A2 domain of human recombinant-derived factor VIII is required for procoagulant activity but not for thrombin cleavage.
Thrombin treatment of the coagulation factor VIII results in a rapid activation of procoagulant activity with a subsequent first order decay. The structural requirements for thrombin-activated factor VIII were characterized using recombinant-derived human factor VIII and site-directed DNA-mediated mutagenesis. Thrombin-activated human recombinant-derived factor VIII was isolated in an active form by passage over Mono-S fast protein liquid chromatography. The peak fractions had a specific activity of 60,000 U/mg. The subunit composition in the peak fraction contained the 50-Kd A1 domain from the heavy chain, the 73-Kd light chain fragment, and trace amounts of the 43-Kd A2 domain. The requirement of domain A2 for functional activity was shown in several ways. First, the addition of an inhibitory monoclonal antibody that recognizes domain A2 destroyed factor VIIIa activity. Second, addition of a Mono-S FPLC fraction that contained the A2 domain polypeptide back to the peak activity fraction increased activity of the factor VIIIa by 22-fold. The maximum specific activity achieved was 180,000 U/mg. Finally, expression of an A2 domain deletion mutant did not yield procoagulant activity, although the mutant was effectively secreted from the cell, exhibited appropriate heavy and light chain association, and was susceptible to thrombin cleavage. Cotransfection of this A2 domain deletion mutant with an A2 domain expression vector yielded a secreted complex and restored procoagulant activity in the conditioned medium. This result shows that the A2 domain can fold and assemble with A2-deleted factor VIII to yield a functional molecule. We conclude that the A2 domain is required for functional factor VIIIa activity and loss of activity in activated factor VIII may result from dissociation of A2 from the thrombin-activated heterotrimer.
Pittman
D D
DD
Genetics Institute, Cambridge, MA 02140.
Millenson
M
M
Marquette
K
K
Bauer
K
K
Kaufman
R J
RJ
D000911:Antibodies, Monoclonal; D001777:Blood Coagulation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D015944:Factor VIIIa / Q000737:chemistry / Q000502:physiology; D006801:Humans; D007163:Immunosorbent Techniques; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005169:Factor VIII; D013917:Thrombin