Assessment of direct activation of enzymes of the intrinsic pathway of coagulation by RBC-MVs or sPL vesicles in purified systems. (A) FXIIa activity generated after incubation of MVs (red) with 375 nM purified human FXII for 2 hours at 37°C. (B) Kallikrein activity generated after incubation of MVs with 580 nM purified human PK and 670 nM HK for 2 hours at 37°C. (C) FXIa activity generated after incubation of MVs with 30 nM purified human FXI and 150 nM HK for 2 hours at 37°C. (D) FIXa-AT complexes formed after incubation of MVs with 100 nM purified human FIX for 1 hour at 37°C, then addition of human AT (2.5 µM) and 1 U/mL unfractionated heparin. Enzyme activity was measured using fluorogenic substrate: 10 µg/mL kaolin (A-B), 10 nM FXIIa (C), or 0.5 nM FXIa as the positive control (purple) or buffer (blue) as the negative control (D). Lipid sPL vesicles (green) contained 15% PS, 41% PE, and 44% PC. FIXa-AT was measured with an in-house ELISA, capturing FIX and detecting AT. Data are expressed as the mean ± standard deviation of at least 4 independent experiments run as duplicates. Each experimental condition was compared with buffer using Dunnett’s multiple-comparisons test. *P < .05; ***P < .001.