The second exon-encoded factor XII region is involved in the interaction of factor XII with factor XI and does not contribute to the binding site for negatively charged surfaces.

Contact system activation, in vitro, is triggered by activation of factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located within the amino acid residues 1-28 by identifying the epitope recognized by a monoclonal antibody (MoAb), B7C9, which inhibits kaolin-induced clotting activity. To further elucidate the role of the amino terminal binding site in the regulation of FXII activation, we have characterized a FXII recombinant protein (rFXII-triangle up19) deleted of the amino acid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-triangle up19 was constructed and expressed in HepG2 cells by using vaccinia virus. Purified rFXII-triangle up19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharose, thus confirming that it lacked the epitope for this MoAb, and had no amidolytic activity towards the chromogenic substrate S-2302 in the absence of activator. rFXII-triangle up19 specific clotting activity was lower (44%) than that of native FXII. The activation rate of rFXII-triangle up19 by kallikrein in the absence of dextran sulfate was about four times higher than that of full-length FXII and was increased in the presence of dextran sulfate. However, rFXII-triangle up19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-triangle up19 bound to kaolin, which binding was equally well inhibited by either, rFXII-triangle up19 or full-length FXII (IC50 = 7.2 +/- 2.2 nmol/L for both proteins). Accordingly, a synthetic peptide corresponding to FXII amino acid residues 3-19 did not inhibit the binding of labeled full-length FXII to kaolin. rFXII-triangle up19 generated a similar amount of FXIIa- and kallikrein-C1-inhibitor complexes in FXII-deficient plasma in the presence of kaolin, as did full-length FXII; but generated less factor XIa-C1-inhibitor complexes (50%) than full-length FXII. This impaired factor XI activation by rFXII-triangle up19a was also observed in a purified system and was independent of the presence of high molecular weight kininogen. Furthermore, the synthetic peptide 3-19, preincubated with factor XI, inhibited up to 30% activation of factor XI both in the purified system as well as in plasma. These results together indicate that amino acid residues 3-19 of FXII are involved in the activation of factor XI and do not contribute to the binding of FXII to negatively charged surfaces.