An inhibitory monoclonal antibody to factor X that blocks prothrombin
activation but not prothrombinase enzyme assembly
WR Church, TL Messier, MM Tucker and KG Mann
Department of Biochemistry, University of Vermont, Burlington 05405.
A monoclonal antibody (designated alpha BFX-2b) prepared against bovine
factor X inhibited factor X activity in human, bovine, porcine, rabbit, and
canine plasma. In assays using purified prothrombinase components, factor
Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent
active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5-
pentanediyl)amide, the antibody inhibited the conversion of prothrombin to
thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the
macromolecular substrates prethrombin 1 and prethrombin 2 but did not
inhibit factor Xa hydrolysis of the synthetic substrate benzoyl-
Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the
inactivation of factor Xa by antithrombin III but did not prevent the
inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound
factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant
of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase
activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to
factor Va-phospholipid as measured by using fluorescence polarization or
high-pressure liquid gel chromatography with the fluorescent Factor Xa
analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X
following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels
and transfer to nitrocellulose indicated that the antigenic determinant
recognized by antibody alpha BFX-2b was found on the heavy chain of factors
X and Xa. From these observations it can be concluded that antibody alpha
BFX-2b recognizes a highly conserved epitope on the factor X heavy chain
that is remote from the topographic sites required for prothrombinase
complex assembly and substrate hydrolysis but may be located at or near a
portion of the macromolecular substrate binding site.
Volume 72,
Issue 6,
pp. 1911-1921,
12/01/1988
Copyright © 1988 by The American Society of Hematology
