Contribution of Plasma Protease Inhibitors to the Inactivation of Kallikrein in Plasma

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Abstract
Although C̄l-inhibitor (C̄l-INH) and α2-macroglobulin (α2M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in C̄l-INH, or artificially depleted of α2M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min−1. This rate constant was reduced to 0.35, 0.30, and 0.06 min−1, in plasma deficient respectively in C̄l-INH, α2M, or both inhibitors. Thus C̄l-INH (42%) and α2M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, 125I-kallikrein (Mr 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of 125I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (Mr 370,000) with C̄l-INH, while 48% formed a complex (Mr 850,000) with α2M. After inactivation by C̄l-INH-deficient plasma, >90% of the active 125I-kallikrein was associated with α2M. A similar proportion of the label was associated with C̄l-INH in plasma deficient in α2M. After inactivation by plasma deficient in both C̄l-INH and α2M, 125I-kallikrein was found to form a complex of Mr 185,000. This latter complex, which may involve antithrombin III, α1-protease inhibitor, and/or α1-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either C̄l-INH or α2M, even after the addition of heparin (2 U/ml). These observations demonstrate that C̄l-INH and α2M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either C̄l-INH or α2M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.