Apixaban

Activation of protein C variants was monitored using a discontinuous assay under pseudo-first order conditions where the concentration of substrate was maintained below the K_m value. Reactions initiated by thrombin (1–150 nM) were measured in the presence of 10 mM CaCl₂ with or without rabbit thrombomodulin (50–200 nM) or in the presence of 5 mM EDTA. The factor Xa assays were conducted in the presence of 200 μM phospholipids (75% phosphatidylcholine and 25% phosphatidylserine) and included 250 nM hirudin in order to exclude any activity from possible contamination with thrombin. Reactions with thrombin were stopped at specific time intervals with excess hirudin, while those with factor Xa were quenched with the specific inhibitor apixaban (MedChemExpress). Formation of activated protein C at given time intervals was quantified from the cleavage of the chromogenic substrate S-2366 (Diapharma) by monitoring the absorbance at 405 nm. The k_cat/K_m value was obtained after fitting the initial velocities to an exponential equation. Assays were performed at least in duplicates with standard errors lower that 5%. All measurements were conducted under experimental conditions: 20 mM Tris, pH 7.5, 145 mM NaCl, 0.1% PEG 8,000 at 37 °C. Thrombin was purified and activated as described previously^{29 (link)}. Human factor Xa was purchased from Haematologic Technologies Inc.

Apixaban, betrixaban, rivaroxaban, edoxaban, and ²H₄-rivaroxaban were purchased from MedChemExpress (Monmouth Junction, NJ, USA). ²H₃-Apixaban, ²H₆-edoxaban, and ²H₆-betrixaban were obtained from SHANGHAI ZZBIO CO., LTD (Shanghai, China). HPLC-grade methanol and acetonitrile were purchased from Fisher, USA. Ammonium acetate (A.R. grade), ammonium formate (A.R. grade), and ammonium hydroxide (A.R. grade) were bought from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Sigma-Aldrich Corp. (St. Louis, MO, USA) supplied the formic acid (A.R. grade) and dimethyl sulfoxide (HPLC-grade). Deionized water was purified with a Milli-Q^® Ultrapure water system (Millipore Corporation, Bedford, MA, USA).