(A). Interaction of leptospires to Fg.

<p>The binding of soluble human Fg (10 µg/mL) to immobilized strains of leptospires (<i>L. interrogans</i> serovar Copenhageni strain Fiocruz L1-130, <i>L. interrogans</i> serovar Copenhageni strain M-20, and <i>L. biflexa</i> serovar Patoc strain Patoc 1) was accessed by ELISA. After 2 h interaction, plates were washed and the detection of bound Fg was verified by incubation with goat anti-Fg (1∶25,000) followed by incubation with HRP-conjugated rabbit anti-goat IgG (1∶50,000). The binding of Fg to each leptospiral strain was performed in triplicate and the data represent the mean ± the standard deviation from one representative experiment. Fg+ and Fg− stand for the presence and absence of Fg, respectively, while in the no-cell control, BSA replaced leptospires. For statistical analyses, the attachment of Fg to leptospiral strains was performed by one-way ANOVA followed by Tukey post-test for pairwise comparison, asterisks above the bars refer to comparison to BSA (no cell control) (**P<0.01). The comparison among leptospiral strains is also shown (#P<0.05 and ## P<0.01). (<b>B</b>) Inhibition of fibrin clot formation by leptospires. 1 mg/mL of Fg plus live leptospires, pre-incubated for 2 h at 37°C, and 10 U/mL of thrombin were placed into ELISA plates. The positive control of the reaction employed Fg (1 mg/mL) plus thrombin (10 U/mL) while in the negative control thrombin was omitted. BSA was used as a negative control of binding. The fibrin clot formation was measured every 1 min for 10 min and then every 5 min for 35 min. Each point was performed in triplicate and expressed as the mean absorbance value at 595_nm ± SD for each point.