rHuEPO enhances the production of plasminogen activator inhibitor-1 in cultured endothelial cells

Abstract

The direct effects of recombinant human erythropoietin(rHuEPO) on coagulation and fibrinolysis factors were evaluated in a cultured endothelial cell (EC) system. Confluent quiescent ECs were incubated with or without 5.0 U/ml rHuEPO for 1, 6, and 18 hours, and supernatant concentrations of plasminogen activator inhibitor-1 (PAI-1):antigen (Ag), tissue plasminogen activator at and thrombomodulin, and supernatant activities of tissue factor pathway inhibitor and von Willebrand factor were measured. The results showed that only PAI-1 levels were increased by the presence of rHuEPO. In order to assess the effect of rHuEPO on PAI-1 production by EC more precisely, confluent ECs were incubated with various doses of rHuEPO (0, 1.0, 2.5, 5.0, 10.0 U/ml) for 1, 6, 12, and 18 hours, and PAI-1:Ag concentrations in the supernatants of media were measured, PAI-1:Ag in the supernatants were increased by the presence of rHuEPO at all incubation times (P < 0.01) and the increase in PAI-1:Ag was dependent on rHuEPO concentration. The increases in PAI-1:Ag by 5.0 U/ml rHuEPO were comparable to those by 0.1 U/ml tumor necrosis factor-α, 1.0 μg/ml lipopolysaccharide, and 0.5 U/ml thrombin. The increase in PAI-1:Ag by rHuEPO was suppressed by pre-incubation with 10 μg/ml cycloheximide (P < 0.01) or 0.2 μg/ml actinomycin D (P < 0.01). These results indicate that rHuEPO directly stimulates PAI-1 production in cultured EC via de novo protein and RNA and syntheses.