Active Tissue Plasminogen Activator (tPA)

Figure. The binding activity of tPA with PAI1.
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. Besides, Plasminogen Activator Inhibitor 1 (PAI1) has been identified as an interactor of tPA, thus a binding ELISA assay was conducted to detect the interaction of recombinant mouse tPA and recombinant mouse PAI1. Briefly, tPA were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100uL were then transferred to PAI1-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-tPA pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of tPA and PAI1 was shown in Figure 1, and this effect was in a dose dependent manner.