Human factor xa

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.

All DNA oligos were purchased from Integrated DNA Technologies Inc. Modified RNA oligos were synthesized in house using a Mermade oligo synthesizer followed by HPLC purification. Chemicals were purchased from Sigma without specification. Dabigatran (DAB) and apixaban-COOH (APX) were purchased from AK Scientific Inc. Human alpha thrombin, human factor Xa, and human fibrinogen were purchased from Haematologic Technologies Inc. Recombinant human factor VIII was purified from Kogenate FS (Bayer) and quantified by UV abosorbance. Pooled normal human plasma was purchased from George King Bio-Medical. Human whole blood samples in this work were obtained under Duke IRB protocol (Pro00007265).

prothrombin activation by eryptotic erythrocytes was determined using a previously described assay 30. Briefly, untreated (Control) and pyocyanin‐treated erythrocytes (4.5% haematocrit) incubated for 48 hrs were treated with human factor Xa (2 nM; Haematologic Technologies, Essex Junction, VT, USA), human factor Va (0.2 nM; Haematologic Technologies) and 2 mM CaCl₂ for 3 min. at 37°C. The erythrocytes were then treated with prothrombin (1.4 μM; Haematologic Technologies) for 5 min. and the reaction was stopped by the addition of 10 mM ethylenediaminetetraacetic acid. The samples were then centrifuged (3 min. at 400 × g), diluted fivefold and kinetically evaluated at 405 nm following addition of the chromogenic substrate S2238 (100 μM; Diapharma, West Chester, OH, USA). As a negative control, the coagulation factors and other agents were added to the solution in the absence of erythrocytes.