Human plasma kallikrein and C1 inhibitor form a complex possessing an epitope that is not detectable on the parent molecules: demonstration using a monoclonal antibody.

Abstract

The inactivation of human plasma kallikrein (EC 3.4.21.8) by the inhibitor of activated complement component 1 (Ci inhibitor) induces the formation of a 1:1 stoichiometric kallikrein-Ci inhibitor complex and a proteolytically modified form of Ci inhibitor. We have produced a monoclonal antibody that recognizes the kallikrein-Cl inhibitor complex as well as modified C1 inhibitor but fails to react with virgin Ci inhibitor or native plasma kallikrein. This observation constitutes an unequivocal demonstration that the reaction between plasma kallikrein and Ci inhibitor leads to the emergence of an epitope that is undetectable on the parent enzyme and inhibitor molecules.Complexes formed between serine proteases and protease inhibitors of the a1-protease inhibitor class seem to possess a specific domain that is not present on the parent enzyme and inhibitor molecules. This suggestion is based on the following: (i) immunochemical studies showing that neoantigenic determinants are formed during the inactivation of thrombin and factor Xa by antithrombin III, or when plasmin reacts with a2-antiplasmin (1-4); and (ii) in vivo investigations indicating that the complexes between thrombin and antithrombin III, trypsin and a1-protease inhibitor, or involving a2-antiplasmin are rapidly cleared from the circulation by receptor-mediated pathways, whereas this is not the case for the respective virgin inhibitor molecules (5-7).Plasma kallikrein (EC 3.4.21.8) is a serine protease that results from proteolytic cleavage of the zymogen prekallikrein (8-10). Kallikrein plays a central role in the activation of the contact system of blood coagulation, fibrinolysis, and inflammation (11,12). Once formed in the plasma milieu, kallikrein reacts with its substrates, as well as with plasma protease inhibitors. In normal plasma, the predominant inhibitors of kallikrein are the inhibitor of activated complement component 1 (Ci inhibitor) and a2-macroglobulin (13,14). The mechanism by which kallikrein is inactivated by Ci inhibitor has been examined in purified systems. Kallikrein and Ci inhibitor form stable 1:1 stoichiometric enzyme-inhibitor complexes, which do not exhibit enzymatic or inhibitory activity (15-18). Thus, Ci inhibitor inactivates kallikrein by the same mechanism as that reported for the reaction of a1-protease inhibitor, antithrombin III, or a2-antiplasmin with their respective target serine proteases (19). Because of these mechanistic similarities, the reaction between kallikrein and Ci inhibitor may also induce the formation of a complex-specific domain. In this study, we have examined the feasibility of producing monoclonal antibodies (mAbs) that would specifically react with such a domain but would fail to detect the parent kallikrein and Ci inhibitor molecules. This attempt was successful, and relevant mAbs will be made available to other investigators for research purposes. MATERIALS AND METHODSProteins. Human plasma kallikrein (9) and Ci inhibitor (20) were purified by using published procedures. The complex ...

Characterization of common neoantigenic epitopes generated in plasminogen activator inhibitor‐1 after cleavage of the reactive center loop or after complex formation with various serine proteinases

“…This indicates that the epitopes recognized by the antibodies are not located in the near vicinity of the active site residues Arg346-Met347 of PAI-I. de Agostini et al [28,29] nor Nuijens et al [30] provided information on the functional effects of their monoclonal antibodies.…”

Proteolytic inactivation of plasma C1- inhibitor in sepsis.

“…Second, about one-third of native Cl-Inh in plasma is cleaved in vitro to iCl-Inh upon activation of the contact system by kaolin (53) and DXS (45). Third, analytic gel (SDS-PAGE) studies concerning the interaction of Cl-Inh with either kallikrein, Factor XIa, or plasmin have demonstrated the formation of stable high-molecular weight complexes and the appearance of iCl-Inh species (22,(27)(28)(29)56).…”

“…Several studies have shown that the formation of high-molecular weight target proteinase-C1-Inh complexes is accompanied by the generation of modified (cleaved), inactive Cl-Inh (iCl-Inh) species with a lower apparent molecular weight than native Cl-Inh (23,24,(27)(28)(29). In addition, a number ofendogenous (e.g., neutrophil elastase) and bacterial nontarget proteinases (i.e., proteinases that are not inhibited by Cl-Inh) generate iCl-Inh species by catalytical cleavage ofthe inhibitor in its reactive center (30,31).…”

Formation and properties of C1‐inhibitor polymers

“…TPCK-treated bovine trypsin was from Worthington (Freehold, N J) and was active site-titrated with para-nitrophenol guanidinobenzoate [12]. Monoclonal antibodies 3C7 and 4C3 were prepared as described previously [9].…”

“…Monoclonal antibody 4C3 binds to cleaved and complexed Cl-inhibitor but not to the native protein [9,10] and is, therefore, an indicator of conformational changes involving the reactive centre loop. Thus, it was tested if polyinerisation could induce the formation of the epitope.…”