Human factor VIIIa subunit structure. Reconstruction of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit.
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.
Fay
P J
PJ
Department of Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Haidaris
P J
PJ
Smudzin
T M
TM
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
D046911:Macromolecular Substances; D010446:Peptide Fragments; D012965:Sodium Chloride; D005169:Factor VIII; D013917:Thrombin; D002122:Calcium Chloride