Rational design of a potent anticoagulant thrombin
Rational design of a potent anticoagulant thrombin
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DOI：
10.1074/jbc.c000751200
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发表时间：
2000-12-22
影响因子：
4.8
通讯作者：
Di Cera, E
中科院分区：
文献类型：
--
作者：
Cantwell, AM;Di Cera, E
凝血酶在切割纤维蛋白原并促进纤维蛋白凝块形成时充当促凝血剂，并且在辅因子血栓调节蛋白的协助下激活蛋白C时充当抗凝剂。凝血酶在血液中的双重功能提出了挑战，通过选择性地消除纤维蛋白原切割将酶转化为有效的抗凝剂。使用功能和结构数据，我们合理地设计了凝血酶突变体W215 A/E217 A，其以比野生型慢约20，000倍的k（cat）/K-m值切割纤维蛋白原，但在血栓调节蛋白存在下以与野生型相当的特异性激活蛋白C。该突变体首次证明凝血酶对纤维蛋白原和蛋白C的相对特异性可以完全逆转。
Thrombin acts as a procoagulant when it cleaves fibrinogen and promotes the formation of a fibrin clot and functions as an anticoagulant when it activates protein C with the assistance of the cofactor thrombomodulin. The dual function of thrombin in the blood poses the challenge to turn the enzyme into a potent anticoagulant by selectively abrogating fibrinogen cleavage. Using functional and structural data, we have rationally designed a thrombin mutant, W215A/E217A, that cleaves fibrinogen with a value of k(cat)/K-m about 20,000-fold slower than wild-type but activates protein C in the presence of thrombomodulin with a specificity comparable with wild-type. This mutant demonstrates for the first time that the relative specificity of thrombin toward fibrinogen and protein C can be completely reversed.