Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ.
Recombinant factor VIII SQ (r-VIII SQ), ReFacto, is a recombinant factor VIII product similar to the smallest active factor VIII protein 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 activity 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 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 calcium 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 4 x 10(6) M(-1)s(-1). Two dissociation rate constants were found, 1 X 10(-2)s(-1) and 4 x 10(-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.
Sandberg
H
H
Biochemistry, Plasma Products, Pharmacia, Stockholm, Sweden. helena.sandberg@eu.pnu.com
Almstedt
A
A
Brandt
J
J
Gray
E
E
Holmquist
L
L
Oswaldsson
U
U
Sebring
S
S
Mikaelsson
M
M
D001665:Binding Sites; D001780:Blood Coagulation Tests; D015153:Blotting, Western; D002942:Circular Dichroism; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D015951:Factor Xa / Q000096:biosynthesis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D020349:Surface Plasmon Resonance; D014841:von Willebrand Factor / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C427184:recombinant factor VIII SQ; D005169:Factor VIII; D015951:Factor Xa