Mapping of the Discontinuous H-kininogen Binding Site of Plasma Prekallikrein

Abstract

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K D ‫؍‬ 1.2 ؋ 10 ؊8 M). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 Ϸ A1 > A3. Removal of single apple domains in prekallikrein deletion mutants reduced kininogen binding by 21 (A1), 64 (A2), and 24% (A4), respectively, whereas deletion of A3 was without effect. Transposition of homologous A2 domain from prekallikrein to factor XI conferred high-affinity kininogen binding from the former to the latter. The principal role of A2 for H-kininogen docking to the prekallikrein heavy chain was further substantiated by the finding that cleavage of a single peptide bond in A2 drastically diminished the H-kininogen binding affinity. Furthermore, the epitope of monoclonal antibody PKH6 which blocks kallikrein-kininogen complex formation with an IC 50 of 8 nM mapped to the center portion of domain A2. Our data indicate that domain A2 and two flanking sequence segments of A1 and A4 form a discontinuous binding platform for H-kininogen on the prekallikrein heavy chain. Domain-specific antibodies directed to these critical sites efficiently interfered with contact phase-induced bradykinin release from H-kininogen.

Characterization of the H-kininogen-binding Site on Factor XI

“…The generation and characterization of murine monoclonal antibodies against FXI (␣F1, ␣F2, and ␣F4), PPK (␣P1, ␣P2, and ␣P4), and tPA have been described (30,32,33). The properties of monoclonal anti-HK antibody HKH14 have been published (25). Polyclonal antisera AS199 and AS176 against human FXI and PPK, respectively, were raised in New Zealand White rabbits.…”

“…The names of some antibodies used in previous studies were changed to comply with the new nomenclature system. Specifically, anti-PPK antibodies ␣P1, ␣P2, and ␣P4 were formerly designated PKH19, PHK6, and PKH1, respectively (25), and anti-FXI antibodies ␣F1, ␣F2, and ␣F4 were previously named XI-5, XI-3, and XI-1, respectively (30). For chimeric FXI/PPK proteins, the molecule that makes up the majority of the chimera is listed first, while the abbreviation for the apple domain substituted into the protein is listed second.…”

“…Human factor XII was purchased from Enzyme Research Laboratories (South Bend, IN) and activated by incubation with glass beads for 30 min at 37°C. To activate FXI, PPK, and chimeric proteins (see below), protein was incubated with FXIIa in a 250:1 molar ratio in 6.5 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 2.7 mM KCl, 150 mM NaCl, pH 7.4 (PBS) for 72 h at 37°C as described (25,31). The generation and characterization of murine monoclonal antibodies against FXI (␣F1, ␣F2, and ␣F4), PPK (␣P1, ␣P2, and ␣P4), and tPA have been described (30,32,33).…”

“…Therefore, it appears that there are similarities in the mechanism by which HK binds to FXI and to PPK. A series of studies using recombinant whole molecules and individual apple domains, as well as monoclonal antibodies, have determined that PPK binding to HK requires the first, second, and fourth apple domains (P1, P2, an P4, respectively) but not the third apple domain (P3) (23)(24)(25). The P2 domain appears to be most important for this process (25).…”

“…A series of studies using recombinant whole molecules and individual apple domains, as well as monoclonal antibodies, have determined that PPK binding to HK requires the first, second, and fourth apple domains (P1, P2, an P4, respectively) but not the third apple domain (P3) (23)(24)(25). The P2 domain appears to be most important for this process (25). In contrast, studies of FXI binding to HK using conformationally constrained peptides suggest that HK is bound through a relatively small area between amino acids Phe 56 and Ser 86 within the first apple domain (F1).…”