Evaluating tPA Activity Inhibition

The commercially available tPA Activity Assay Kit (Abcam, ab108905) was used to assess whether senicapoc and TRAM-34 affect the ability of human tPA to cleave its substrate plasminogen similar to protocols published by Lapchak and Boitano [38 (link)] and Lapchak et al. [39 (link)]. Reactions were run in optically clear flat-bottom 96-well plates (Corning, Catalog #3904). Human plasminogen activator inhibitor-1 (PAI-1; Sigma-Aldrich CC4075) was used as a positive control. Human plasminogen, the chromogenic plasmin substrate, and human tPA were included in the tPA Activity Assay Kit. PBS with 10% human serum was used as assay medium; senicapoc or TRAM-34 was added at 500 nM and 5 mM with a final DMSO concentration of 0.1%. The reactions were pre-incubated at 37 °C for 5 min prior to the addition of the chromogenic plasmin substrate and subsequently placed in a heated SpectraMax M5 spectrophotometer. Absorbances at 405 nm were measured every 30 s for 30 min.