Urano S, Metzger A R, Castellino F J
Department of Chemistry, University of Notre Dame, IN 46556.
Proc Natl Acad Sci U S A. 1989 Apr;86(8):2568-71. doi: 10.1073/pnas.86.8.2568.
A rapid and quantitative fibrinolytic assay has been used to measure the overall activity of a recombinant tissue plasminogen activator (rTPA) preparation for dissolution of a fibrin clot by its ability to activate [Glu1]plasminogen (containing glutamic acid at position 1) to plasmin. A standard curve constructed for wild-type two-chain rTPA that contains, from the amino terminus, the finger (F)-growth factor (E)-kringle 1 (K1)-kringle 2 (K2)-serine protease (P) domains was used to assess the overall fibrin-dissolving abilities of variant recombinant molecules. Two-chain deletion mutants lacking the E domain, the F-E domains, the F-E-K1 domains, and the K1-K2 domains yielded activities ranging from 22% to 35% of the overall activity of wild-type two-chain rTPA, suggesting that both the K2 and F domains are individually responsible for a portion of the function of the molecule. Comparison of variant molecules containing F-K1-K2-P and F-K2-K2-P domains showed that the latter variant possessed a 4-fold higher activity (1.4-fold greater than that of wild-type two-chain rTPA), indicating that, for the activity measured, the presence of K2 leads to a greater effectiveness than that of K1. A plasmin cleavage-resistant mutant (Arg-275----Ser) has been used to assess possible differences in one- and two-chain rTPA in this overall activity, the former displaying 86% of the activity of the latter, suggesting that such differences are indeed small. Finally, the proper covalent attachment of the light and heavy chains of two-chain rTPA are very important to its overall fibrinolytic activity, since replacement of Cys-264 with glycine and concomitant disruption of one of the covalent attachment sites of the two chains provides a variant of rTPA with less than 2% of the activity of the wild-type two-chain molecule. The effector molecule, epsilon-amino hexanoic acid (epsilon Ahx; epsilon-aminocaproic acid), inhibits the overall fibrinolytic effect of rTPA in this system, with an effective Ki of approximately 1.5 mM. Its efficacy, as measured by the Ki, is independent of the presence of the epsilon Ahx binding regions of plasminogen and rTPA and is similar to the efficacy obtained when urokinase was the activator in place of wild-type two-chain rTPA or when activation of plasminogen was bypassed as a result of provision of preformed plasmin to the assay. The results suggest that in the overall clot lysis system, an important epsilon Ahx binding site may exist on fibrin that inhibits its dissolution by plasmin.
一种快速定量的纤维蛋白溶解测定法已被用于通过重组组织型纤溶酶原激活剂（rTPA）制剂激活[Glu1]纤溶酶原（第1位含谷氨酸）转化为纤溶酶的能力来测量其溶解纤维蛋白凝块的总体活性。构建了野生型双链rTPA的标准曲线，该rTPA从氨基末端起包含指状（F）-生长因子（E）-kringle 1（K1）-kringle 2（K2）-丝氨酸蛋白酶（P）结构域，用于评估变体重组分子的总体纤维蛋白溶解能力。缺乏E结构域、F-E结构域、F-E-K1结构域和K1-K2结构域的双链缺失突变体产生的活性为野生型双链rTPA总体活性的22%至35%，这表明K2和F结构域各自对分子的部分功能负责。比较含有F-K1-K2-P和F-K2-K2-P结构域的变体分子表明，后一种变体的活性高4倍（比野生型双链rTPA高1.4倍），这表明对于所测量的活性，K2的存在比K1更有效。一种抗纤溶酶裂解突变体（Arg-275----Ser）已被用于评估单链和双链rTPA在这种总体活性方面可能存在的差异，前者显示出后者活性的86%，这表明这种差异确实很小。最后，双链rTPA轻链和重链的正确共价连接对其总体纤维蛋白溶解活性非常重要，因为用甘氨酸取代Cys-264并同时破坏两条链的一个共价连接位点会产生一种rTPA变体，其活性不到野生型双链分子的2%。效应分子ε-氨基己酸（εAhx；ε-氨基己酸）在该系统中抑制rTPA的总体纤维蛋白溶解作用，有效Ki约为1.5 mM。通过Ki测量，其效力与纤溶酶原和rTPA的εAhx结合区域是否存在无关，并且与用尿激酶替代野生型双链rTPA作为激活剂或由于在测定中提供预先形成的纤溶酶而绕过纤溶酶原激活时获得的效力相似。结果表明，在总体凝块溶解系统中，纤维蛋白上可能存在一个重要的εAhx结合位点，该位点抑制纤溶酶对其的溶解。
