FXIa inhibits the anticoagulant role of platelet TFPI. (A) Supernatant from activated platelets (2.5 × 10⁸) was incubated with increasing concentrations of FXIa for 1 hour. The extent of TFPI present in the supernatant was analyzed by western blotting with a polyclonal anti-TFPI antibody, with levels of fibrinogen serving as a loading control. (B) FXa generation after initiation with tissue factor was determined in the absence or presence of supernatant from activated platelets. In selected experiments, platelet supernatant (3 × 10⁷) was pretreated with 5 nM FXIa for 60 minutes in the presence of 1A6 (20 µg/mL), 10C9 (50 µg/mL), 12F5 (50 µg/mL), or aprotinin (50 µM). In separate experiments, platelet supernatant was incubated with blocking anti-TFPI K1 and K2 antibodies (10 µg/mL) ,or 5 nM FXIa was added immediately before assaying FXa generation (0 minutes). Aprotinin (50 µM) was added to stop the reaction. *P < .05 with respect to vehicle in the presence of supernatant from activated platelets. **P < .05 with respect to vehicle in the presence of supernatant from activated platelets and FXIa. (C) FXa activity was determined in the presence of supernatant from quiescent or activated platelets pretreated with or without 5 nM FXIa or blocking anti-TFPI K1 and K2 antibodies (10 µg/mL). After 1 hour of incubation with FXIa, aprotinin (50 µM) was added to stop the reaction. Data are mean ± standard error (n = 3). *P < .05 with respect to vehicle in the presence of supernatant from activated platelets. Mann-Whitney U test was used for statistical comparisons.