Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII

• Published in: Experimental & molecular medicine 1999-06, Vol.31 (2), p.95-100
• Main Authors: Oh, S H, Lee, M Y, Song, D W
• Format: Article
• Language: English
• Subjects: Animals; Baculoviridae - genetics; Blotting, Western; Cell Line; Factor VIII - biosynthesis; Factor VIII - chemistry; Factor VIII - genetics; Factor VIII - metabolism; Genetic Vectors; Humans; Mutagenesis, Site-Directed; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spodoptera
• ISSN: 1226-3613; 2092-6413; 2092-6413
• DOI: 10.1038/emm.1999.16

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.