THE 2ND EXON-ENCODED FACTOR-XII REGION IS INVOLVED IN THE INTERACTIONOF 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 fac tor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located wit hin 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 characteri zed a FXII recombinant protein (rFXII-Delta 19) deleted of the amino a cid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-Delta 19 was constructed and expressed in HepGZ cells by using vaccinia virus. Purified rFXII-Delta 19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)polyacry lamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharo se, 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-Delta 19 specific clotting activity was l ower (44%) than that of native FXII. The activation rate of rFXII-Delt a 19 by kallikrein in the absence of dextran sulfate was about four ti mes higher than that of full-length FXII and was increased in the pres ence of dextran sulfate. However, rFXII-Delta 19 underwent autoactivat ion in the presence of dextran sulfate. Labeled rFXII-Delta 19 bound t o kaolin, which binding was equally well inhibited by either, rFXII-De lta 19 or full-length FXII (IC50 = 7.2 +/- 2.2 nmol/L for both protein s). 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-Delta 19 generated a similar amount of FXIIa- and kal likrein-C1-inhibitor complexes in FXII-deficient plasma in the presenc e 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-Delta 19a was also observed in a purified syst em and was independent of the presence of high molecular weight kinino gen. 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 amin o acid residues 3-19 of FXII are involved in the activation of factor XI and db not contribute to the binding of FXII to negatively charged surfaces. (C) 1998 by The American Society of Hematology.