Parallel mechanisms of high molecular weight kininogen action as a cofactor in kallikrein inactivation and prekallikrein activation reactions

• Published in: Biochemistry (Easton) Vol. 32; no. 45; pp. 12148 - 12159
• Main Authors: Olson, Steven T, Francis, Ann Marie, Sheffer, Roberta, Choay, Jean
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 16.11.1993
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Glycoproteins; Heparin - chemistry; Heparin - metabolism; Humans; Kallikreins - antagonists & inhibitors; Kinetics; Kininogens - chemistry; Kininogens - metabolism; Molecular Weight; Prekallikrein - metabolism; Proteins; Zinc; Molecular form; Kallikrein kinin system; Activation; Glycoproteins; Inactivation; Antithrombin; Cofactor; Molecular weight; Proteins; Kininogen; Binding capacity; Kinetics; Heparin
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00096a027

The mechanism by which high molecular weight kininogen (H-kininogen) potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin [Olson, S. T., Sheffer, R., & Francis, A. M. (1993) Biochemistry (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 not heavy-chain, forms of H-kininogen were similarly effective in potentiating the heparin-accelerated antithrombin-kallikrein reaction, indicating 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 potentiation increased in a saturable manner with increasing kininogen concentration, reflecting a KD (23 +/- 8 nM) similar to that previously measured for H-kininogen binding to kallikrein by equilibrium methods. Both 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 (KD = 10-30 nM). However, at higher heparin concentrations, the stimulated but not the unstimulated heparin rate enhancement decreased in parallel with the saturation of a protein-heparin interaction with a KD (0.4 +/- 0.2 microM) comparable to that directly measured for the H-kininogen-heparin interaction (2.0 +/- 0.2 microM). These results implied that H-kininogen stimulation required the formation of a quaternary complex in which antithrombin and H-kininogen-kallikrein complex were bound to the same heparin chain. In keeping with this interpretation, a synthetic heparin pentasaccharide representing the antithrombin binding sequence accelerated the antithrombin-kallikrein reaction to an extent similar to that of full-length heparin chains containing this sequence, but the pentasaccharide acceleration was not stimulated by H-kininogen. The importance of H-kininogen-kallikrein complex binding to heparin for kininogen stimulation was further indicated from the marked salt dependence of the second-order rate constant for the association of H-kininogen-kallikrein complex but not free kallikrein with antithrombin-heparin complex, under conditions where saturation of the two binary complexes was maintained. Kinetic analyses of antithrombin-kallikrein reactions as a function of the inhibitor concentration indicated that the KD for an initial antithrombin-kallikrein encounter complex was decreased 20-fold by heparin binding to antithrombin and an additional 200-fold by H-kininogen also binding to kallikrein. By contrast, rate constants for the conversion of the encounter complex to a stable complex were comparable for all reactions.