A monoclonal antibody to factor VIII inhibits von Willebrand factor binding
and thrombin cleavage
JW Precup, BC Kline and DN Fass
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation,
Rochester, MN 55905.
To study the interaction of human factor VIII (FVIII) with its various
ligands, select regions of cDNA encoding FVIII light chain were cloned into
the plasmid expression vector pET3B to overproduce FVIII protein fragments
in the bacterium Escherichia coli. Partially purified FVIII protein
fragments were used to produce monoclonal antibodies. One monoclonal
antibody, 60-B, bound both an FVIII protein fragment (amino acid residues
1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This
antibody prevented FVIII-vWF binding and acted as an inhibitor in both the
activated partial thromboplastin time (APTT) assay and a chromogenic
substrate assay that measured factor Xa generation. The ability of the
antibody to inhibit FVIII activity was diminished in a dose-dependent
fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound
to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino
acid residues 1674 through 1684. The 60-B antibody did not react with a
peptide in which the aspartic acid residue at 1681 (underlined) was changed
to a glycine, which is the amino acid present at this position in porcine
FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human
FVIII showed that the 60-B antibody prevented thrombin cleavage at light
chain residue 1689. The coagulant inhibitory activity of the 60-B antibody
may be due, in part, to the prevention of thrombin activation of FVIII
light chain.
Volume 77,
Issue 9,
pp. 1929-1936,
05/01/1991
Copyright © 1991 by The American Society of Hematology
