BO2C11 inhibits fVIIIa. Plasma-derived fVIII (5 IU/mL; 1 μg/mL) was incubated with bovine thrombin (0.1 U/mL) for 30 seconds at 37°C, and the reaction was stopped by the addition of hirudin. fVIIIa was then mixed for 90 seconds at 37°C with factor IXa, factor X, PL, and CaCl₂, in the absence or presence of different concentrations of BO2C11. The final concentration of fVIII in the assay was 1 IU/mL (200 ng/mL). After 90 seconds, the generation of factor Xa was evaluated by the addition of a chromogenic substrate for a further 5 minutes of incubation. OD was read at 405 nm and results were expressed as percentages of OD measured in the absence of BO2C11.