Identification of a putative binding site for negatively charged surfaces in the fibronectin type II domain of human factor XII - An immunochemical and homology modeling approach

Monoclonal antibodies directed against functional sites of proteins provide useful tools for structure-function studies. Here we describe a mAb, KOK5, directed against the heavy chain region of human coagulation factor XII (F XII), which inhibits kaolin-induced clotting activity by preventing the bin ding of FXII to kaolin. Furthermore, mAb KOK5 enhances FXII susceptibility for cleavage by kallikrein and supports FXII autoactivation. Hence, mAb KOK 5 likely is directed against the binding site of FXII for negatively charge d surfaces. Screening of two phage-displayed random peptide libraries with mAb KOK5 selected phages that could be grouped on the basis of two amino ac id consensus sequences: A) FXFQTPXW and B) HQ/LCTHR/KKC. Sequence A contain s two motifs: one shares homology with FXII amino acid residues 30-33 (FPFQ ), the second one with residues 57-60 (TPNF); both amino acid stretches bel onging to the fibronectin type II domain of FXII. Sequence B also reveals h omology with part of the fibronectin type II domain, i.e. the stretch 40-47 (HKCTHKGR). A three-dimensional model of FXII residues 28-65, obtained by homology modeling, indicated that the three amino acid stretches 30-33, 40- 47 and 57-60 are close to each other and accessible for the solvent, i.e. i n a form available for interaction with the monoclonal antibody, suggesting that mAb KOK5 recognizes a discontinuous epitope on the fibronectin type I I domain of FXII. Peptides corresponding to FXII sequences 29-37 (FXII29-37 ) or 39-47 (FXII39-47), were synthesized and tested for the capability to i nhibit FXII binding to negatively charged surfaces. Peptide FXII39-47 inhib ited the binding of labeled FXII to kaolin and effectively prevented both d extran sulfate- and kaolin-induced activation of the contact system in plas ma. Hence, we suggest that the fibronectin type IT domain of FXII, in parti cular residues 39 to 47, contribute to the binding site of FXII for negativ ely charged surfaces.