Human factor VIII procoagulant protein. Monoclonal antibodies define precursor-product relationships and functional epitopes.

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.

Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine351-serine365 of the factor VIII heavy chain.

“…The purification of FVIII from commercial factor VIII concentrates (Armour Pharmaceutical Co., Kankakee, IL) by immunoadsorbent chromatography has previously been described (5,6,13). FVIII preparations prepared by this method had specific activities of 2,900-3,800 U/mg.…”

“…FVIII purified from plasma by means of immunoaffinity chromatography consists of heterodimers of an 80-kD2 light chain in association with a heavy chain-containing species of 92-210 kD (5)(6)(7)(8)(9)(10). The two chains are presumed to be associated via a calcium linkage (7,(9)(10)(11)(12).…”

“…Thrombin digestion of the 80-kD light chain results in cleavage at Arg'689 to form a carboxyl-terminal 72-kD peptide (4,(6)(7)(8)13). Thrombin digestion of the 92-2 10-kD polypeptides results in cleavage at Arg74 with formation of the 92-kD amino-terminal heavy chain (14) and subsequently cleavage at Arg372 with formation of a 54-kD amino-terminal subunit and a 44-kD carboxyl-terminal subunit (4,(6)(7)(8)13).…”

“…Thrombin digestion of the 92-2 10-kD polypeptides results in cleavage at Arg74 with formation of the 92-kD amino-terminal heavy chain (14) and subsequently cleavage at Arg372 with formation of a 54-kD amino-terminal subunit and a 44-kD carboxyl-terminal subunit (4,(6)(7)(8)13). Further thrombin digestion cleaves the 54-kD peptide into a 30-and a 20-kD peptide (4).…”

“…It has also been demonstrated that alteration in the proposed activated protein C and factor Xa cleavage site at Arg336 results in a FVIII molecule with higher specific activity than normal, possibly due to resistance to proteolytic inactivation (16 (4,(6)(7)(8)10; see also references 15, 36, and 37). For the purpose of discussion in this paper, the molecular FVIII molecule, indicating the heterogeneous nature of the epitopes of these antibodies (6,8,(21)(22)(23)(24)(25).…”