HIGH-MOLECULAR-WEIGHT KININOGEN POTENTIATES THE HEPARIN-ACCELERATED INHIBITION OF PLASMA KALLIKREIN BY ANTITHROMBIN - ROLE FOR ANTITHROMBININ THE REGULATION OF KALLIKREIN
St. Olson et al., HIGH-MOLECULAR-WEIGHT KININOGEN POTENTIATES THE HEPARIN-ACCELERATED INHIBITION OF PLASMA KALLIKREIN BY ANTITHROMBIN - ROLE FOR ANTITHROMBININ THE REGULATION OF KALLIKREIN, Biochemistry, 32(45), 1993, pp. 12136-12147
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.