Proteolytic activity of IgG purified from the plasma of patients with acquired hemophilia. (A-B) IgG-mediated proteolysis of FVIII and FIX. Biotinylated human recombinant FVIII (A, 185nM) or FIX (B, 185nM) was incubated alone for 0 or 24 hours, or in the presence of IgG (67nM) for 24 hours at 37°C. IVIg was used as a source of normal IgG and as a control. Samples were subjected to 10% SDS-PAGE and transferred onto a nitrocellulose membrane, before revelation of biotinylated fragments. Vertical lines between lanes indicate a repositioned gel lane (A). (C) IgG-mediated proteolysis of FVIII versus proteolysis of FIX. The graph shows the rates of IgG-mediated proteolysis of FVIII plotted as a function of the rates of IgG-mediated proteolysis of FIX (values in Table 2). The correlation between the 2 parameters was not significant as computed using the Spearman rank correlation test. (D) Proteolysis of ¹²⁵I-labeled FIX by IgG in the presence of increasing amounts of unlabeled FIX. ¹²⁵I-Labeled FIX (4.55 ng) was incubated for 24 hours with IgG (25 μg/mL) from patient 16 in the presence of increasing concentrations of unlabeled FIX (0-20μM). Proteolysis of FIX was analyzed on a 7.5% SDS-PAGE, and autoradiographs were scanned. Rates of proteolysis of labeled FIX were calculated by densitometric analysis of the 45-kDa protein band that corresponds to activated FIX. Data are the mean of 3 independent experiments (mean ± SEM); ○ indicates empirical data. Curve indicates data fitted to the Michaelis-Menten equation (R = 0.88). (Insets) Reciprocal of the substrate concentration versus that of the velocity (R = 0.99) for the 5 highest concentrations of unlabeled FIX.