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Structural and Functional Characteristics of the B-domain-deleted Recombinant Factor VIII Protein, r-VIII SQ

Journal: Thrombosis and Haemostasis
ISSN: 0340-6245
Issue: 2001: 85/1 (Jan) pp.1-190
Pages: 93-100

Structural and Functional Characteristics of the B-domain-deleted Recombinant Factor VIII Protein, r-VIII SQ

Helena Sandberg, Annelie Almstedt, Jörgen Brandt, Eva Gray, Leif Holmquist, Ulla Oswaldsson, Stefan Sebring, Marianne Mikaelsson
Pharmacia, Stockholm, Sweden

Summary

Recombinant factor VIII SQ (r-VIII SQ), ReFacto ® , is a recombinant factor VIII product similar to the smallest active factor VIII pro-tein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide 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 asparagine 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 by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount 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 activ-ity of about 14,000 IU VIII:C/mg as determined by use of a chromogen-ic 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 substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and cal-cium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles 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 real-time biospecific interaction analysis (BIA) with use of the BIAcore ® instrument. The apparent association rate constant was 4x106 M-1 s-1 . Two dissociation rate constants were found, 1x10-2 s-1 and 4x10-4 s-1 . The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.

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