High Molecular Weight Kininogen-binding Site of Prekallikrein Probed by Monoclonal Antibodies*

A panel of monoclonal antibodies against human pre- kallikrein was raised in mice and characterized with respect to the major antigenic epitopes. Of 18 antibod- ies, nine were directed against the light chain portion performing the proteolytic function of activated kalli- krein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen. Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentration- dependent manner the activated partial thrombo- plastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent K,.. of 6.8 2 0.44.10’ M-’ for prekallikrein. Functional analyses revealed that PK6 does not interfere with prekallikrein activation by activated Hageman factor (/3-F XII,),

A panel of monoclonal antibodies against human prekallikrein was raised in mice and characterized with respect to the major antigenic epitopes.
Of 18 antibodies, nine were directed against the light chain portion performing the proteolytic function of activated kallikrein, and nine recognized the heavy chain mediating the binding of prekallikrein to high molecular weight (H-)kininogen.
Among the anti-heavy chain antibodies, one (PK6) interfered with the procoagulant activity of prekallikrein, and prolonged in a concentrationdependent manner the activated partial thromboplastin time of reconstituted prekallikrein-deficient plasma (Fletcher type). Antibody PK6 was subtyped IgG1,k and had an apparent K,.. of 6.8 2 0. 44 Mammalian blood coagulation is a well-orchestrated process of cellular and molecular events designed for the rapid occlusion of leaky blood vessels by thrombus formation (1). Two major cascades, i.e. the endogenous (2) and the extrinsic system, serve in fibrin generation thereby involving a large number of proteins, including the non-enzymatic cofactor high molecular weight kininogen (H-kininogen)l*' and the zymogen prekallikrein (Fletcher factor) (for reviews, see refs. 3, 4). In concert with two other proenzymes, i.e. factor XII (Hageman factor) and factor XI (plasma thromboplastin antecedent), they trigger the endogenous blood coagulation cascade and the kinin-forming pathway on subendothelial surfaces ("contact phase") (4). Local assembly of the various factors is facilitated by direct binding of factor XII and Hkininogen to the contact phase, and by non-covalent complex formation (5) between H-kininogen and prekallikrein (K,,, = 8.3. lo7 M-l) or H-kininogen and factor XI (K,,, = 3.4. lo7 M-l), respectively (6, 7). The binding site for prekallikrein contained in H-kininogen has been mapped to the extreme COOH-terminal portion of the kininogen light chain (8) corresponding to positions 565-595 of mature human H-kininogen (9, 10). This binding segment overlaps the corresponding binding region of factor XI covering positions 556-613 of the H-kininogen light chain sequence (11). Synthetic peptides encompassing the predicted prekallikrein-binding site of H-kininogen effectively mimic the binding of H-kininogen to prekallikrein (11). The corresponding binding site(s) for H-kininogen contained in prekallikrein and factor XI are known to be harbored by their heavy chains (7, 12); however, their precise localizations remain to be determined.
In the study of protein-protein interactions and protein functions, monoclonal antibodies which selectively interfere with functional role(s) expressed by their target proteins have proven to be useful tools (13-15). A monoclonal anti-human prekallikrein antibody was selected which inhibits activation of prekallikrein by factor XII. on a surface without competing for H-kininogen binding of prekallikrein or impairing the enzymatic activity of activated kallikrein (16). This antibody allowed identification of a previously unrecognized epitope of the prekallikrein heavy chain portion required for the interaction with factor XII, on the contact surface (16). Furthermore, a monoclonal antibody against factor XII was raised which inhibits the surface-mediated coagulant activity of fac- tor XII, and its target epitope mapped to a sequence of 20 amino acid residues of the factor XII heavy chain (17, 18). Yet another monoclonal antibody directed toward a neoantigenie determinant of the factor XII heavy chain has enabled the analysis of the molecular mechanisms driving contact phase activation (19). Similar studies addressing structurefunction relationships by virtue of monoclonal antibody probes were carried out with human factor XI (20). Taking advantage of the unique property of an antibody which selectively inhibits complex formation of H-kininogen and factor XI (21,22), the kininogen-binding site of factor XI was roughly mapped to the NH,-terminal portion (residues l-102) of its heavy chain (23,24).
Here A competitive ELISA was set up using prekallikrein as the coating antigen, and biotinylated H-kininogen as the probe, and the biotin-avidin-peroxidase system as the reporter system. Binding of biotinylated H-kininogen was scrutinized in the presence of increasing amounts of antibody competitor. Specificity of the assay was demonstrated by the application of native H-kininogen which displaced biotinylated H-kininogen from the complex almost at equimolar concentrations whereas Lkininogen devoid of a prekallikrein-binding site failed to interfere with complex formation (data not shown). Among the monoclonal anti-heavy chain antibodies tested, PK6 (class C) was the only one capable of competing with H-kininogen for prekallikrein binding (Fig. 5). PK6 was even more effective in prekallikrein binding than native H-kininogen (factor of 16 at A.,,,5 ,,,,, = 0.75). Furthermore, Fab' and F(ab')g fragments of PK6 were equipotent in preventing kininogen binding to prekallikrein (not shown) thus excluding the possibility that displacement of kininogen was due to the Fc part of the intact immunoglobulin molecule. Polyclonal anti-prekallikrein IgG were equally effective in displacing H-kininogen (Fig. 5). Unlike PK6, monoclonal anti-heavy chain antibodies PK2 (class A), PK4 (class B) as well as anti-light chain antibodies PKl4 (class D), and PK18 (class E) did not affect complex formation (exemplified for PK2 in Fig. 5).
" Portions of this paper (including "Materials and Methods," part of "Results," The presence of PK6 did not change the relative mobility of prekallikrein in this system (Fig. 6, top panel; note that the pH of the system was chosen such that antibodies lack a net charge). The preformed complex of prekallikrein and H-kininogen applied in equimolar ratio gave a single precipitation line positioned intermediate to those of the single components whereas precipitation lines corresponding to the single components were not detected. Incubation prior to electrophoresis of the complex with a 2.5fold molar excess of PK6 resulted in the formation of two non-fusing precipitation lines at the relative positions of the single components, whereas the corresponding precipitation line of the complex was absent (Fig. 6, bottom panel). This finding indicated that dissociation of the heterodimeric noncovalent complex had occurred in the presence of PK6. Antiheavy chain antibodies other than PK6 had no effect on complex dissociation (not shown). XI (Fig. 8A). Titer nlate-bound factor XI was readilv detected Identification of the Antigenic Epitope of PK6-Interference of PK6 with prekallikrein-kininogen complex formation might suggest that PK6 binds to an epitope closely neighbored to or even incorporated in the H-kininogen-binding site of prekallikrein.
To examine this possibility, a synthetic peptide of 31 amino acid residues (HK31) known to encompass the structural elements of the prekallikrein-binding site of Hkininogen was allowed to compete with PK6 for prekallikrein binding. This peptide combines with prekallikrein at a K,,, similar to that of native H-kininogen (11). The competitive ELISA detailed above was employed, except that biotinylated HK31 instead of biotinylated H-kininogen was used as the probe. Specificity of the assay was demonstrated by application of increasing concentrations of nonderivatized peptide HK31 or native H-kininogen which efficiently competed for prekallikrein binding (Fig. 7) while an unrelated peptide of 24 residues was ineffective (not shown). Moreover, polyclonal anti-prekallikrein IgG were effective competitors of HK31 binding to prekallikrein whereas monoclonal antibodies PK2, PK4, PK14, and PK18 had no effect (exemplified for PK2 in Fig. 7). The most dramatic effect was observed with antibody PK6 which competed with biotinylated HK31 for prekallikrein binding in a concentration-dependent manner (Fig.  7). At the highest concentrations of PK6, binding of biotinylated HK31 was almost nullified.
Conversely, HK31 was capable of displacing biotinylated PK6 from immobilized prekallikrein at a high molar excess of the peptide (not shown). Cross-reactivity of Antibody PK6 with Factor XI-Because plasma prekallikrein and factor XI are homologous proteins of closely related structure (46) which compete for overlapping binding sites on the H-kininogen light chain (8, ll), we tested for the cross-reactivity of antibody PK6 with human factor by pol&lonal antibodies against purified human factor XI. Application of monoclonal anti-prekallikrein antibody PK6 resulted in a strong signal indicating that PK6 effectively cross-reacted with factor XI. Among the other monoclonal anti-prekallikrein antibodies, only PK2 showed a weak crossreactivity whereas antibodies PK4, PK14, and PK18 failed to bind to factor XI thus excIuding the possibility that the factor XI preparation used for titer plate coating contained trace amounts of contaminating prekailikrein. Furthermore, affinity purified polyclonal IgG against prekallikrein barely recognized factor XI (Fig. 8A). To examine the possibility that PK6 interferes with H-kininogen binding to factor XI, a competitive ELISA was established with purified factor XI as the coating antigen and biotinylated H-kininogen as the probe (Fig. 8B) with H-kininogen (47) is activated on the contact phase by factor XII, (2). Specific cleavage of a single Arg-Ile bond (positions 371 and 372) results in the formation of a twochain molecule consisting of a heavy chain of 371 residues derived from the NH2 terminus of the zymogen, and a light chain of 248 residues from the COOH terminus (48). The kallikrein heavy chain which combines with H-kininogen is composed of four tandemly arranged repeats of 90-91 residues (48). These repeated sequence segments are characterized by a well-conserved arrangement of 6 half-cysteine residues each, except for the fourth repeat which carries 2 extra half-cysteine residues, most probably engaged in the bridging of the heavy and light chains (46,48). Extensive sequence similarity among prekallikrein and factor XI was reported (46). In particular, the four heavy chain tandem repeats are well conserved among the two proteins (46). To date, the heavy chain domain(s) involved in the complex formation of prekallikrein and Hkininogen has (have) not been identified. As a first step to localize the target site of prekallikrein for H-kininogen, we have raised a panel of monoclonal antibodies against native prekallikrein and characterized their corresponding epitopes. Among 18 anti-prekallikrein antibodies tested, PK6 was chosen as the candidate antibody because it effectively blocked H-kininogen binding to prekallikrein, dissociated preformed complexes of the two proteins, and interfered with the procoagulant activity of prekallikrein. The corresponding F(ab')* and Fab' fragments were also efficient PK 2 PKL 11213 . PK 6 11213 PK IL PK I8 --i 1