St. Olson et al., PARALLEL MECHANISMS OF HIGH-MOLECULAR-WEIGHT KININOGEN ACTION AS A COFACTOR IN KALLIKREIN INACTIVATION AND PREKALLIKREIN ACTIVATION REACTIONS, Biochemistry, 32(45), 1993, pp. 12148-12159
The mechanism by which high molecular weight kininogen (H-kininogen) p
otentiates the heparin-accelerated inhibition of plasma kallikrein by
antithrombin [Olson, S. T., Sheffer, R., & Francis, A. M. (1993) Bioch
emistry (preceding paper in this issue)] was investigated at I = 0.15,
pH 7.4, 25-degrees-C. Single-chain, two-chain, and light-chain, but n
ot heavy-chain, forms of H-kininogen were similarly effective in poten
tiating the heparin-accelerated antithrombin-kallikrein reaction, indi
cating that the light-chain region of the protein was responsible for
promoting kallikrein inactivation and that cleavage of H-kininogen did
not significantly affect this promoting activity. H-kininogen potenti
ation increased in a saturable manner with increasing kininogen concen
tration, reflecting a K(D) (23 +/- 8 nM) similar to that previously me
asured for H-kininogen binding to kallikrein by equilibrium methods. B
oth H-kininogen-stimulated and unstimulated heparin rate enhancements
initially increased with increasing heparin concentration in a manner
corresponding to the saturation of antithrombin with heparin (K(D) = 1
0-30 nM). However, at higher heparin concentrations, the stimulated bu
t not the unstimulated heparin rate enhancement decreased in parallel
with the saturation of a protein-heparin interaction with a K(D) (0.4
+/- 0.2 muM) comparable to that directly measured for the H-kininogen-
heparin interaction (2.0 +/- 0.2 muM). These results implied that H-ki
ninogen stimulation required the formation of a quaternary complex in
which antithrombin and H-kininogen-kallikrein complex were bound to th
e same heparin chain. In keeping with this interpretation, a synthetic
heparin pentasaccharide representing the antithrombin binding sequenc
e accelerated the antithrombin-kallikrein reaction to an extent simila
r to that of full-length heparin chains containing this sequence, but
the pentasaccharide acceleration was not stimulated by H-kininogen. Th
e importance of H-kininogen-kallikrein complex binding to heparin for
kininogen stimulation was further indicated from the marked salt depen
dence of the second-order rate constant for the association of H-kinin
ogen-kallikrein complex but not free kallikrein with antithrombin-hepa
rin complex, under conditions where saturation of the two binary compl
exes was maintained. Kinetic analyses of antithrombin-kallikrein react
ions as a function of the inhibitor concentration indicated that the K
(D) for an initial antithrombin-kallikrein encounter complex was decre
ased 20-fold by heparin binding to antithrombin and an additional 200-
fold by H-kininogen also binding to kallikrein. By contrast, rate cons
tants for the conversion of the encounter complex to a stable complex
were comparable for all reactions. Together, these results indicate a
heparin accelerating mechanism involving mostly heparin activation of
antithrombin in the absence of H-kininogen, with an additional mechani
sm involving heparin bridging antithrombin and kallikrein contributing
in the presence of H-kininogen due to kininogen facilitating the bind
ing of kallikrein to heparin. Zinc ions, at levels which promoted H-ki
ninogen binding to heparin, diminished kininogen potentiation at I = 0
.15, but increased this potentiation at I = 0.3, suggesting that antag
onism of inhibitor or kallikrein-kininogen complex binding to heparin
by free kininogen can offset the promoting effect of kininogen dependi
ng on the kininogen binding affinity for heparin.