Coagulation assay

The aPTT and PT were determined using a Thrombotimer (Behnk Elektronik, Norderstedt, Germany) as per the manufacturer's instructions and as described previously 19. Briefly, citrated normal human plasma (90 μl) was mixed with 10 μl of heparin or of each compound and was incubated for 1 min. at 37°C. Subsequently, the aPTT assay reagent (100 μl) was added and the plasma sample was incubated for an additional 1 min. at 37°C, followed by the addition of 20 mM CaCl₂ (100 μl). The clotting times were recorded. For the PT assays, citrated normal human plasma (90 μl) was mixed with 10 μl of each compound's stock solution and was incubated for 1 min. at 37°C. The PT assay reagent (200 μl), which had been pre‐incubated for 10 min. at 37°C, was subsequently added and the clotting time was recorded. The PT results were expressed in seconds and as international normalized ratios (INR): INR = (PT sample/PT control)^ISI, where ISI = international sensitivity index. The aPTT results were expressed in seconds. All experimental protocols (KNUH 2012‐01‐010) were approved by the Institutional Review Board of Kyungpook National University Hospitals (Daegu, Republic of Korea).