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A2 domain of human recombinant-derived factor VIII is required for
procoagulant activity but not for thrombin cleavage
DD Pittman, M Millenson, K Marquette, K Bauer and RJ Kaufman
Genetics Institute, Cambridge, MA 02140.
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.
Volume 79,
Issue 2,
pp. 389-397,
01/15/1992
Copyright © 1992 by The American Society of Hematology
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