A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage.
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.
Precup
J W
JW
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905.
Kline
B C
BC
Fass
D N
DN
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D000941:Antigens / Q000276:immunology; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005169:Factor VIII / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D008969:Molecular Sequence Data; D010314:Partial Thromboplastin Time; D010446:Peptide Fragments / Q000276:immunology; D010957:Plasmids; D013917:Thrombin / Q000378:metabolism*; D014357:Trypsin; D014841:von Willebrand Factor / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000941:Antigens; D010446:Peptide Fragments; D014841:von Willebrand Factor; D005169:Factor VIII; D014357:Trypsin; D013917:Thrombin