Human factor VIII procoagulant protein. Monoclonal antibodies define precursor-product relationships and functional epitopes.
The human Factor VIII procoagulant protein (VIII:C) purified from commercial Factor VIII concentrate consisted of a polypeptide doublet of 80,000 mol wt, a 92,000-mol wt polypeptide, and additional polypeptides of up to 188,000 mol wt. Thrombin digests contained a doublet of 72,000 mol wt, as well as 54,000- and 44,000-mol wt fragments. Proteolysis studies of purified VIII:C using thrombin and activated protein C have suggested that the 92,000- and 80,000 (or 72,000)-mol wt polypeptides comprise activated VIII:C. We have now used seven monoclonal antibodies raised against purified VIII:C to construct a preliminary epitope map of these VIII:C polypeptides. The specific VIII:C polypeptides with which the monoclonal antibodies reacted were determined by immunoblotting of VIII:C onto nitrocellulose sheets after reduced NaDodSO4-polyacrylamide gel electrophoresis. A minimum of five distinct epitopes were defined by these monoclonal anti-VIII:C antibodies. Identification of polypeptides bearing these epitopes allowed localization of distinct thrombin cleavage sites to the 92,000- and 80,000-mol wt chains, helped define polypeptide chain precursor-product relationships, and suggested that both the 92,000- and 80,000-mol wt polypeptides are necessary for VIII:C function. These data and their interpretation are consistent with the published description of the complete primary structure of VIII:C and its thrombin cleavage products. The 92,000- and 80,000-mol wt chains have been located at the amino- and carboxy-terminal ends of the molecule, respectively.
Fulcher
C A
CA
Roberts
J R
JR
Holland
L Z
LZ
Zimmerman
T S
TS
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000939:Epitopes; D005169:Factor VIII / Q000276:immunology*; D006801:Humans; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008970:Molecular Weight; D011498:Protein Precursors / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D011498:Protein Precursors; D005169:Factor VIII