4.7. Thrombin Generation Assay

Tumor cell-induced thrombin generation was evaluated using a fluorogenic thrombin generation assay. To eliminate the effects of the intrinsic coagulation system, platelet-rich-plasma was supplemented with Corn Trypsin Inhibitor (Santa Cruz Biotechnology). Tumor cells were added to a final concentration of 1 × 10⁴ cells/mL. After adding the fluorogenic substrate Technothrombin TGA SUB (Technoclone GmbH, Vienna, Austria) and recalcification, the kinetic of the subsequent thrombin mediated Z-Gly-Gly-Arg-AMC cleavage was measured immediately and fluorescence was converted into thrombin concentrations using the evaluation software provided by Technoclone GmbH.