Ixolaris increases the amidolytic activity of Fxa; effects of FX and des-Gla-FXa.

(A) Buffer (control; curve a), Ixolaris (0.8 nM, curve b), or human TFPI (20 nM, curve c) and chromogenic substrate (S2222, 250 μM) were incubated at 37°C for 15 minutes before addition of FXa (125 pM). (B) FXa (125 pM) and Ixolaris (0-1600 pM) were incubated at 37°C for 15 minutes followed by addition of S2222 (250 μM). Substrate hydrolysis was continuously recorded at 405 nm for 1 hour at 37°C. Inset shows progress curves of the effects of Ixolaris on FXa amidolytic activity: Ixolaris (a) 0 nM; (b) 40 pM; (c) 80 pM; (d) 160 pM; (e) 300 pM; (f) 600 pM; and (g) 1600 pM. (C) Ixolaris (1.6 nM) was incubated with FXa (125 pM), des-Gla-FXa (600 pM), or thrombin (150 pM), followed by addition of S2222, or S2238 for thrombin. Substrate hydrolysis was continuously recorded at 405 nm for 1 hour at 37°C. (D) Ixolaris (400 pM) was added to a mixture containing FXa (640 pM) and (▪) DEGR-FXa (0-3.2 nM), or (●) des-Gla-DEGR-FXa (0-3.2 nM), or (▴) DEGR-FX (0-3.2 nM). After a 15-minute incubation, reactions were initiated with S2222 (250 μM). Inset: Ixolaris was incubated with (▪) DEGR-FXa (0-3.2 nM), or (●) des-Gla-DEGR-FXa (0-3.2 nM), or (▴) DEGR-FX (0-3.2 nM), and S2222 (250 μM) for 15 minutes followed by addition of FXa (640 pM) (n = 3). Reactants were diluted in buffer A. Substrate hydrolysis was continuously recorded at 405 nm at 37°C.