STUDIES ON THE EFFECT OF SERINE PROTEASE INHIBITORS ON ACTIVATED CONTACT FACTORS - APPLICATION IN AMIDOLYTIC ASSAYS FOR FACTOR-XIIA, PLASMA KALLIKREIN AND FACTOR-XIA
STUDIES ON THE EFFECT OF SERINE PROTEASE INHIBITORS ON ACTIVATED CONTACT FACTORS - APPLICATION IN AMIDOLYTIC ASSAYS FOR FACTOR-XIIA, PLASMA KALLIKREIN AND FACTOR-XIA
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DOI：
10.1111/j.1432-1033.1987.tb11174.x
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发表时间：
1987-05-04
期刊：
影响因子：
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通讯作者：
GRIFFIN, JH
中科院分区：
文献类型：
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作者：
TANS, G;JANSSENCLAESSEN, T;GRIFFIN, JH
在含有不同量的每种酶的混合物中，已经开发出了测定因子XIIa、因子XIa和血浆钾激肽的酶解测定法。对市售的显色对硝基苯胺底物pro - ph - arg - nh - np （S2302或染色体PK）、Glp-Pro-Arg-NH-Np （S2366）、Ile-Glu-（胡椒酰基）- gly - arg - nh - np （S2337）和Ile-Glu- gly - arg - nh - np （S2222）进行了测试，以确定它们作为底物的适用性。研究了S2303、S222、S2337和S2366转化的动力学参数。因子XIIa，因子XIa和血浆钾激肽肽表明每种活性酶对许多这些底物表现出相当大的活性。当存在大量其他活化接触因子时，这就排除了对单个酶的直接定量。已经测试了几种丝氨酸蛋白酶抑制剂有选择性地抑制那些可能干扰被测试因子的接触因子的能力。大豆胰蛋白酶抑制剂在中等浓度下能很好地抑制钾激肽，抑制因子XIIa，但不影响因子XIIa的酶解活性。因此，该抑制剂可用于在因子XIIa测定中消除钾激肽和因子XIa的贡献。本文报道了三种不同的氯甲基酮对接触活化因子的抑制速率常数。d - ph - pro - arg - ch2cl对接触因子有中等活性（k = 2.2倍）。103 M-1 s-1, pH 8.3)，但特异性无差异。d - ph - ph - arg - ch2cl是一种非常有效的血浆钾激肽抑制剂（k = 1.2倍）。105 M-1 s-1, pH 8.3)，而缓慢抑制因子XIIa （k = 1.4倍）。103 M-1 s-1)和因子XIa (k = 0.11。103 M-1 s-1)。Dns-Glu-Gly-Arg-CH2Cl对钾激肽的反应性更强（k = 1.6倍）。104 M-1 s-1)比因子XIIa (k = 4.6。102 M-1 s-1)和因子XIa (k = 0.6。102 M-1 s-1)。由于ph - ph - arg - ch2cl对血浆钾激肽具有高度特异性，因此可用于因子XIa测定中选择性地抑制钾激肽。基于显色底物转化的催化效率和丝氨酸蛋白酶抑制剂和氯甲基酮的抑制特性，我们能够在接触活化因子的混合物中建立因子XIIa，因子XIa和钾激肽的定量分析。
Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p-nitroanilide substrates Pro-Phe-Arg-NH-Np (S2302 or chromozym PK), Glp-Pro-Arg-NH-Np (S2366), Ile-Glu-(piperidyl)-Gly-Arg-NH-Np (S2337), and Ile-Glu-Gly-Arg-NH-Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2303, S222, S2337 and S2366 by .beta. factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D-Phe-Pro-Arg-CH2Cl was moderately active against contact factors (k = 2.2 .times. 103 M-1 s-1 at pH 8.3) but showed no differences in specificity. D-Phe-Phe-Arg-CH2Cl was a very efficient inhibitor of plasma kallikrein (k = 1.2 .times. 105 M-1 s-1 at pH 8.3) whereas it slowly inhibited factor XIIa (k = 1.4 .times. 103 M-1 s-1) and factor XIa (k = 0.11 .times. 103 M-1 s-1). Also Dns-Glu-Gly-Arg-CH2Cl was more reactive towards kallikrein (k = 1.6 .times. 104 M-1 s-1) than towards factor XIIa (k = 4.6 .times. 102 M-1 s-1) and factor XIa (k = 0.6 .times. 102 M-1 s-1). Since Phe-Phe-Arg-CH2Cl is highly specific for plasma kallikrein it can be used in a factor XIa assay selectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.