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 defici ency whose plasma level of F.VII was < 1.0%. However, the biochemical natur e of the inhibitor has not yet been clarified. In the present study, we pur ified the F.VII inhibitor from the patient's plasma by using activated F.VI I (F.Wa)-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.Wa affinity gel. SDS-polyacrylamide gel electrophoresis (SDS- PAGE) and immunoblotting analysis of these inhibitors demonstrated that bot h antibodies had features of immunoglobulin G1 (IgG1) with kappa and h-ligh t chains. Antibody 1 bound to the immobilized F.VIIa with a high affinity i n the presence of calcium ion, while antibody 2 bound to the F.VIIa very we akly and the binding was independent of calcium ion. Immunoblotting analysi s demonstrated that antibody 1 bound to the light chain of F.VIIa after red uction with 2-mercaptoethanol, while it did not react with either the gamma -carboxyelutamic acid (Gla)-domainless light chain of F.VIIa or the heavy c hain with the protease domain. Antibody markedly inhibited the activity of tissue factor-F.VIIa complex. Based on these observations, it is suggested that F.VIIa autoantibody (antibody I)recognizes the calcium-dependent confo rmation within or near the Gla domain and inhibits F.VIIa activity by inter acting with the light chain.