S 2251

In order to evaluate the ability of both antigenic extracts to activate plasminogen and promote plasmin generation, a chromogenic assay was carried out according to the methodology described by González-Miguel et al. [19 (link)] and Fernandes et al. [22 (link)] with some modifications. One µg of AsL3C or AsL3ES was incubated in the presence of 1 µg of human plasminogen (Acris Antibodies), 3 µg of the plasmin chromogenic substrate S-2251 (Sigma-Aldrich) and a plasminogen activator [15 ng of tPA (Sigma-Aldrich) or 10 ng of uPA (Sigma-Aldrich)] in a final volume of 100 µl of PBS in multi-well microplates (Corning). In some wells the antigenic extract was replaced by BSA as negative control and in others the presence of plasminogen activators was omitted to study the ability of antigenic extracts to generate plasmin on their own. All the mixtures were maintained at 37 °C for 2 h 30 min and the hydrolysis of the substrate was analysed at 415 nm in a Microplate Absorbance Reader iMark (Bio-Rad). Each sample was analysed in triplicate.

Activation of Tuf bound plasminogen to plasmin was investigated using the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide dihydrochloride (S-2251, Sigma-Aldrich). MaxiSorp 96-well microtiter plates (Nunc) were coated with 100 μl of recombinant proteins or BSA (5 μg/ml) in PBS at 4°C overnight. Wells were blocked with blocking buffer III BSA (AppliChem) for 2 h at RT and after washing with PBS-T, plasminogen (10 μg/ml) was added. Following incubation for 1 h at RT, wells were washed three times with PBS-T and incubated with 96 μl of a reaction mixture containing 50 mM Tris/HCl, pH 7.5, 300 mM NaCl, 0.003% Triton X-100, and 0.3 mg/ml S-2251. Finally, 4 μl of 2.5 μg/ml urokinase plasminogen activator (uPA) were added to activate bound plasminogen to plasmin. Microtiter plates were then incubated at 37°C and absorbance was measured every 30 mins at 405 nm for a period of 18 h. In controls, either plasminogen or uPA were omitted from the reaction mixtures, or plasminogen was added together with 50 mM tranexamic acid.

This assay is an adaptation of the PLG activation assay described in the previous section including incubation with live FhNEJ. FhNEJ were obtained by stimulating the excystment of F. hepatica metacercariae as described (see section “In vitro excystment of F. hepatica metacercariae”) and were left to recover in RPMI-1640 culture media (Thermo Fisher) for three hours at 37 °C in a 5% CO₂ atmosphere after excystment. In every well, 2 μg of PLG (Origene) were diluted in PBS together with 3 μg of S-2251 (Sigma) and 20 FhNEJs in the presence or absence of 15 ng t-PA (Sigma) plus 10 ng u-PA (Sigma). Some wells contained FhNEJ-Teg in place of live FhNEJ as a positive control for plasmin generation. Wells containing PBS only, FhNEJ in PBS or PLG plus t-PA and u-PA (without FhNEJ) were used as negative controls. Microplates were incubated for three hours at 37 °C and substrate cleavage was assessed by measuring absorbance at 405 nm every 30 min in a Multiskan GO spectrophotometer (Thermo Fisher). All the reactions were performed in biological quadruplicate.