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Comparison of activated protein C/protein S-mediated inactivation of human
factor VIII and factor V
D Lu, M Kalafatis, KG Mann and GL Long
Department of Biochemistry, College of Medicine, University of Vermont,
Burlington, VT 05405, USA.
The proteolytic cleavage and subsequent inactivation of recombinant human
factor VIII (rhFVIII) and human factor VIIIa (rhFVIIIa) by recombinant
human activated protein C (rAPC) was analyzed in the presence and absence
of human protein S and human factor V (FV). Membrane-bound rhFVIIIa
spontaneously looses most of its initial cofactor activity after 15 minutes
of incubation at pH 7.4. The remaining activity can be eliminated after
incubation with rAPC. Complete inactivation of the membrane-bound rhFVIII
and rhFVIIIa by APC correlates with cleavage at Arg336. The inactivation of
rhFVIII and human plasma FV by rAPC were also compared. Under similar
experimental conditions, complete inactivation of membrane-bound FVIII (60
nmol/L) by rAPC (10 nmol/L) requires 4 hours of incubation, in contrast to
5 minutes for FV (60 nmol/L). The presence of protein S (100 nmol/L)
enhances rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by
twofold, whereas membrane-bound FV showed no protein S dependence during
inactivation. The addition of human FV to the APC/protein S inactivation
mixture increases by approximately twofold the rate of inactivation of
rhFVIII. The effect of FV on the rhFVIII inactivation by APC is protein
S-dependent, because FV alone has no effect on the inactivation rate of
rhFVIII by APC. Western blotting using a monoclonal antibody that
recognizes an epitope between amino acid residues 307 and 506 of human FV
showed that FV was completely cleaved by APC at the beginning of the
rhFVIII inactivation process. These data suggest that FV fragments derived
from the B region of the procofactor after incubation of the membrane-bound
procofactor with APC, but not intact single-chain FV, stimulate APC
activity in the presence of protein S. rhFVIII, FV, and rhFVIIIa were not
inactivated by Glu20-- >Ala-substituted rAPC (rAPCgamma20A), and
membrane-bound factor Va was only partially inactivated. Our data suggest
that (1) FV and FVa are the physiologically significant substrates for APC
inactivation and (2) membranes-bound APC-treated FV is a cofactor for the
APC inactivation of rhFVIII only in the presence of the intact form of
protein S.
Volume 87,
Issue 11,
pp. 4708-4717,
06/01/1996
Copyright © 1996 by The American Society of Hematology
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