The interaction of the calcium- and integrin-binding protein (CIBP) with the coagulation factor VIII

The gene encoding the C-terminal part of A1-domain of human blood coagulati on factor VIII (FVIII), a 110-amino acid fragment from Ala(227) to Arg(336) , namely A1(Delta1-226), was cloned and used as a 'bait' to screen a protei n, which might interact with this region by using the yeast two-hybrid syst em. A gene coding for a related protein of FVIII named calcium- and integri nbinding protein (CIBP) was isolated from the normal human liver cDNA libra ry. The results were confirmed by using the mammalian two-hybrid system and coimmunoprecipitation. The gene coding for CIBP was constructed by polymer ase chain reaction (PCR) and then cotransfected with the B-domain-deleted F VIII gene into mammalian cell lines using the expression vector of FVIII fo r transient or stable expression. The culture supernatant was collected and analyzed both by enzyme-linked immunosorbent assay (ELISA) for FVIII antig en level and by one-stage method for procoagulant activity. Coexpressed wit h CIBP, the antigen level df FVIII in the mammalian cell line baby hamster kidney (BHK) cells increased up to about 170% and its bioactivity rose acco rdingly. (C) 2001 Elsevier Science Ltd. All rights reserved.