To determine gene expression (CXCL8, VEGFA), 300 000 cells were seeded per 6‐well in regular culture medium. The following day, the medium was aspirated and replaced by serum‐free medium. After 24 h of serum starvation, cells were stimulated with FXa (Haematologic Technologies Inc., Essex Junction, VT) or thrombin (Enzyme Research Laboratories, South Bend, IN) in the presence or absence of rivaroxaban (Alsachim, Ilkirch Graffenstaden, France) or dabigatran (Alsachim). Cell lysate was collected in 1 mL Trisure (Trisure, Bioline, Taunton, MA).
Total RNA of all in vitro and in vivo samples was isolated from Trisure and converted to cDNA using Super Script II using manufacturer's instructions (ThermoFisher Scientific). The qPCRs were performed in a 10‐μL reaction, with SYBRselect master mix (Thermofisher Scientific), 20 ng cDNA template, and 200 nmol/L primers (see TableS1 for primer sequences), in a 384‐well‐qPCR plate on a CFX384 Touch Real‐Time PCR detection system (BioRad, Veenendaal, The Netherlands). After 10 minutes preheating at 95°C, 40 cycles were run of 15 seconds 95°C and 60 seconds 60°C. The C(t)‐values were obtained using a threshold of 150 relative units using BioRad CFX‐software. Technical triplicates were run for every sample.
Total RNA of all in vitro and in vivo samples was isolated from Trisure and converted to cDNA using Super Script II using manufacturer's instructions (ThermoFisher Scientific). The qPCRs were performed in a 10‐μL reaction, with SYBRselect master mix (Thermofisher Scientific), 20 ng cDNA template, and 200 nmol/L primers (see Table