title: Proteolysis of factor VIII heavy chain polypeptides in plasma and concentrates.
abstract: Factor VIII heavy chain (FVIII HC) polypeptides have been studied in both normal plasma and FVIII concentrates on exposure to three coagulation proteases. FVIII samples were incubated with labelled affinity-purified anti-FVIII Fab' fragments, immunocomplexes formed were visualized by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and apparent relative molecular masses (Mr) of each band assigned. FVIII HC polypeptides were detected in all types of samples, including plasma, without further purification. Normal plasma contained a range of polypeptides with the largest dominant band at a net apparent Mr of 250-300 kD, and the smallest at 80-90 kD: the bands visualized correspond to the 90-210 kD HC species seen on conventional analysis of purified FVIII. No bands were produced from samples of haemophilic plasma. Treatment of plasma or FVIII concentrate with low concentrations (1 IU/ml) of thrombin removed the 250-300 kD and other intermediate bands, intensified then removed the 80-90 kD polypeptide and produced a band at 40-50 kD. Thrombin-associated rise and fall in FVIII clotting activity by one-stage assay correlated with intensity of the 80-90 kD polypeptide. A polypeptide of Mr 40-50 kD was also produced after incubation with activated factor X: activated factor VII plus thromboplastin had no effect on HC structure. FVIII polypeptides were visualized in prothrombin complex concentrates, with a more degraded profile seen in a deliberately 'activated' product.
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Hertfordshire.
authors.forename: G
authors.initials: G
authors.lastname: Kemball-Cook
authors.forename: S J
authors.initials: SJ
authors.lastname: Edwards
authors.forename: T W
authors.initials: TW
authors.lastname: Barrowcliffe
mesh_terms: D001688:Biological Products / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D006467:Hemophilia A / Q000201:enzymology; D006801:Humans; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001688:Biological Products; D010455:Peptides; D013925:Thromboplastin; D010447:Peptide Hydrolases; D015942:Factor VIIa; D013917:Thrombin; D015951:Factor Xa