Thrombin generation assay

Thrombin generation initiated by mononuclear cells was performed as previously described[9]. Briefly, 6×10⁴ mononuclear cells in 80 μl of 1640 medium were added into a TGA cuvette, 100ng/ml LPS or equal volume of PBS was added in and incubated for 4 hours at 37°C [12–14], followed by addition of 40μl of plasma. Thrombin generation was initiated by automatic dispensation of 30μl of fluorescent thrombin substrate containing 100 mM Ca²⁺, and assessed in real time by Ceveron® Alpha TGA (Technoclone, Vienna, Austria). In some experiments, after stimulated by LPS, cells were incubated with 50 μg/ml inhibitory anti-tissue factor antibody 4501, 100μg/ml PDI-wt recombinant protein, or various concentration of PACMA31[15], before addition of plasma and thrombin substrate. The change of fluorescent signal was transformed into thrombin generation curve in real time by the Ceveron® Alpha TGA automatically.