MONOCLONAL-ANTIBODY F1 BINDS TO THE KRINGLE DOMAIN OF FACTOR-XII AND INDUCES ENHANCED SUSCEPTIBILITY FOR CLEAVAGE BY KALLIKREIN

In a previous study we have shown that monoclonal antibody F1 (MoAb F1 ), directed against an epitope on the heavy chain of factor XII distin ct from the binding site for anionic surfaces, is able to activate fac tor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). H ere, we studied in detail the mechanism underlying the activation of f actor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromog enic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with M oAb F1 alone, factor XII was auto-activated in a time-dependent fashio n, activation being maximal after 30 hours. Factor XII incubated in th e absence of MoAb F1 was hardly activated by kallikrein. whereas in th e presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Max imal activation of factor XII with hallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of hallikrein on the cleavag e of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator cou ld not substitute for kallikrein. Also, trypsin could easily activate factor XII. but in contrast to kallikrein, this activation was indepen dent of MoAb F1. SDS-PAGE analysis showed that the appearance of amido lytic activity correlated well with cleavage of factor XII. MoAb F1-in duced activation of factor XII in this purified system was not depende nt on the presence of high-molecular-weight kininogen (HK), in contras t to the activation of the contact system in plasma by MoAb F1. Experi ments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study s hows that binding of ligands to the kringle domain. which does not con tribute to the proposed binding site for negatively charged surfaces, may induce activation of factor XII. Therefore, these findings point t o the existence of multiple mechanisms of activation of factor XII. (C ) 1995 by The American Society of Hematology.