Procoagulant activity of purified human kallikrein in a purified system and in plasma. (A) FIXa-AT complexes generated after incubation of 100 nM purified human FIX with increasing concentrations of purified human kallikrein (red), FXIIa (blue), or plasmin (green) for 1 hour at 37°C, then addition of 2.5 µM purified human AT and 1 U/mL unfractionated heparin. Human FXIa (1.2 nM) was used as positive control (brown). TG in normal pooled (B), XII-deficient (C), XI-deficient (D), or IX-deficient (E) plasma spiked with increasing concentrations of purified human PKa. (F) FIXa-AT complexes formed after incubation for 150 minutes at 37°C of 100 nM purified human FIX with RBC-MVs in the presence (red) or absence (green) of 580 nM PK and 670 nM HK. Kaolin was used as positive control (purple bar). Buffer was used as negative control (B-F; blue). FIXa-AT was measured using an in-house ELISA capturing FIX and detecting AT. Data are expressed as means ± SD of at least 3 independent experiment run in duplicate. **P < .05; ***P < .001.