Time course of plasmin and thrombin cleavage of mutant ₂AP. Mutant ₂AP was assessed for its sensitivity to cleavage by plasmin (A) and thrombin (B) using SDS-PAGE under nonreducing conditions. Mutant ₂AP (3.5 μg) was incubated with 1.2 μg plasmin or 1.2 μg thrombin at 37°C in 15 μL of 40 mmol/L Tris·HCl, pH 7.5 containing 0.15 mol/L NaCl. An aliquot (3 μL) of the incubation mixture was taken at each time point and analyzed on 10% to 20% polyacrylamide gradient gels. Lanes 1 and 2 show molecular weight markers and untreated mutant ₂AP, respectively. Lanes 3 through 7 show samples incubated for 0, 0.5, 1, 4, and 10 minutes, respectively.