Structural and functional characterization of Factor VIII-delta II, a new recombinant Factor VIII lacking most of the B-domain.
A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.
Bihoreau
N
N
T.M. Innovation (Centre National de Transfusion Sanguine-Institut Mérieux), Les Ulis, France.
Paolantonacci
P
P
Bardelle
C
C
Fontaine-Aupart
M P
MP
Krishnan
S
S
Yon
J
J
Romet-Lemonne
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin