HIGH-MOLECULAR-WEIGHT KININOGEN POTENTIATES THE HEPARIN-ACCELERATED INHIBITION OF PLASMA KALLIKREIN BY ANTITHROMBIN - ROLE FOR ANTITHROMBININ THE REGULATION OF KALLIKREIN

The effects of previously characterized interactions of high molecular weight kininogen (H-kininogen) with plasma kallikrein and with hepari n 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 anti thrombin inhibition of kallikrein produced by heparin with high affini ty 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-kallikr ein reaction in the absence of heparin, nor did the protein enhance th e 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 alpha2- macroglobulin, respectively, in the absence or presence of heparin. Co nsistent with these results, SDS gel electrophoresis of the I-125-labe led kallikrein-inhibitor complexes formed in a mixture of these kallik rein inhibitors at their relative plasma concentrations indicated that antithrombin effectively competed with C1-inhibitor and alpha2-macrog lobulin for kallikrein, accounting for 54% of the total kallikrein com plexes, 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 plasm as enhanced the rate of inactivation of added kallikrein by 2.3-fold a nd significantly altered the partitioning of radiolabeled kallikrein f rom predominantly C1-inhibitor and alpha2-macroglobulin complexes (86- 92%) to mostly antithrombin complexes (50-53%). Experiments in antithr ombin-deficient and H-kininogen-deficient plasmas confirmed that the e nhanced kallikrein inactivation rate and predominant formation of anti thrombin-kallikrein complexes in heparinized plasma were dependent on antithrombin and H-kininogen. The contribution of antithrombin to kall ikrein inhibition in plasma remained significant (approximately 40-70% ) at optimal concentrations of unfractionated or size- and antithrombi n affinity-fractionated heparin, in the presence of plasma levels of c alcium and zinc ions, at 37-degrees-C, and with minimal plasma dilutio n. These results suggest that antithrombin and H-kininogen may play im portant roles in the regulation of kallikrein activity in the presence of heparin or heparin-like glycosaminoglycans.