Binding and activation of FXII and HK processing at the surface of Leptospira.
Bacteria were incubated with human plasma in the presence or absence of the contact system inhibitor N-1210 and washed. Bound proteins were eluted from the bacteria and subjected to Western blot analysis using antibodies against FXII (A) and HK (B). Human plasma (lane 7) and human plasma activated with kaolin (lane 8) served as controls. Lanes 1 and 2: virulent L. interrogans serovar Copenhageni. Lanes 3 and 4: culture-attenuated L. interrogans serovar Copenhageni. Lanes 5 and 6: L. biflexa. Experiments were performed in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4 and 6) of N-1210. Lane 9: purified FXIIa. The arrows indicate full length FXII and HK and their respective cleavage products (FXIIa and HKa). The binding experiments were performed twice with similar results. (C) Virulent L. interrogans serovar Copenhageni were incubated with human plasma, washed, and resuspended in buffer. After incubation, bradykinin contents in the supernatants were measured by ELISA. Peptide N-1210 was added to the reaction mixture at 25 μg/mL and 100 μg/mL and the cysteine proteinase inhibitor, E64 (100 μg/mL), was used as control. To determine background levels, bacteria were incubated with plasma kallikrein (PK) deficient plasma or buffer alone. The bars represent the media ± standard deviation of three replicates and are representative of three independent experiments. *P > 0.005 in comparison to Plasma samples.