Serine 380 (P14) → glutamate mutation activates antithrombin as an inhibitor of factor Xa
Serine 380 (P14) → glutamate mutation activates antithrombin as an inhibitor of factor Xa
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DOI：
10.1074/jbc.275.6.4092
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发表时间：
2000-02-11
影响因子：
4.8
通讯作者：
Gettins, PGW
中科院分区：
文献类型：
--
作者：
Futamura, A;Gettins, PGW
肝素调节抗凝血酶的抑制活性。已经提出，残基P15和P14通过肝素结合从抗凝血酶的β-折叠A中排出，允许反应中心环与因子Xa更好地相互作用。我们已经制备了P14抗凝血酶变体（S380 E）以产生抗凝血酶的活化抑制形式，其中P14已经从β折叠A排出。与对照抗凝血酶相比，S380 E抗凝血酶荧光增强35 +/-5%。肝素结合后，抗凝血酶荧光进一步增加最小。该变体具有比对照抗凝血酶低5 ℃的T-m。该变体是蛋白酶的抑制剂，并且在校正增加的抑制化学计量后，具有增加近200倍的Xa因子抑制的基础速率。这与高亲和力肝素五糖激活的抗凝血酶相当。全长高亲和力肝素仅导致速率增加7倍，抑制化学计量大幅增加。相比之下，凝血酶的基础抑制率与对照抗凝血酶相似，但肝素使其增加300倍。这些发现表明，天然状态的S380 E变体存在于环驱逐构象，因此对因子Xa具有高度反应性。
Heparin regulates the inhibitory activity of antithrombin. It has been proposed that residues P15 and P14 are expelled from beta-sheet A of antithrombin by heparin binding, permitting better interaction of the reactive center loop with factor Xa. We have made a P14 antithrombin variant (S380E) to create an activated inhibitory form of antithrombin in which P14 is already expelled from beta-sheet A. S380E antithrombin fluorescence is enhanced 35 +/- 5% compared with control antithrombin. There is minimal further increase in antithrombin fluorescence upon heparin binding. The variant has a 5 degrees C lower T-m than control antithrombin. The variant is an inhibitor of proteinases and has a nearly 200-fold increased basal rate of inhibition of factor Xa, after correction for an increased stoichiometry of inhibition. This is comparable to that of antithrombin activated by high affinity heparin pentasaccharide. Full-length high affinity heparin causes only a 7-fold additional increase in rate and a large increase in stoichiometry of inhibition. In contrast, the basal rate of inhibition of thrombin is similar to that of control antithrombin but is increased 300-fold by heparin. These findings suggest that the native state of the S380E variant exists in a loop-expelled conformation that is consequently highly reactive toward factor Xa.