Analysis of inhibition of thrombin generation by PC derivatives in coupled assays. (A) FV (2.5 nM), thrombin (1 nM), protein S (100 nM), sTM (10 nM), PC derivatives (0-100 nM), and PC/PS vesicles (25 μM) incubated in TBS/Ca²⁺ for 1 minute before adding prothrombin (1 μM) and FXa (1 nM) and measuring thrombin generation for 1 minute as described in “Materials and methods.” (B) FVIII (5 nM), thrombin (1 nM), protein S (100 nM), sTM (10 nM), PC derivatives (0-100 nM), and PC/PS vesicles (50 μM) were incubated in TBS/Ca²⁺ for 8 minutes before adding FX (0.2 μM) and factor IXa (2 nM) and measuring the rate of FXa generation for 10 minutes as described in “Materials and methods.” (C) The same as panel A except that thrombin generation was directly started with 0.1 nM FXa. (D) The same as panel A except that the zymogen FX (135 nM) was included in the assay and thrombin generation was initiated with the FVIIa (0.2 nM) in complex with tissue factor (10 pM) as described in “Materials and methods.”