Effect of PZ on the inhibition of factor Xa by antithrombin III. Reaction mixtures containing factor Xa (5 nM), CaCl₂ (4 mM), with or without PZ (40 nM), and with or without cephalin (15 μM) were incubated 5 min at 22°C before the addition of antithrombin III (3.4 μM). At the indicated times thereafter, samples were removed, diluted in HSA with 1 mM EDTA, and assayed for factor Xa activity by coagulation assay. (▴), Without cephalin, without PZ; (○), without cephalin, with PZ; (■), with cephalin, without PZ; (•), with cephalin, with PZ.

SDS/PAGE of purified ZPI. ZPI (5 μg) was analyzed with (lane 2) or without (lane 1) reduction with 5% 2-mercaptoethanol. Protein was stained with Coomassie brilliant blue. The position of molecular mass standards in kDa is shown on the left.

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.

Effect of PZ and anti-ZPI antibodies on factor Xa inhibition in serum. Factor Xa (5 nM), CaCl₂ (4 mM), and cephalin (15 μM) with or without PZ (40 nM) were incubated 5 min at 22°C before the addition of barium-adsorbed serum (25%, vol/vol), which had been treated previously for 30 min with rabbit preimmune or immune anti-ZPI IgG (300 μg/ml). At the specified times thereafter, samples of the reaction mixtures were diluted in HSA with 1 mM EDTA and assayed for factor Xa activity by coagulation assay. (•), With PZ and preimmune IgG; (▾), with PZ and anti-ZPI IgG; (○), without PZ and with preimmune IgG; (▴), without PZ and with anti-ZPI IgG.

Effect of anti-ZPI antibodies on factor Xa-induced coagulation of plasma. Reaction mixtures (200 μl) containing factor Xa (0.125 nM), CaCl₂ (5 mM), and cephalin (18.75 μM), with or without PZ (50 nM), were incubated in the sample cup of a fibrometer. After 2 min at 37°C, 50 μl of factor X-deficient plasma that had been treated with rabbit preimmune IgG (600 μg/ml) or various concentrations of immune anti-ZPI IgG for 30 min was added and the clotting time was measured. Apparent factor Xa inhibition (76%) produced by the inclusion of PZ during the preincubation period and using plasma treated with preimmune IgG is listed as maximal PZ-dependent inhibition (100% on the ordinate). The concentration of anti-ZPI IgG used to treat the plasma is listed on the abscissa. Note that the range of “% Maximal Inhibition” listed on the ordinate is plotted from 40 to 100.