂。全长TFPI-2或其分离的第一个Kunitz结构域（KD 1）也抑制纤溶酶;因此，已提出将其用作抗纤维蛋白溶解剂。然而，TFPI-2或KD 1的抗凝特性会降低其抗纤溶功能。在这项研究中，基于结构的调查和分析的丝氨酸蛋白酶的档案显示，凝血酶更喜欢在其底物/抑制剂的P2'位置的疏水残基，而纤溶酶更喜欢在其底物/抑制剂的相应位置的带正电荷的精氨酸残基。基于这一观察，我们将KD 1中的P2'残基Leu-17改变为Arg（KD 1-L17 R），并将其抑制特性与野生型KD 1（KD 1-WT）进行比较。WT和KD 1-L17 R均在大肠杆菌中表达，折叠并纯化至均一。N-末端序列和质谱证实了KD 1-WT和KD 1-L17 R的正确表达。与KD 1-WT相比，KD 1-L17 R不抑制因子XIa、血浆激肽释放酶或因子VIIa/组织因子。此外，KD 1-L17 R抑制纤溶酶的亲和力增加了约6倍，并有效地阻止了组织纤溶酶原激活剂诱导的血浆凝块纤溶。类似地，在小鼠肝裂伤出血模型中，KD 1-L17 R在预防失血方面的有效性类似于KD 1-WT的8倍。重要的是，在该出血模型中，KD 1-L17 R与抑肽酶或氨甲环酸同等或更有效，抑肽酶或氨甲环酸已被用作抗纤维蛋白溶解剂以防止大手术/创伤期间的失血。此外，与抑肽酶相比，使用KD 1-L17 R未观察到肾毒性。
Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2' position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2' residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with similar to 6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was similar to 8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.