Incubation of ¹²⁵I-prothrombin with the autoantibody cleaves prothrombin in the absence of antithrombin III or makes a high-molecular-weight complex in the presence of antithrombin III.

(A) Nonlabeled (1 μM) and ¹²⁵I-labeled prothrombin (10 nM) were incubated with 750 μg/mL normal (▪) or patient IgG (●) in the absence of antithrombin III at 37°C for 0 to 24 hours. Each sample was separated by 10% SDS-PAGE under reducing conditions, followed by autoradiography (inset). Percentage residual prothrombin at each time point was determined by counting the 72-kd band and total radioactivity of each lane. (B) Nonlabeled (1 μM) and¹²⁵I-labeled prothrombin (10 nM) were incubated with 750 μg/mL normal (▪) or patient IgG (●) in the presence of 5 μM antithrombin III at 37°C for 0 to 24 hours. Each sample was separated by 10% SDS-PAGE under reducing conditions, followed by autoradiography (inset). TAT′ generation (asterisk) was determined by counting radioactivity and was expressed as the percentage of total radioactivity of each lane. (C) Prothrombin (1 μM) was incubated with 0 to 500 μg/mL normal (▪) or patient IgG (●) in the presence of 5 μM antithrombin III at 37°C for 3 hours. The amount of TAT′ complex generated in each sample was measured by ELISA for normal TAT.