Figure 2
Figure 2. Activation of FIX by IgG from patients with acquired hemophilia. (A) Cleavage sites for hydrolytic IgG on FIX. Human recombinant FIX (lane 1), IgG purified from the plasma of patients 16 and 32 (lanes 2 and 3), human recombinant activated FIX (lane 4), and FIX incubated in the presence of patients' IgG (lanes 5 and 6) were subjected to 4% to 12% SDS-PAGE. Proteins were stained by colloidal Coomassie blue. (B) Activation of FIX by IgG from 65 patients with acquired hemophilia. FIX (1μM) was incubated alone (FIX) or in the presence of IVIg, IgG from a patient with congenital hemophilia B (HJC), or IgG (67nM) purified from the plasma of 65 patients with FIX-proteolyzing IgG (Table 2) for 24 hours at 37°C. Rates of formation of FIX were calculated based on the ability of the generated activated FIX to activate FX. The data are expressed as millimoles of activated FIX formed per min per mole of IgG. The data represent the means and SDs of 3 individual experiments. Purified IgG neither directly activated FX (data not shown) nor hydrolyzed the chromogenic substrate for activated FX (rates < 10 fmol/min per mole of IgG). (C) IgG-mediated FIX activation correlates with FIX proteolysis. The graph shows the rates of IgG-mediated activation of FIX plotted as a function of the rates of IgG-mediated proteolysis of FIX (Table 2), both expressed in terms of millimoles of activated FIX/min per mole of IgG. The significance of correlation between the 2 parameters was computed using the Spearman rank correlation test. (D) Generation of thrombin by activated FIX in the presence of FVIII. The thrombin generation assay probes the whole intrinsic coagulation cascade from contact activation to the formation of thrombin as well as the inactivation of activated coagulation factors by plasma protease inhibitors. Tissue factor-independent thrombin generation curves were determined in human plasma that is devoid of platelets (PPP). The generation of thrombin was monitored during 120 minutes in FVIII-deficient plasma supplemented with exogenous FVIII at 0%, 3%, 10%, or 30% of the level found in normal plasma and in the presence of 0.3nM of activated FIX. The time to reach the peak of thrombin generation (referred to as time-to-peak) was computed. Results are representative of 2 independent experiments. Indicated percentage values represent adjusted FVIII levels in test plasma. (E) Relevance of IgG-mediated activation of FIX. The 52 acquired hemophilia patients with a documented survival status were divided into 2 groups based on the survival status 1 year after diagnosis. Cumulative average rates of IgG-mediated activation of FIX in deceased patients differed, although not significantly (P = .067), from those of surviving patients, as assessed using a 2-sided unpaired t test followed by Welch correction.

Activation of FIX by IgG from patients with acquired hemophilia. (A) Cleavage sites for hydrolytic IgG on FIX. Human recombinant FIX (lane 1), IgG purified from the plasma of patients 16 and 32 (lanes 2 and 3), human recombinant activated FIX (lane 4), and FIX incubated in the presence of patients' IgG (lanes 5 and 6) were subjected to 4% to 12% SDS-PAGE. Proteins were stained by colloidal Coomassie blue. (B) Activation of FIX by IgG from 65 patients with acquired hemophilia. FIX (1μM) was incubated alone (FIX) or in the presence of IVIg, IgG from a patient with congenital hemophilia B (HJC), or IgG (67nM) purified from the plasma of 65 patients with FIX-proteolyzing IgG (Table 2) for 24 hours at 37°C. Rates of formation of FIX were calculated based on the ability of the generated activated FIX to activate FX. The data are expressed as millimoles of activated FIX formed per min per mole of IgG. The data represent the means and SDs of 3 individual experiments. Purified IgG neither directly activated FX (data not shown) nor hydrolyzed the chromogenic substrate for activated FX (rates < 10 fmol/min per mole of IgG). (C) IgG-mediated FIX activation correlates with FIX proteolysis. The graph shows the rates of IgG-mediated activation of FIX plotted as a function of the rates of IgG-mediated proteolysis of FIX (Table 2), both expressed in terms of millimoles of activated FIX/min per mole of IgG. The significance of correlation between the 2 parameters was computed using the Spearman rank correlation test. (D) Generation of thrombin by activated FIX in the presence of FVIII. The thrombin generation assay probes the whole intrinsic coagulation cascade from contact activation to the formation of thrombin as well as the inactivation of activated coagulation factors by plasma protease inhibitors. Tissue factor-independent thrombin generation curves were determined in human plasma that is devoid of platelets (PPP). The generation of thrombin was monitored during 120 minutes in FVIII-deficient plasma supplemented with exogenous FVIII at 0%, 3%, 10%, or 30% of the level found in normal plasma and in the presence of 0.3nM of activated FIX. The time to reach the peak of thrombin generation (referred to as time-to-peak) was computed. Results are representative of 2 independent experiments. Indicated percentage values represent adjusted FVIII levels in test plasma. (E) Relevance of IgG-mediated activation of FIX. The 52 acquired hemophilia patients with a documented survival status were divided into 2 groups based on the survival status 1 year after diagnosis. Cumulative average rates of IgG-mediated activation of FIX in deceased patients differed, although not significantly (P = .067), from those of surviving patients, as assessed using a 2-sided unpaired t test followed by Welch correction.

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