Characterization of recombinant human factor VIII.
Recently, complete human factor VIII DNA clones have been obtained and subsequently expressed in baby hamster kidney cells (Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., and Lawn, R. M. (1984) Nature 312, 330-337). The recombinant factor VIII (rVIII) protein secreted from these cells has now been purified allowing its structural analysis and comparison to plasma-derived factor VIII (pdVIII). Analysis of purified rVIII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with relative mobilities (Mr) ranging from 80,000-210,000. The same pattern of polypeptides is also observed for pdVIII resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins associated with rVIII are recognized by pdVIII antibodies in a Western blot. When rVIII and pdVIII are subjected to isoelectric focusing they are resolved into a similar pattern of protein bands. Thrombin, factor Xa, and activated protein C, which modulate factor VIII activity by proteolysis, process rVIII in the same manner they do pdVIII. As is the case for pdVIII, thrombin activation of rVIII coagulant activity correlates with the generation of subunits with Mr of 73,000, 50,000 and 43,000. These subunits appear to form a metal-(perhaps Ca2+) linked complex. EDTA inactivates thrombin-activated rVIII and pdVIII, with the activity being regenerated after the addition of a molar excess of MnCl2. The results suggest that rVIII is structurally and functionally very similar to pdVIII.
D000818:Animals; D001777:Blood Coagulation; D002460:Cell Line; D006224:Cricetinae; D005169:Factor VIII / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D015944:Factor VIIIa; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa; D006801:Humans; D007159:Immunologic Tests; D007525:Isoelectric Focusing; D007668:Kidney; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011486:Protein C / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
D010446:Peptide Fragments; D011486:Protein C; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005169:Factor VIII; D005170:Factor X; D013917:Thrombin; D015951:Factor Xa