Thrombin cleavage analysis of a novel antihaemophilic factor variant, factor VIII delta II.
Factor VIII delta II is a genetically engineered deletion variant of factor VIII expressed by recombinant Chinese hamster ovary cells, in which a major portion of the central (B) domain and a part of the light chain (Pro771-Asp1666) are missing. After immunoaffinity purification, the kinetics of thrombin cleavage of the novel molecule was analysed by SDS/PAGE, Western blotting and N-terminal amino acid sequencing. Thrombin first cleaves factor VIII delta II at Arg740-Ser741 to generate the 90-kDa heavy chain and an 80-kDa fusion polypeptide consisting of the remaining portion of the B domain and the 73-kDa light chain. The 90-kDa fragment is further cleaved, giving rise to 50-kDa and 40-kDa fragments while the 80-kDa fragment generates a 71/73-kDa doublet. The 71/73-kDa doublet, 50-kDa and 40-kDa fragments were further analysed by N-terminal amino acid sequencing and found to correspond to the predicted amino acid sequences. Our study shows that, in spite of the 900 amino acid deletion present in factor VIII delta II, the essential structural elements required for thrombin activation are conserved.
Krishnan
S
S
TM-Innovation, Les Ulis, France.
Kolbe
H V
HV
Lepage
P
P
Faure
T
T
Sauerwald
R
R
de la Salle
H
H
Muller
C
C
Bihoreau
N
N
Paolantonacci
P
P
Roitsch
C
C
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D005167:Factor VII / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013917:Thrombin / Q000378:metabolism*; D014162:Transfection
D010446:Peptide Fragments; D011994:Recombinant Proteins; C067605:factor VIII delta II; D005167:Factor VII; D005169:Factor VIII; D013917:Thrombin