Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity
Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity
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DOI：
10.4049/jimmunol.168.10.5170
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发表时间：
2002-05-15
影响因子：
4.4
通讯作者：
Levy, GA
中科院分区：
文献类型：
--
作者：
Chan, CWY;Chan, MWC;Levy, GA
FG 1 2凝血酶原酶通过其产生凝血酶的能力，已被证明是病毒诱导的肝炎、精氨酸诱导的胎儿丢失综合征以及异种和同种异体移植排斥的发病机制的关键。在这匹种马中，详细检查了FGL 2凝血酶原酶活性的分子基础。在杆状病毒表达系统中产生的纯化的fgl 2蛋白没有可测量的凝血酶原酶活性，而当将纯化的蛋白重构到含磷脂-L-丝氨酸的囊泡中时，活性恢复。重构的fgl 2催化人凝血酶原裂解为凝血酶，动力学符合一级反应，表观V-max值为6 mol/min/mol fgl 2，凝血酶原的表观K-m值为8.3 μ M。催化活性vas完全依赖于钙，并且因子Va（500 nM）通过将表观V-max值增加到3670 mol/min/mol fgl 2和将凝血酶原的表观K-m值降低到7.2 μ M来增强fgl 2的催化效率。通过定点诱变和截短蛋白质的产生的组合，清楚地表明残基Ser（89）对于fgl 2的凝血酶原酶活性是关键的。此外，fgl 2凝血酶原酶活性不被抑制抗凝血酶III，大豆胰蛋白酶抑制剂，4-氨基苯甲脒，抑肽酶，或苯甲基磺酰氟，而二异丙基氟磷酸完全废除的活动。在这项工作中，我们提供了直接的证据表明，fgl 2切割凝血酶原凝血酶与丝氨酸蛋白酶活性一致，并需要钙，磷脂，和因子Va，其全部活动。
fgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this stud., the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidl-L-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent V-max value of 6 mol/min/mol fgl2 and an apparent K-m value for prothrombin of 8.3 muM. The catalytic activity vas totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent V-max value to 3670 mol/min/mol fgl2 and decreasing the apparent K-m value for prothrombin to 7.2 muM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser(89) was critical for the prothrombinase activity of fgl2. Furthermore,fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.