Active Tissue Plasminogen Activator (tPA)

Plasminogen activators are serine proteases that catalyze the activation of plasmin via proteolytic cleavage of its zymogen form plasminogen. Plasmin is an important factor in fibrinolysis, the breakdown of fibrin polymers formed during blood clotting. There are two main plasminogen activators: urokinase (uPA) and tissue plasminogen activator (tPA). Tissue plasminogen activators (tPA) are used to treat medical conditions related to blood clotting including embolic or thrombotic stroke, myocardial infarction, and pulmonary embolism. The activity assay of tPA was measured by its ability to cleave a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC). The reaction was performed in 50 mM Tris, 0.01% Tween-20, pH 8.5 ( Assay Buffer), ainitiated by addition 50 μL of various concentrations of tPA (diluted by Assay Buffer) to 50 µL of 200 uM Substrate. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes. The specific activity of recombinant human tPA is 10-60 pmol/min/µg.