Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties
Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties
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DOI：
10.1074/jbc.273.4.2127
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发表时间：
1998-01-23
影响因子：
4.8
通讯作者：
Nesheim, ME
中科院分区：
文献类型：
--
作者：
Boffa, MB;Wang, W;Nesheim, ME
凝血酶活化纤维蛋白溶解抑制剂（TAFI）是一种类似于胰腺前羧肽酶B的人血浆酶原，凝血酶/血栓调节蛋白对酶原的裂解产生酶，激活TAFI (TAFIa)，在体外延缓纤维蛋白凝块溶解，并可能在体内调节纤维蛋白溶解。在本研究中，我们在幼鼠肾细胞中稳定表达重组TAFI，从条件无血清培养基中纯化其至均匀性。并将其与血浆TAFI （pTAFI）在糖基化和凝血酶/凝血调节素激活动力学方面进行了比较。尽管rTAFI比pTAFI糖化有点不同,乳沟产品与凝血酶/ thrombomodulin无法辨认,和激活动力学非常相似的参数k (cat) = 0.55(1),为= 0.54μM和k d = 6.0 nM rTAFI和k (cat) = 0.61(1),为= 0.55μM和k d = 6.6 nM pTAFI,各自的塔非亚酒物种也准备并与热稳定性和酶的性质,包括抑制纤维蛋白溶解,酶的半衰期在37摄氏度是大约10分钟,和相关酶活性的衰变是淬火(类似于62%的初始值在60分钟)的固有荧光酶,稳定高度随温度而变的,根据过渡态理论,表明高焓和熵的变化与失活(δH-o双匕首全等45千卡每摩尔和δ)双匕首全等80大卡/ mol / K),两种TAFIa都被竞争性抑制剂2-胍基乙基巯基琥珀酸和epsilon-氨基己酸稳定，rTAFIa和pTAFIa在小底物的裂解动力学，对抑制剂的敏感性以及延缓tpa诱导和纤溶酶介导的纤溶的能力方面非常相似，这些研究为TAFIa的热不稳定性提供了新的见解，这一特性可能是其体内活性的重要调节因素；此外，他们表明rTAFI和rTAFIa是天然血浆来源物种的优秀替代品，这是未来通过位点特异性诱变研究结构和功能的必要前提。
Thrombin-activable fibrinolysis inhibitor (TAFI) is a human plasma zymogen similar to pancreatic pro-carboxypeptidase B, Cleavage of the zymogen by thrombin/thrombomodulin generates the enzyme, activated TAFI (TAFIa), which retards fibrin clot lysis in vitro and likely modulates fibrinolysis in vivo, In the present work we stably expressed recombinant TAFI in baby hamster kidney cells, purified it to homogeneity from conditioned serum-free medium, and compared it to plasma TAFI (pTAFI) with respect to glycosylation and kinetics of activation by thrombin/thrombomodulin. Although rTAFI is glycosylated somewhat differently than pTAFI, cleavage products with thrombin/thrombomodulin are indistinguishable, and parameters of activation kinetics are very similar with k(cat) = 0.55 s(-1), K-m = 0.54 mu M, and K-d = 6.0 nM for rTAFI and k(cat) = 0.61 s(-1), K-m = 0.55 mu M, and K-d = 6.6 nM for pTAFI, The respective TAFIa species also were prepared and compared with respect to thermal stability and enzymatic properties, including inhibition of fibrinolysis, The half-life of both enzymes at 37 degrees C is about 10 min, and the decay of enzymatic activity is associated with a quenching (to similar to 62% of the initial value at 60 min) of the intrinsic fluorescence of the enzyme, Stability was highly temperature-dependent, which, according to transition state theory, indicates both high enthalpy and entropy changes associated with inactivation (Delta H-o double dagger congruent to 45 kcal/mol and Delta S-o double dagger congruent to 80 cal/mol/K), Both species of TAFIa are stabilized by the competitive inhibitors 2-guanidinoethylmercaptosuccinic acid and epsilon-aminocaproic acid, rTAFIa and pTAFIa are very similar with respect to kinetics of cleavage of small substrates, susceptibility to inhibitors, and ability to retard both tPA-induced and plasmin-mediated fibrinolysis, These studies provide new insights into the thermal instability of TAFIa, a property which could be a significant regulator of its activity in vivo; in addition, they show that rTAFI and rTAFIa are excellent surrogates for the natural plasma-derived species, a necessary prerequisite for future studies of structure and function by site-specific mutagenesis.