Effect of HRPII on the AT-heparin inhibition of FXa. Reaction buffer alone as control or reaction buffer containing indicated combinations of heparin (0.01 U/mL), AT (100nM), and HRPII (300nM) was added to a fixed amount of FXa (5nM). The resulting mixture was incubated for 1 minute at room temperature, and the FXa activity remaining was then determined by amidolytic assay using the chromogenic substrate S-2222 as described in “Amidolytic assays.” Four reaction conditions were used: (A) no divalent cations; (B) 5mM CaCl₂ present; (C) 15μM ZnCl₂ present; and (D) both 5mM CaCl₂ and 15μM ZnCl₂ present. (B,D) The AT-heparin inhibition of FXa is enhanced by Ca²⁺. (E) Same as panel D, except that native HRPII (5nM) was used. Assays were done in triplicate, and data are mean ± SE. Hep indicates heparin; P-HRPII, AT-hep + HRPII.