Olson S T, Sheffer R, Francis A M
Division of Biochemical Research, Henry Ford Hospital, Detroit, Michigan 48202.
Biochemistry. 1993 Nov 16;32(45):12136-47. doi: 10.1021/bi00096a026.
The effects of previously characterized interactions of high molecular weight kininogen (H-kininogen) with plasma kallikrein and with heparin on the regulation of kallikrein by the heparin-activated inhibitor, antithrombin, were investigated. H-kininogen, at levels sufficient to fully complex kallikrein, greatly potentiated the acceleration of antithrombin inhibition of kallikrein produced by heparin with high affinity for antithrombin. At I = 0.15, pH 7.4, 25 degrees C, kininogen thus maximally increased the heparin enhancement of the second-order rate constant for the antithrombin-kallikrein reaction from 13-fold (1.6 x 10(2) M-1 s-1 to 2.1 x 10(3) M-1 s-1) to 1200-fold (1.9 x 10(5) M-1 s-1). In contrast, H-kininogen had no effect on the antithrombin-kallikrein reaction in the absence of heparin, nor did the protein enhance the rate constants of 1.7 x 10(4) and 3.4 x 10(4) M-1 s-1 for kallikrein reactions with its primary plasma inhibitors C1-inhibitor and alpha 2-macroglobulin, respectively, in the absence or presence of heparin. Consistent with these results, SDS gel electrophoresis of the 125I-labeled kallikrein-inhibitor complexes formed in a mixture of these kallikrein inhibitors at their relative plasma concentrations indicated that antithrombin effectively competed with C1-inhibitor and alpha 2-macroglobulin for kallikrein, accounting for 54% of the total kallikrein complexes, only when both heparin and H-kininogen were present. Similarly, the presence of therapeutic levels of heparin (approximately 1 unit/mL) in normal, factor XII-deficient, and prekallikrein-deficient plasmas enhanced the rate of inactivation of added kallikrein by 2.3-fold and significantly altered the partitioning of radiolabeled kallikrein from predominantly C1-inhibitor and alpha 2-macroglobulin complexes (86-92%) to mostly antithrombin complexes (50-53%). Experiments in antithrombin-deficient and H-kininogen-deficient plasmas confirmed that the enhanced kallikrein inactivation rate and predominant formation of antithrombin-kallikrein complexes in heparinized plasma were dependent on antithrombin and H-kininogen. The contribution of antithrombin to kallikrein inhibition in plasma remained significant (approximately 40-70%) at optimal concentrations of unfractionated or size- and antithrombin affinity-fractionated heparin, in the presence of plasma levels of calcium and zinc ions, at 37 degrees C, and with minimal plasma dilution. These results suggest that antithrombin and H-kininogen may play important roles in the regulation of kallikrein activity in the presence of heparin or heparin-like glycosaminoglycans.
研究了先前已表征的高分子量激肽原（H - 激肽原）与血浆激肽释放酶以及与肝素的相互作用对肝素激活的抑制剂抗凝血酶调节激肽释放酶的影响。H - 激肽原在足以使激肽释放酶完全形成复合物的水平下，极大地增强了对肝素具有高亲和力的抗凝血酶对激肽释放酶抑制作用的加速效果。在I = 0.15、pH 7.4、25℃条件下，激肽原因此将抗凝血酶 - 激肽释放酶反应的二级速率常数的肝素增强倍数从13倍（1.6×10² M⁻¹ s⁻¹ 提高到2.1×10³ M⁻¹ s⁻¹）最大化提高到1200倍（1.9×10⁵ M⁻¹ s⁻¹）。相比之下，H - 激肽原在无肝素时对抗凝血酶 - 激肽释放酶反应没有影响，并且在无肝素或有肝素存在的情况下，该蛋白质也未增强激肽释放酶与其主要血浆抑制剂C1 - 抑制剂和α2 - 巨球蛋白反应的速率常数，分别为1.7×10⁴ 和3.4×10⁴ M⁻¹ s⁻¹。与这些结果一致，在这些激肽释放酶抑制剂以其相对血浆浓度混合形成的混合物中，对125I标记的激肽释放酶 - 抑制剂复合物进行SDS凝胶电泳表明，仅当肝素和H - 激肽原都存在时，抗凝血酶才有效地与C1 - 抑制剂和α2 - 巨球蛋白竞争激肽释放酶，占总激肽释放酶复合物的54%。同样，在正常、因子XII缺乏和前激肽释放酶缺乏的血浆中，治疗水平的肝素（约1单位/mL）的存在使添加的激肽释放酶的失活速率提高了2.3倍，并显著改变了放射性标记激肽释放酶的分配，从主要是C1 - 抑制剂和α2 - 巨球蛋白复合物（86 - 92%）转变为主要是抗凝血酶复合物（50 - 53%）。在抗凝血酶缺乏和H - 激肽原缺乏的血浆中进行的实验证实，肝素化血浆中激肽释放酶失活速率的增强和抗凝血酶 - 激肽释放酶复合物的主要形成依赖于抗凝血酶和H - 激肽原。在37℃、存在血浆水平的钙和锌离子且血浆稀释最小的情况下，在未分级或按大小和抗凝血酶亲和力分级的肝素的最佳浓度下，抗凝血酶对血浆中激肽释放酶抑制的贡献仍然显著（约40 - 70%）。这些结果表明，在存在肝素或类肝素糖胺聚糖的情况下，抗凝血酶和H - 激肽原可能在激肽释放酶活性的调节中发挥重要作用。
