Ixolaris inhibits FXa generated by LPS-stimulated HUVECs.

A mixture (180 μL) containing FX (200 nM) and Ixolaris (0-4 nM) that have previously incubated at 37°C for 15 minutes was added to confluent LPS-stimulated HUVECs. This was followed by addition of FVIIa (1 nM) to start reactions. After 30 minutes, 100 μL was removed and added to a 96-well plate containing 100 μL S2222 (500 μM) diluted in 50 mM Hepes, 100 mM NaCl, 50 mM EDTA, BSA 0.5%, pH 7.4. Absorbance reading at 405 nm (substrate hydrolysis) was continuously recorded for 1 hour and FXa concentration was estimated using a standard curve, using known concentrations of FXa. Appropriate controls were run in parallel: HUVECs that have not been exposed to LPS, FXa concentration is 68.4 ± 8.7 pM. LPS-exposed HUVECs in the absence of FVIIa, FXa concentration is 78.08 ± 6.59 pM. Data are the mean ± SE of triplicate experiments. Experiments were performed with HUVECs in the second or third passages.