Reversibility of the factor Xa–ZPI interaction.

Mixtures containing factor Xa (20 nmol/L), phospholipids (25 μmol/L), and CaCl₂ (4 mmol/L), with and without PZ (80 nmol/L), ZPI (56 nmol/L), and factor Va (20 nmol/L), were incubated in HSA at room temperature. After 3 minutes, a one-tenth volume of EDTA (100 mmol/L) or HS buffer was added to certain reactions, and the mixtures were transferred to a 37°C water bath. At various times thereafter, samples were removed for factor Xa assay and Western blot analysis with rabbit anti-ZPI antibodies. (A) Factor Xa activity. ■, factor Xa alone; ▪, factor Xa and factor Va; ●, factor Xa, PZ, and ZPI; ▾, factor Xa, factor Va, PZ, and ZPI; ▿, factor Xa, PZ, ZPI, with EDTA. (B) Anti-ZPI Western blots of reactions containing factor Xa, PZ, and ZPI. (C) Anti-ZPI Western blots of reactions containing factor Xa, factor Va, PZ, and ZPI. (D) Anti-ZPI Western blots of reactions containing factor Xa, PZ, and ZPI with EDTA.