The K2 domain of TFPIα enhances prothrombinase inhibition. (A) FVa_Xa (0.5 nM) was incubated with phospholipid vesicles (20 μM), the thrombin inhibitor DAPA (3 μM), and TFPIα (open circle), TFPIΔK2 (open square), or K3C (filled square). Reactions were initiated by addition of prothrombin (1.4 μM) and FXa (5 nM). After dilution to reduce the effect of DAPA, thrombin was quantified by the rate of cleavage of a chromogenic substrate (0.32 mM). The initial rate of thrombin generation is shown as a percentage of control (mean ± standard deviation; n ≥ 3). Lines represent best-fit inhibition curves. (B-F) Thrombin generation was measured in TFPI-depleted plasma (B-C) or PRP (D-F). Reactions were initiated with a mixture of FXa (0.1 nM) and phospholipid vesicles (4 μM) (B-C) or FXa and collagen (15 µg/mL) (D-F), in the presence of the indicated concentrations of TFPIα (B,D), TFPIΔK2 (C,E), or K3C (F). Shown are the average thrombin generation curves from experiments performed in triplicate using TFPI-depleted plasma (B-C) or using PRP from 4 different donors (D-F).