Zhao L, Morser J, Bajzar L, Nesheim M, Nagashima M
Department of Cardiovascular Research, Berlex Biosciences, Richmond, California 94804, USA.
Thromb Haemost. 1998 Dec;80(6):949-55.
Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and is thought to circulate in plasma as a plasminogen-bound zymogen. When it is activated by the thrombin/thrombomodulin complex, activated TAFI exhibits carboxypeptidase B-like activity. To study the structure-function relationship of TAFI, we expressed recombinant human TAFI in insect cells. During the cloning of TAFI cDNA from several human liver cDNA libraries, we identified a second TAFI cDNA which differed from the published sequence at 2 positions. One of these sequences resulted in a substitution of alanine for threonine at residue 147, the other was a silent mutation. These substitutions were found in several cDNA libraries from different sources. Using Southern blot analysis, we confirmed the existence of this TAFI polymorphism in the population. In order to compare the activation and activity of TAFI isoforms, we expressed both isoforms in the baculovirus expression system, and compared the enzyme kinetics of the purified proteins. The molecular weight of recombinant TAFI is lower than plasma TAFI due to differences in glycosylation. The two recombinant TAFI isoforms had similar activation kinetics and the activated enzymes had similar carboxypeptidase B-like activity towards small molecule substrates. Their ability to retard clot lysis was found to be similar in a plate clot lysis assay.
凝血酶可激活纤溶抑制物（TAFI）由肝脏合成，被认为以纤溶酶原结合的酶原形式在血浆中循环。当它被凝血酶/血栓调节蛋白复合物激活时，活化的TAFI表现出羧肽酶B样活性。为了研究TAFI的结构-功能关系，我们在昆虫细胞中表达了重组人TAFI。在从几个人类肝脏cDNA文库克隆TAFI cDNA的过程中，我们鉴定出了另一种TAFI cDNA，它与已发表序列在两个位置上有所不同。其中一个序列导致第147位残基处的苏氨酸被丙氨酸取代，另一个是沉默突变。这些取代在来自不同来源的几个cDNA文库中都被发现。通过Southern印迹分析，我们证实了该TAFI多态性在人群中的存在。为了比较TAFI同工型的激活和活性，我们在杆状病毒表达系统中表达了这两种同工型，并比较了纯化蛋白的酶动力学。由于糖基化的差异，重组TAFI的分子量低于血浆TAFI。两种重组TAFI同工型具有相似的激活动力学，并且活化的酶对小分子底物具有相似的羧肽酶B样活性。在平板凝块溶解试验中发现它们延缓凝块溶解的能力相似。
