Figure 1. Comparison of FVIIa-AT complex formation in vitro and ex vivo conditions.

(A) Blood from wild-type mice was drawn into rivaroxaban as an anticoagulant. Plasma was separated by centrifugation at 6,000×g for 5 min. To a reaction system not containing plasma or blood, a plasma concentration of AT (125 µg/ml) was added to Tris-buffered saline (TBS) containing 1 mg/ml bovine serum albumin (BSA), 5 mM CaCl₂ and 1 mM MgCl₂. rFVIIa was added to all three reaction systems (buffer, plasma and blood) to a final concentration of 1 µg/ml. At various time points, an aliquot was removed from the reaction mixtures and diluted 1∶10 in TBS/BSA containing 10 mM EDTA, and frozen immediately until they were used for the assay. Levels of FVIIa-AT complex was measured in an ELISA as described in Methods (n = 6 to 9). (B) Plasma obtained from HTF and low TF mice was divided into two equal aliquots, and one of the aliquots was subjected to centrifugation 20,000×g for 1 h at 4°C to remove microparticles. rFVIIa (1 µg/ml) was added to both the plasmas, and FVIIa-AT complex generated at 60 min was determined. (C) rFVIIa (1 µg/ml) was incubated for varying times with AT (125 µg/ml) in TBS/BSA buffer containing 5 mM CaCl₂+1 mM MgCl₂ in the presence or absence of heparin (1 U/ml) and relipidated TF (100 pg/ml). FVIIa-AT levels were determined in an ELISA (n = 4). The concentration of FVIIa-AT (ng/ml) reflects ng of FVIIa complexed with AT. *Indicates that the compared values differ in statistically significant manner (P<0.05).