Abstract
In an earlier study, a site directed mutant rFVIII (rFVIII(m), Arg(336) --> Gln(336)) expressed in baculovirus-insect cell (Sf9) system was found to sustain high level activity during incubation at 37 degrees celsius for 24 h while the cofactor activity of normal plasma was declined steadily. In this study, a mutant B-domain deleted rFVIII(m), Arg(336) --> Gln(336) expressed in baculovirus-insect cell (Sf9) system was characterized for its enzymatic and chemical properties. The expressed rFVIII(m) and plasma FVIII (pFVIII) were purified by immunoaffinity column chromatography and identified by Western blot analysis. The partially purified rFVIII(m) exhibited cofactor specific activity of 2.01 x 10(3)units/mg protein. The molecular weight of rFVIII(m) ranged between 40 to 150 kDa with a major band at 150 kDa. Treatment of both rFVIII(m) and pFVIII with thrombin increased their cofactor activity in a similar pattern. Treatment of both the activated rFVIII(m) and native FVIII with APC decreased their cofactor activities, however, the former exhibited a slower decrease than the latter, although no significant difference was present. rFVIII(m) formed a complex with vWF, resulting in a stabilized form, and the lag period of thrombin-mediated activating was extended by vWF association. These results implicated that rFVIII(m) expressed in baculovirus-insect cell system had a comparable capacity as FVIII cofactor activity and might be a good candidate for the FVIII replacement therapy for hemophilia A patients.
Highlights
Factor VIII (FVIII) is a glycoprotein required for the activation of factor X (FX) by activated factor IX (FIXa) in blood coagulation cascade
An activated protein C (APC)-resistant B-domain deleted recombinant FVIII protein expressed in baculovirusinsect cell system was examined for its biological characteristics
The presently described result indicates that a mutant recombinant blood coagulation factor VIII (rFVIIIm) produced in insect cells are functional they are smaller in size than their counterparts produced in mammalian cells
Summary
Factor VIII (FVIII) is a glycoprotein required for the activation of factor X (FX) by activated factor IX (FIXa) in blood coagulation cascade. For the treatment of hemophilia A, replacement of deficient human blood clotting factor VIII molecules in the form of either plasma or recombinantderived FVIII (pFVIII) preparations is necessary. Inactivation of FVIII proceeds by proteolysis of A1 subunit by activated protein C (APC) which hydrolizes subunit peptides at Arg336 in the heavy chain and at Arg1721 in the light chain (Vehar et al, 1984). Recent observation suggests that cleavage at Arg1719 or Arg1721 in the light chain may be unrelated to FVIII inactivation (Fay, 1993). We prepared a mutant cDNA of FVIII heavy chain by site-directed mutagenesis (G1074 A1074) and substitution of Arg336 with Gln336 for the production of rFVIII with high stability(Oh et al, 1999).
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