PARALLEL MECHANISMS OF HIGH-MOLECULAR-WEIGHT KININOGEN ACTION AS A COFACTOR IN KALLIKREIN INACTIVATION AND PREKALLIKREIN ACTIVATION REACTIONS

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.