Pixley R A, Schapira M, Colman R W
J Biol Chem. 1985 Feb 10;260(3):1723-9.
Studies of the inactivation of factor XIIa by plasma protease inhibitors in purified systems and in plasma were initiated to determine the relative importance of these inhibitors to the neutralization of factor XIIa. Factor XIIa was measured by the amidolysis of H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride or by coagulant activity. C1 inhibitor (C1INH), alpha 2-antiplasmin (alpha 2AP), alpha 2-macroglobulin (alpha 2M), and antithrombin III (ATIII) inhibited factor XIIa with second-order rate constants of 2.2 X 10(5), 1.1 X 10(4), 5.0 X 10(3), and 1.3 X 10(3) M-1 min-1. Factor XIIa activity was not affected by alpha 1-proteinase inhibitor. Incubation of 125I-radiolabeled factor XIIa resulted in 1:1 stoichiometric complexes with C1INH (Mr 190,000), ATIII (Mr 125,000), and alpha 2AP (Mr 150,000 and 125,000) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of 125I-Factor XIIa with alpha 2M resulted in a component of Mr 85,000 on a reduced sodium dodecyl sulfate-polyacrylamide gel, indicating that a subunit of factor XIIa was covalently bound to a proteolyzed portion of alpha 2M. The relative effectiveness of each inhibitor at plasma concentrations was 61:2:3:1 for C1INH, alpha 2AP, alpha 2M, and ATIII, respectively. Kinetic studies of the inactivation of purified factor XIIa added to various plasmas containing different concentrations of C1INH verified the predictions from the purified systems. Gel filtration of radiolabeled factor XIIa incubated with plasma confirmed that factor XIIa-C1INH was the major complex. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complexes in plasma had the same molecular size as those with purified inhibitors. C1INH functions as the predominant inhibitor of factor XIIa in plasma.
开展了关于血浆蛋白酶抑制剂在纯化系统和血浆中使因子XIIa失活的研究，以确定这些抑制剂对中和因子XIIa的相对重要性。通过H-D-脯氨酰-L-苯丙氨酰-L-精氨酸对硝基苯胺二盐酸盐的酰胺水解或凝血活性来测定因子XIIa。C1抑制剂（C1INH）、α2-抗纤溶酶（α2AP）、α2-巨球蛋白（α2M）和抗凝血酶III（ATIII）抑制因子XIIa的二级速率常数分别为2.2×10⁵、1.1×10⁴、5.0×10³和1.3×10³ M⁻¹ min⁻¹。α1-蛋白酶抑制剂不影响因子XIIa的活性。使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，125I放射性标记的因子XIIa孵育后与C1INH（分子量190,000）、ATIII（分子量125,000）和α2AP（分子量150,000和125,000）形成1:1化学计量比的复合物。125I-因子XIIa与α2M孵育后，在还原型十二烷基硫酸钠-聚丙烯酰胺凝胶上出现分子量为85,000的条带，表明因子XIIa的一个亚基与α2M的一个蛋白水解部分共价结合。在血浆浓度下，每种抑制剂的相对效力分别为C1INH:α2AP:α2M:ATIII = 61:2:3:1。对添加到含有不同浓度C1INH的各种血浆中的纯化因子XIIa失活的动力学研究证实了来自纯化系统的预测。对与血浆孵育的放射性标记因子XIIa进行凝胶过滤证实因子XIIa-C1INH是主要复合物。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明血浆中的复合物与纯化抑制剂形成的复合物具有相同的分子大小。C1INH在血浆中作为因子XIIa的主要抑制剂发挥作用。
