Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ

Recombinant factor VIII SQ (r-VIII SQ), ReFacto(R), is a recombinant factor VIII product similar to the smallest active factor VIII protein found in p lasma-derived factor VIII (p-WI) concentrates. The protein comprises two po lypeptide chains' of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637, r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparag ine residues 41, 239, 582, 1685, 1810 and 2118. We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown b y SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mappi ng, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amou nt of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific a ctivity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrat e or a two-stage clotting assay, in interactions with thrombin, and with ac tivated protein C (APC) in combination with Protein S. The ability of r-VII I SQ to participate as a cofactor in factor Xa generation in a mixture of f actors IXa and X, phospholipid and calcium was in conformity with that of p -VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid v esicles and von Willebrand factor (VWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by rear-time biospecific interaction analysis (BIA) with use of the BIAcore(R) instrument. The apparent association rate constant was 4 X 10(6) M(-1)s(-1). Two dissociation rate constants were fou nd, 1 x 10(-2)s(-1) and 4 X 10(-4)s(-1). The results extend the present kno wledge that the factor VIII B-domain is dispensable for the factor VIII cof actor function in hemostasis.