NOVEL FORMS OF B-DOMAIM-DELETED RECOMBINANT FACTOR-VIII MOLECULES - CONSTRUCTION AND BIOCHEMICAL-CHARACTERIZATION

Recombinant molecules similar to the smallest active plasma-derived fa ctor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide ch ain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80+90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor Vm we re designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII. In the type 1 constructs, ei ther Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creat ing a link of 14 amino acids between the C-terminus (Arg740) of the 90 -kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VII I RH, Arg747 was fused to His1646. In the type 3 constructs, the B-dom ain was completely removed or replaced with 1-4 Arg residues. After ex pression in Chinese hamster ovary cells, the type 1 derivatives and th e type 3 derivatives with 0-2 Arg residues inserted were found to be o nly partially processed and contained a large amount of the 170-kDa pr imary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VI II R5 containing three or four extra Arg residues preceding the N-term inus of the 80-kDa chain were processed into the desired 80+90-kDa cha in complexes. The feature common to the most efficiently processed fac tor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like p rotease furin, which is expressed in most types of cells; that is, bas ic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N- and C-termini of the 80-kDa and 90-kDa chains corresponding to those f ound in plasma-derived factor VIII.