IC₅₀ for HRPII reversal of AT-heparin and HCII-heparin activities. (A) IC₅₀ for HRPII reversal of the AT-heparin inhibition of FXa. Heparin (0.01 U/mL), AT (100nM), and increasing amounts of HRPII (0-300nM) were added to a fixed amount of FXa (5nM) in the presence of zinc acetate (15μM) and CaCl₂ (5mM). The reaction was incubated for 1 minute at room temperature, and the FXa activity remaining was then determined by amidolytic assay using S-2222 as described in “Amidolytic assay.” (B) IC₅₀ for HRPII reversal of the AT-heparin inhibition of thrombin. The assay was performed similarly as described for FXa in panel A. Final concentrations were heparin (0.02 U/mL), AT (100nM), HRPII (0-600nM), thrombin (2.5nM), zinc acetate (15μM), and CaCl₂ (5mM). The thrombin activity remaining was determined by amidolytic assay using S-2238 as described in “Amidolytic assay.” (C) IC₅₀ for HRPII neutralization of the HCII-heparin inhibition of thrombin. Reaction mixtures in 100 μL contained HCII (300nM), heparin (0.05 U/mL), thrombin (2.5nM), CaCl₂ (5mM), zinc acetate (15μM), and various HRPII (0-400nM). After 1 minute incubation at room temperature, the thrombin activity remaining was determined as in panel B. (A-B) Data are the percentage of activity in the absence of AT-heparin. (C) Data are the percentage of activity in the absence of HCII-heparin.