Human factor VIII procoagulant protein. Monoclonal antibodies define precursor-product relationships and functional epitopes.
Open Access
- 1 July 1985
- journal article
- research article
- Published by American Society for Clinical Investigation in JCI Insight
- Vol. 76 (1), 117-124
- https://doi.org/10.1172/jci111933
Abstract
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.This publication has 23 references indexed in Scilit:
- Molecular cloning of a cDNA encoding human antihaemophilic factorNature, 1984
- Structure of human factor VIIINature, 1984
- Protein antigens of normal and malignant human cells identified by immunoprecipitation with monoclonal antibodies.Journal of Biological Chemistry, 1980
- Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.The Journal of Immunology, 1980
- [11] Radioiodination of proteins by the use of the chloramine-T methodMethods in Enzymology, 1980
- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, 1979
- Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-SepharoseImmunochemistry, 1978
- D-3-Phosphoglycerate dehydrogenase from chicken liver. I. Purification.Journal of Biological Chemistry, 1978
- Letter: A more uniform measurement of factor VIII inhibitors.1975
- Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin GImmunochemistry, 1971