Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles.
Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles.
复制标题
人因子 Xa 重组变体的结构/功能分析：合成磷脂囊泡无法模拟因子 Xa 掺入凝血酶激活的血小板表面上的凝血酶原酶。
DOI：
10.1021/bi972428p
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
High,KA
中科院分区：
文献类型：
--
作者：
Larson,PJ;Camire,RM;Wong,D;Fasano,NC;Monroe,DM;Tracy,PB;High,KA
本报告描述了一系列重组因子Xa变体的表达、纯化和特性，这些变体对通常经历γ-羧基化的谷氨酸残基进行了天冬氨酸替代。因子X在人胚胎肾细胞中表达，并通过免疫亲和和羟基磷灰石层析从条件培养基中纯化。用Russell’s viper venom Factor X活化剂活化因子X，通过尺寸排斥层析法从活化因子X中去除单链未活化因子X。重组野生型因子Xa在凝血试验中具有正常的活性，而玻璃残基16、26和29被天冬氨酸取代的突变体没有可检测到的凝血活性。在纯化组分分析中，这些玻璃变体在合成磷脂囊泡上组装的凝血酶原复合物中基本上没有可检测到的活性，但当凝血酶原组装在凝血酶活化的血小板上时，它们具有显著的活性。此外，gla 32变体在血小板凝血酶原中具有正常活性，但在合成PSPC囊泡上组装的凝血酶原活性降低。这些差异并不是由凝血酶激活的血小板膜的总磷脂组成来解释的。我们已经生产出了完全活性的重组人因子Xa，并证明了玻璃残基16、26和29对因子Xa的正常活性至关重要。更重要的是，本研究提供了与维生素k依赖性凝血蛋白的玻璃变体形成的大分子酶复合物的广泛表征，并提供了凝血酶原复合物在凝血酶活化的血小板上的组装不等同于在合成磷脂囊泡上的组装的证据。数据表明，凝血酶活化的血小板含有一些元素（30%磷脂酰丝氨酸或Va因子除外），可能是蛋白质或磷脂，作为Xa因子结合位点的组成部分。
This report describes the expression, purification, and characterization of a series of recombinant factor Xa variants bearing aspartate substitutions for each of the glutamate residues which normally undergo γ-carboxylation. Factor X was expressed in human embryonic kidney cells and purified from conditioned media by immunoaffinity and hydroxylapatite chromatography. Factor X was activated with Russell's viper venom factor X activator, and single-chain unactivated factor X was removed from activated factor X by size-exclusion chromatography. Recombinant wild-type factor Xa had normal activity in a clotting assay, and mutants with aspartate substitutions for glas residues 16, 26, and 29 had no detectable clotting activity. In purified component assays, these gla variants had essentially no detectable activity in the prothrombinase complex assembled on synthetic phospholipid vesicles but had significant activity when the prothrombinase was assembled on thrombin-activated platelets. In addition, the gla 32 variant had normal activity in the platelet prothrombinase but diminished activity in prothrombinase assembled on synthetic PSPC vesicles. These differences were not accounted for by the total phospholipid composition of the thrombin-activated platelet membrane. We have produced fully active recombinant human factor Xa and demonstrated that gla residues 16, 26, and 29 are critical for normal activity of factor Xa. More importantly, this study provides an extensive characterization of macromolecular enzyme complex formation with gla variants of a vitamin K-dependent coagulation protein and provides evidence that prothrombinase complex assembly on thrombin-activated platelets is not equivalent to assembly on synthetic phospholipid vesicles. The data suggest that thrombin-activated platelets possess some element(s) (other than 30% phosphatidyl serine or factor Va), presumably either protein or phospholipid, that serves as a component of the factor Xa binding site.