Purification and characterization of factor VII inhibitor found in a patient with life threatening bleeding.
We recently observed a patient with acquired inhibitor-induced F.VII deficiency whose plasma level of F.VII was < 1.0%. However, the biochemical nature of the inhibitor has not yet been clarified. In the present study, we purified the F.VII inhibitor from the patient's plasma by using activated F.VII (F.VIIa)-conjugated gel and characterized the inhibitor. The results showed that the inhibitor comprised two kinds of antibodies: one was eluted with EDTA (antibody 1) and the other with glycine-HCl buffer (pH 2.3) (antibody 2) from the F.VIIa affinity gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of these inhibitors demonstrated that both antibodies had features of immunoglobulin G1 (IgG1) with kappa and lambda-light chains. Antibody 1 bound to the immobilized F.VIIa with a high affinity in the presence of calcium ion, while antibody 2 bound to the F.VIIa very weakly and the binding was independent of calcium ion. Immunoblotting analysis demonstrated that antibody 1 bound to the light chain of F.VIIa after reduction with 2-mercaptoethanol, while it did not react with either the gamma-carboxyglutamic acid (Gla)-domainless light chain of F.VIIa or the heavy chain with the protease domain. Antibody 1 markedly inhibited the activity of tissue factor-F.VIIa complex. Based on these observations, it is suggested that F.VIIa autoantibody (antibody 1) recognizes the calcium-dependent conformation within or near the Gla domain and inhibits F.VIIa activity by interacting with the light chain.
Kamikubo
Y
Y
The Chemo-Sero-Therapeutic Research Institute, Kyokushi Kikuchi, Kumamoto, Japan. kamikubo@kaketsuken.or.jp
Miyamoto
S
S
Iwasa
A
A
Ishii
M
M
Okajima
K
K
D000906:Antibodies / Q000097:blood / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D005167:Factor VII / Q000276:immunology*; D015942:Factor VIIa / Q000276:immunology*; D006470:Hemorrhage / Q000097:blood / Q000276:immunology*; D006801:Humans
D000906:Antibodies; D005167:Factor VII; D015942:Factor VIIa