Binding of a Monoclonal Anti-Human Plasma Prekalliprein Antibody to the Complexes of Kallikrein with Cl-Inhibitor and α2-Macroglobulin Analyzed by Immunoblot and “Sandwich” Assays

Studies in vitro have shown that factor XII and prekallikrein associated with high molecular weight kininogen (HK) interact with a negatively charged surface for optimal activation, with the formation of factor XIIa and kallikrein (1-4), and subsequent release of bradykinin from HK hydrolyzed by kallikrein (5). Prekallikrein activated in the fluid phase by an active fragment of factor XII (factor XIIf) (6) or other proteases such as trypsin also cleave HK with the formation of the potent peripheral vasodilator bradykinin. These proteases may be released into the circulation in vivo under pathological conditions such as release of trypsin in acute pancreatitis (7) or of proteases from invading microorganisms (8). Such enzymes share the same inhibitor α2-macroglobulin (α2 M) with kallikrein. In addition, when prekallikrein activation occurs in the presence of other proteins (e.g. in plasma), the kallikrein formed or the products of kallikrein activity such as factor XIIa or factor XIIf, can interact with other plasma proteins or blood cells. Complement activation, fibrinolysis, prorenin activation and neutrophil activation may result from these interactions (9, 10). Therefore, the concentrations of prekallikrein and the products of its activation in plasma will depend not only on prekallikrein biosynthesis in liver but also on the interplay between the rate of activation of this zymogen, inactivation of the kallikrein formed by active inhibitors available, and clearance of kallikrein inhibitors from the circulation, which are also dependent on the activation of other plasma proteins. Two major inhibitors, C1-inhibitor (C1-Inh) and α2M and minor inhibitors such as antithrombin III (11) have been described to inhibit kallikrein (12-15). Since kallikrein is rapidly associated with these inhibitors in the formation of complexes and the concentration of prekallikrein (0.3 uM) is much lower than that of the inhibitors, Cl-Inh (2 uM), and α2M (2.5 uM), under normal conditions almost all the kallikrein formed is inactivated by Cl-Inh and α2M (11, 16) and, in small amounts, by other inhibitors. The kallikrein-inhibitor complexes are rapidly removed from the circulation. However, increase of zymogen activation and/or changes in the concentration of inhibitors of the plasma contact activation may change the pattern of the distribution of prekallikrein antigen among zymogen, active enzyme and complexes. For example, activation of factor XII by endotoxin (17, 18), a condition which may lead to hypotensive shock (19, 20) has been associated with a decrease of plasma kininogen, increase of bradykinin and a decrease in plasma prekallikrein and kallikrein inhibitors (19, 21); increased concentrations of kallikrein-Cl-Inh complexes have been reported in bacterial infections such as typhoid fever (22); depletion of prekallikrein and HK with increased concentrations of bradykinin have been also observed during attacks of angioedema, a condition in which the concentration of Cl-Inh is lower than in normal plasma (23). Anaphylaxis (24), Rochy Mountain spotted fever (25), nephrotic syndrome (26) and adult respiratory distress syndrome (27) have also been reported to be related with alterations in the concentrations of the proteins of the contact activation system. Although increased concentrations of kallikrein-Cl-Inh complexes in plasma indicates abnormal prekallikrein activation, values of kallikrein-α2M in patients have not been reported. However, in situations when Cl-Inh is depleted or inactivated, complexes of kallikrein with other inhibitors such as α2M are expected to be formed in larger amounts better reflecting abnormal activation of the contact system.