Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies.
Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.
F
F
Rotblat
D P
DP
O'Brien
F J
FJ
O'Brien
A H
AH
Goodall
E G
EG
Tuddenham
D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation; D000939:Epitopes / Q000032:analysis; D005169:Factor VIII / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D013917:Thrombin / Q000502:physiology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005169:Factor VIII; D013917:Thrombin