Figure 4

Penicillin activates the contact system in an FXII-dependent manner. (A) Penicillin-induced CSA in standard or FXII-deficient human plasma. 100 μL of plasma was pretreated with 100 μL of penicillin at various concentrations (diluted by Tris buffer: 50 mM Tris–HCl, 0.117 M NaCl, pH 7.8) at 37 °C. Ten minutes later, 100 μL of the chromogenic substrate S-2302 (1.5 mg/mL) was added and further incubated at 37 °C for 30 min. The reaction mix was centrifuged at 3,000 × g for 5 min. Supernatant absorbance was monitored at 405 nm. Kaolin was used as a positive control of the contact system. Buffer alone was included as the negative control. *P < 0.05 and **P < 0.01 vs. negative control. (B) Plasma prototypical FXII level decreased after penicillin treatment. Standard human plasma was incubated with or without 4 KU/mL penicillin at 37 °C for 30 min and analyzed for FXII determination by western blotting. Transferrin was used as the internal reference. Full-length blots and the detailed information of the used antibodies were presented in Supplementary file 1. (C) Penicillin induced BK release in PI-HUVEC. HUVEC were incubated with 10% standard human plasma in the presence of 20 μM Zn²⁺ at 37 °C for 1 h. The plasma was removed and the cells were washed twice. PI-HUVEC and non-PI-HUVEC were further incubated with penicillin at 37 °C for 30 min. Supernatant BK was determined by ELISA. **P < 0.01.