Thrombin enhances release of tissue plasminogen activator from bovine corneal endothelial cells

The effect of thrombin on release of plasminogen activators (PAs) was studied using cultivated endothelial cells of the bovine cornea. Species of PAs released into the conditioned medium were determined by fibrin autography and immunological analyses. Chromogenic peptide (S-2251) microassay was used for a quantitative estimation of the PA activity in conditioned medium and enzyme-linked immunosorbent assay (ELISA) for tissue plasminogen activator concentration. Fibrin autography revealed that cultured bovine corneal endothelial cells released into the conditioned medium tissue plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA). Addition of increasing concentrations (0.1 to 10.0 U/ml) of thrombin to the confluent cultures led to a dose-dependent increase in the rate of release of t-PA, while there was no significant increase in the release of u-PA. About a 2-fold increase in the t-PA concentration occurred when 10.0 U/ml thrombin was to the confluent cultures for 24 hr. Thrombin induced an increase in the release of t-PA, in a time-dependent manner. The addition of cycloheximide or actinomycin D to the thrombin-treated cultures resulted in a reduction of t-PA levels in the media. These findings indicate that the enhancing effect of thrombin is due to an increase in t-PA production, via protein synthesis. Thrombin inactivated with diisopropylfluorophosphate (DFP) did not induce an increase in t-PA levels. A 100-fold excess of DFP-treated thrombin did not inhibit the thrombin-induced increase. These findings indicate that binding ability and the effect of t-PA release depend on the enzymatically active site of thrombin.