Figure 8. Effect of Ixolaris on antithrombin-mediated factor Xa inactivation by heparin or sulfated galactan.

(A) Dependence on the concentration of sulfated polysaccharide: Increasing concentrations of heparin (squares) or sulfated galactan (circles) were incubated with 0.5 nM plasma-derived factor Xa and 5 nM antithrombin, in the absence (open symbols) or in the presence (closed symbols) of 10 nM Ixolaris, in TS/PEG buffer containing 10 mM CaCl₂. The total volume of reaction mixtures was 100 μL. After 60 s, the remaining factor Xa activity activity was determined using S-2765 substrate. (B) Dependence on the concentration of Ixolaris: The assays were as described for Panel A, except that increasing concentration of Ixolaris and fixed concentrations of heparin (1 μg/ml) or sulfated galactan (10 μg/ml) were used. (C) Sulfated galactan-affinity chromatography: One mL of plasma-derived factor Xa (100 nM), before (○) or after (●) incubation with 200 nM Ixolaris in TS/PEG buffer, containing 50 mM CaCl₂ was applied to a sulfated galactan-Sepharose column (1 mL), connected to a HPLC system. The column was washed with 5 mL of the TS/PEG buffer and eluted with a linear gradient of 0.15 - 3 M NaCl, at a flow rate of 0.5 mL/min. Fractions of 0.5 mL were collected and checked by factor Xa activity using S-2765 substrate. *p < 0,05 for ■ vs. □ or ○ vs. ●; NS, not significant (p > 0,05).