Inhibition of FVIIa/TF amidolytic activity by Ixolaris and DEGR-FXa/Ixolaris.

(A) Ixolaris (0-4.8 μM) was incubated with FVIIa/TF (1 nM) for 15 minutes at 37°C, followed by addition of S2288 (1 mM). Ixolaris:a, 0 μM; b, 0.48 μM; c, 1.6 μM;d, 4.8 μM. (B) Ixolaris (5 nM) was incubated with DEGR-FXa (a) 0 nM; (b) 0.1 nM; (c) 0.25 nM; (d) 0.5 nM; (e) 1 nM; (f) 2.5 nM; (g) 5 nM for 15 minutes at 37°C, followed by addition of FVIIa/TF (1 nM). Fifteen minutes later, S2288 (1 mM) was added. (C) Progress curves depicted in panel B are expressed as Vs/Vo, yielding a IC₅₀ of 0.41 ± 0.04 nM in the presence of DEGR-FXa. DEGR-FXa did not affect FVIIa/TF amidolytic activity in the absence of Ixolaris (straight line). (D) In (●), DEGR-FXa (5 nM) was incubated with Ixolaris (0-5 nM) for 15 minutes, followed by addition of FVIIa/TF (1 nM). Reactions were initiated 15 minutes later by S2288 (1 mM). In (▪), Ixolaris without DEGR-FXa was incubated for 15 minutes with FVIIa/TF (1 nM) and reactions were initiated by S2288 (1 mM). In panels A through D, substrate hydrolysis was continuously recorded at 405 nm for 30 minutes at 37°C.