The Activated Partial thromboplastin time (APTT), Prothrombin Time (PT), Thrombin Time (TT) and Fibrinogen (FIB) Assays in Vitro

Blood samples were drawn from Rabbit’s Auricular Vein (NO: 2015–035). Before collection, the sodium citrate (38 mg/mL, 400 μL) was placed in a 4 mL centrifuge tube to prevent blood clotting. Plasma was then separated from the blood by centrifugation at 3000 rpm 5 °C for 15 min. APTT and PT were determined according to the method described previously [16]. In brief, plasma (100 μL) was mixed with 20 μL of samples, APTT assay reagent (100 μL) was added and incubated for 5 min at 37 °C, and then 25 mM CaCl₂ (100 μL) was added. Clotting times were recorded. For PT assays, plasma (100 μL) was mixed with 20 μL of samples and incubated at 37 °C for 3 min. PT assay reagent (200 μL), which was hatched at 37 °C for 3 min, was then added and clotting time was recorded. TT and FIB were determined according to the manufacturer’s recommendations (Shanghai Sun Biotech Co., Ltd, China).

For all the tests mentioned above, blank solvent (dimethyl sulphoxide: Tween 80: normal saline = 2:1:17) was used as negative control group, while the drugs of breviscapine (13.3 mg/mL) and Vitamin K₁ (5 mg/mL) used in clinical were used as positive control groups. All the samples were dissolved in blank solvent. The concentrations of compounds were 5 mg/mL and all the extract samples were 15 mg/mL. PT, APTT, TT and FIB tests were conducted by Semi-Automated Coagulation Analyzer (CPC Diagnostics Pvt. Ltd, India).