Human Factor VIII coagulant protein (FVIII:C) has been prepared in milligram amounts from commercial FVIII concentrate by immunoadsorbent chromatography. Although this FVIII:C was partially proteolyzed, it was used to identify an amino terminal Msubr 92,000 and a carboxyl terminal Msubr 72,000 polypeptide and provide evidence for their functional importance. More recently, native, unproteolyzed FVIII:C has been partically purified on an analytical scale. Nine monoclonal antibodies have been raised against purified FVIII:C and used in Western blotting to construct a preliminary epitope map of FVIII:C: three of these antibodies reacted with the amino terminal Msubr 54,000 thrombin-derived fragment of the Msubr 92,000 chain; one reacted with the Msubr 44,000 thrombin fragment of the Msubr 92,000 chain; two had epitopes in the middle section of the molecule; and three had epitopes on a short peptide segment adjacent to the carboxyl terminal Msubr 72,000 chain. Two of these antibodies, one against the Msubr 54,000 and one against the segment adjacent to the Msubr 72,000 chain were strong neutralizers of FVIII:C activity. Similar Western blotting experiments using three inhibitor plasmas have been performed. Two inhibitors react with the Msubr 72,000 carboxyl terminal FVIII:C chain. The other inhibitor reacts with the Msubr 44,000 thrombin fragment and the Msubr 72,000 chain. The goals of this project are: determination of the reactivity of inhibitors from individuals with hemophilia A with purified FVIII:C and thrombin-degraded purified FVIII:C, using Western blotting and HPLC peptide separation; further localization of inhibitor-reactive regions of FVIII:C by proteolytic or chemical cleavage of FVIII:C or its thrombin products; further definition of inhibitor-reactive FVIII:C fragments by competition of inhibitors with monoclonal anti-FVIII:C antibodies or polyclonal anti-FVIII:C peptide antibodies; and use of synthetic peptides of FVIII:C and antibodies to them to define inhibitor binding regions of FVIII:C and their relevance to FVIII:C function.
