Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4198-4206
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
Franca Citarella,
Giorgio Fedele,
Dorina Roem,
Antonio Fantoni, and
C. Erik Hack
From the Dipartimento di Biotecnologie Cellulari ed Ematologia,
Sezione di Genetica Molecolare, Università di Roma "La
Sapienza," Roma, Italy; the Central Laboratory of The Netherlands
Red Cross Blood Transfusion Service, Amsterdam; and the Department of
Internal Medicine, Free University Hospital, Amsterdam, The
Netherlands.
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-
19) deleted of
the amino acid residues 3-19, which are encoded by the second exon of
FXII gene. A plasmid encoding for rFXII-19 was constructed and
expressed in HepG2 cells by using vaccinia virus. Purified rFXII-19
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-19 specific
clotting activity was lower (44%) than that of native FXII. The
activation rate of rFXII-19 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-19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-19 bound to kaolin, which binding was equally well inhibited by either, rFXII-19 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-19 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-19a 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.
