title: Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit.
abstract: Heterodimeric human factor VIII was proteolytically activated by catalytic levels of thrombin to yield the (labile) active cofactor factor VIIIa possessing an initial specific activity of approximately 80 units/microgram. Activation paralleled the generation of fragments A1 and A2 derived from the heavy chain and A3-C1-C2 derived from the light chain. Chromatography of factor VIIIa, on Mono-S buffered at pH 6.0 resulted in separation of the bulk of the A2 fragment from a fraction composed predominantly of A1/A3-C1-C2 dimer plus low levels of A2 fragment. Only the latter fraction contained clotting activity (approximately 20 units/microgram) which was stable and represented a less than 10% yield when compared with the peak activity of unfractionated factor VIIIa. Further depletion of A2 fragment from Mono-S-purified factor VIIIA, achieved using an immobilized monoclonal antibody to the A2 domain, yielded a relatively inactive A1/A3-C1-C2 dimer (less than 0.4 unit/microgram). Factor VIIIa (greater than 40 units/microgram) was reconstituted from the A1/A3-C1-C2 dimer plus the A2 fragment in a reaction that was Me(2+)-independent and inhibited by moderate ionic strength. Reassociation of A2 required the A1 subunit in that the A2 subunit associated weakly if at all to A3-C1-C2 in the absence of A1. These results indicated that human factor VIIIa is a trimer represented by the subunits A1/A2/A3-C1-C2 and that the A2 subunit is required for expression of factor VIIIa activity.
authors.affiliation: Department of Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
authors.forename: P J
authors.initials: PJ
authors.lastname: Haidaris
authors.forename: T M
authors.initials: TM
authors.lastname: Smudzin
mesh_terms: D015153:Blotting, Western; D002122:Calcium Chloride / Q000737:chemistry; D004789:Enzyme Activation; D005169:Factor VIII / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology
chemical_list: D046911:Macromolecular Substances; D010446:Peptide Fragments; D012965:Sodium Chloride; D005169:Factor VIII; D013917:Thrombin; D002122:Calcium Chloride