Thrombin generation assay

Thrombin generation in platelets was measured in the automated analyzer CEVERON alpha with a TGA module (TECHNOTHROMBIN TGA, DiaPharma) based on monitoring the formation of thrombin by means of a fluorogenic substrate upon activation of the coagulation cascade by tissue factor. Blood was withdrawn in the presence of 6× ACD, and PRP was collected after centrifuging at 200 × g for 10 min. The samples were further centrifuged for 15 min at 1500 × g to collect the PPP as controls. Platelets in PRP were counted, and the concentration was adjusted to 5 × 10⁸/ml. For each measurement, 40 μl of each sample, 10 μl of TGA trigger B16F10 tumor cells (1 × 10⁷/ml), and agonists were mixed in 96-well plates in duplicate. The samples were maintained at 33 °C. Once 50 μl of TGA substrate was added into each well, measurements were obtained at 30 s intervals for 90 min. Thrombin generation was calculated by the manufacturer’s software utilizing a standard thrombin calibration curve that was derived separately.