Summary

Recombinant factor VIII SQ (r-VIII SQ), ReFacto ® , is a recombinantfactor VIII product similar to the smallest active factor VIII pro-teinfound in plasma-derived factor VIII (p-VIII) concentrates. The proteincomprises two polypeptide chains of 80 and 90 kDa and lacks themajor part of the heavily glycosylated B-domain i.e. amino acidsGln744 to Ser1637. r-VIII SQ retains six potential glycosylation sitesfor N-linked oligosaccharides at asparagine residues 41, 239, 582,1685, 1810 and 2118.We describe a thorough comparison of the characteristics of r-VIIISQ with those of p-VIII. The primary and secondary structures of r-VIIISQ were in good agreement with that of B-domain-deleted p-VIII(p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactorVIII antibodies, tryptic mapping, amino acid sequence analysisand circular dichroism spectroscopy. A few divergences also existed.Thus r-VIII SQ was shown to contain a small amount of the singlechain primary translation product of 170 kDa and also the product specificsequence of 14 amino acids, the SQ-link, in the C-terminal end ofthe 90 kDa chain. It was shown that r-VIII SQ had a high specific activ-ityof about 14,000 IU VIII:C/mg as determined by use of a chromogen-icsubstrate assay. The r-VIII SQ protein was comparable to p-VIIIforms with a retained B-domain, in terms of potency measured by achromogenic substrate or a two-stage clotting assay, in interactionswith thrombin, and with activated protein C (APC) in combination withProtein S. The ability of r-VIII SQ to participate as a cofactor in factorXa generation in a mixture of factors IXa and X, phospholipid and cal-ciumwas in conformity with that of p-VIII. Furthermore r-VIII SQ hada good binding capacity for phospholipid vesicles and von Willebrandfactor (vWF) as shown in gel filtration studies. The same kinetics inbinding to von Willebrand factor was found for r-VIII SQ and p-VIII asdetermined by real-time biospecific interaction analysis (BIA) with useof the BIAcore ® instrument. The apparent association rate constant was4x10⁶ M^-1 s^-1 . Two dissociation rate constants were found, 1x10^-2 s^-1and 4x10^-4 s^-1 . The results extend the present knowledge that thefactor VIII B-domain is dispensable for the factor VIII cofactor functionin hemostasis.