Thr90Ser Mutation in Antithrombin is Associated with Recurrent Thrombosis in a Heterozygous Carrier
Thr90Ser Mutation in Antithrombin is Associated with Recurrent Thrombosis in a Heterozygous Carrier
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抗凝血酶中的 Thr90Ser 突变与杂合子携带者的复发性血栓形成相关
DOI：
10.1055/s-0040-1710590
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发表时间：
2020-07-01
影响因子：
6.7
通讯作者：
Rezaie，Alireza R.
中科院分区：
文献类型：
--
作者：
Lu，Yeling;Villoutreix，Bruno O.;Rezaie，Alireza R.
抗凝血酶（AT）是一种丝氨酸蛋白酶抑制剂，可调节内源性和外源性凝血蛋白酶的活性。我们确定了一个AT缺陷患者与杂合子Thr 90 Ser（T90 S）突变谁的经验，复发性静脉血栓形成。为了了解凝血缺陷的分子基础，我们在哺乳动物细胞中表达了AT-T90 S，将其纯化至均一，并在已建立的动力学、结合和凝血测定中表征了其特性。还通过分子建模评估了突变对AT结构的可能影响。结果表明，AT-T90 S对凝血酶和因子Xa的抑制活性在肝素存在和不存在的情况下均被削弱了三至五倍。肝素对AT-T90 S的亲和力降低了4 - 5倍。动力学分析显示，在肝素存在和不存在的情况下，凝血酶和因子Xa的AT-T90 S抑制的化学计量均升高了三至四倍，表明凝血蛋白酶与AT-T90 S的反应性在底物途径中升高。在补充AT-T90 S的AT缺陷血浆中分析，AT-T90 S的抗凝活性已显著受损。AT-T90 S的抗炎作用也降低。结构分析预测AT-T90 S中Ser的较短侧链对AT和/或AT-蛋白酶复合物的结构具有不稳定作用，可能增加内部空腔的大小并改变氢键网络，该氢键网络调节变构连接的肝素结合位点和丝氨酸蛋白酶抑制剂的反应中心环的构象。这种突变效应增加了AT-T90 S与底物途径中的凝血蛋白酶的反应性。
Abstract Antithrombin (AT) is a serine protease inhibitor that regulates the activity of coagulation proteases of both intrinsic and extrinsic pathways. We identified an AT-deficient patient with a heterozygous Thr90Ser (T90S) mutation who experiences recurrent venous thrombosis. To understand the molecular basis of the clotting defect, we expressed AT-T90S in mammalian cells, purified it to homogeneity, and characterized its properties in established kinetics, binding, and coagulation assays. The possible effect of mutation on the AT structure was also evaluated by molecular modeling. Results demonstrate the inhibitory activity of AT-T90S toward thrombin and factor Xa has been impaired three- to fivefold in both the absence and presence of heparin. The affinity of heparin for AT-T90S has been decreased by four- to fivefold. Kinetic analysis revealed the stoichiometry of AT-T90S inhibition of both thrombin and factor Xa has been elevated by three- to fourfold in both the absence and presence of heparin, suggesting that the reactivity of coagulation proteases with AT-T90S has been elevated in the substrate pathway. The anticoagulant activity of AT-T90S has been significantly impaired as analyzed in the AT-deficient plasma supplemented with AT-T90S. The anti-inflammatory effect of AT-T90S was also decreased. Structural analysis predicts the shorter side-chain of Ser in AT-T90S has a destabilizing effect on the structure of AT and/or the AT-protease complex, possibly increasing the size of an internal cavity and altering a hydrogen-bonding network that modulates conformations of the allosterically linked heparin-binding site and reactive center loop of the serpin. This mutational effect increases the reactivity of AT-T90S with coagulation proteases in the substrate pathway.