Figure 3.

PZ-dependent inhibition of factor Xa by ZPI. (A) ZPI dose/response. Reaction mixtures containing factor Xa (2.5 or 5.0 nM), CaCl₂ (4 mM), cephalin (15 μM), and PZ (40 nM) were incubated with increasing concentrations of ZPI for 15 min at 22°C before remaining factor Xa activity was determined by amidolytic assay. The molar concentration of ZPI was estimated assuming 1.0 mg/ml ZPI produces an A₂₈₀ of 1.0. (□), Factor Xa, 2.5 nM; (○), factor Xa, 5.0 nM. (B) PZ dose/response. Reaction mixtures containing factor Xa (2.5 or 5.0 nM), CaCl₂ (4 mM), cephalin (15 μM), and ZPI (10 nM) were incubated with increasing concentrations of PZ for 15 min at 22°C before remaining factor Xa activity was determined by amidolytic assay. (□), Factor Xa, 2.5 nM; (○), factor Xa, 5.0 nM. (C) Cephalin dose/response. Reaction mixtures containing factor Xa (2.5 nM), CaCl₂ (4 mM), PZ (40 nM), and ZPI (10 nM) were incubated with increasing concentrations of cephalin for 15 min at 22°C before remaining factor Xa activity was determined by amidolytic assay. (D) Time course of factor Xa inhibition by PZ/ZPI. Reaction mixtures containing factor Xa (5.0 nM), with or without CaCl₂ (4 mM), with or without cephalin (15 μM), and with or without PZ (40 nM) were incubated 5 min at 22°C before the addition of ZPI (10 nM). At the specified times thereafter remaining factor Xa activity was determined by coagulation assay or amidolytic assay. Coagulation assay: (•), with all reactants. Amidolytic assay: (■), with all reactants; (▾), without CaCl₂; (▴), without cephalin; (⧫), without PZ.