FXII activation by EVs derived from cancer cell lines. (A) Activation of FXII induced by EV from cancer cells (L3.6, H1975, HT29, and U937) and HDFs. (B) FXIIa generation by cancer cell–derived EV. EV-induced S-2302 hydrolysis was estimated at 90 minutes after addition of the indicated amount of EV (determined by protein concentration) and comparison with a FXIIa standard curve (supplemental Figure 3). (C) FXIIa generated by cancer cell–derived EV treated with DNase I (10 U/mL), RNase A (10 U/mL), and CIP (1, 10, and 20 U/mL). (D) Effect of CTI on FXII activation induced by L3.6 EV in NHP and FXII-deficient (FXII-def.) plasma. Hydrolysis of S-2302 in plasma incubated with EV in the presence or absence of CTI was compared. (E) Activation of purified FXII (375 nM) by EV. EVs (20 or 40 µg protein) were incubated in the presence of FXII (375 nM) and S-2302 and A₄₀₅ were monitored for 90 minutes. (F) FXII cleavage in the presence of L3.6 EV was assessed by immunoblotting using the reaction mixture in (D). The FXII heavy chain (HC) and light chain (LC) were detected under reducing conditions using goat anti-human FXII polyclonal antibody. Bars represent means ± SEM. *P < .05, **P < .01, ***P < .001, 2-way ANOVA with multiple comparisons. ns, not significant.