Inhibitory effects of antipeptide IgG on APC-catalyzed FVIII inactivation.
(A) FVIII inactivation. FVIII (10 nM) was preincubated with IgG for 1 hour at 37°C, followed by the addition of 5 nM APC, 5 μM PL vesicles, and 2.5 mM CaCl2. At timed intervals, inactivation was immediately stopped by 1 mM PMSF and FVIII activities were measured in a one-stage clotting assay as described in “Materials and methods.” The symbols used are as follows: normal rabbit IgG (0.6 μM), ○; antipeptide IgG (0.6 μM, ●; 0.2 μM, ▪; 0.06 μM, ▴); no APC added, ■. (B) FVIII proteolytic cleavage at Arg336 by APC. The IgG was added to 10 nM FVIII, which was bound to immobilized MoAb JR8 (0.2 μM). Then 5 nM APC, 10 μM PL vesicles, and 2.5 mM CaCl2 were added. At timed intervals, supernatants were removed and APC action was stopped by 2 mM PMSF. FVIII cleavage at Arg336 was detected with peroxidase-conjugated MoAb C5 as described in “Materials and methods.” The symbols used are as follows: normal rabbit IgG (0.6 μM), ○; antipeptide IgG (0.6 μM, ●; 0.2 μM, ▪; 0.06 μM, ▴); no APC added, ■.
