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J. Biol. Chem., Vol. 267, Issue 21, 14759-14766, 07, 1992
MS Hertzberg, O Ben-Tal, B Furie and BC Furie
The prothrombinase complex, which catalyzes the conversion of prothrombin
to thrombin, consists of activated Factor X, Factor Va, a membrane surface
and Ca2+. To examine the structures that support Factor Va binding to
Factor X, we used in vitro mutagenesis to construct a chimeric molecule
that includes regions of Factor IX and Factor X. This chimera
(IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal
growth factor (EGF) and serine protease domains of Factor X linked
downstream from the cDNA encoding the signal peptide, propeptide, Gla
domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor
IX/X chimera and wild-type Factor X were each expressed in Chinese hamster
ovary cells and the secreted proteins purified by affinity chromatography
using polyclonal anti-Factor X antibodies. The chimera migrated as a single
major band corresponding to a molecular weight of 68,000. By Western
blotting, the chimeric protein stained with both polyclonal anti-Factor X
and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis
demonstrated near complete carboxylation of both the wild-type Factor X and
the Factor IX/X chimera. Compared with Factor X, the rate of zymogen
activation of the Factor IX/X chimera was about 50% that of Factor X when
activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme
form of the Factor IX/X chimera, activated Factor IX/X, generated using the
coagulant protein of Russell's viper venom, expressed full amidolytic
activity compared with Factor Xa. The activated Factor IX/X chimera had
about 14% of the activity of Factor Xa when employed in a prothrombinase
assay; this activity reached 100% with increasing concentrations of Factor
Va. A binding assay was employed to test the ability of the active
site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa
to the Factor Va-phospholipid complex, thus inhibiting the activation of
prothrombin to thrombin. In this assay the active site-inactivated form of
the chimera competed with Factor Xa completely but with decreased affinity
for the Factor Va-phospholipid complex. These data indicate that the second
EGF domain and the serine protease domain of Factor Xa are sufficient to
interact with Factor Va. The Factor IX/X chimera is a good substrate for
the tenase complex; the defective enzymatic activity of the activated
Factor IX/X chimera can be accounted for by its decreased affinity for
Factor Va relative to Factor Xa.
Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding
Center for Hemostasis and Thrombosis Research, Tufts University School of Medicine, Boston, Massachusetts 02111.
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