Fibrinolytic system of cultured rabbit aortic endothelial cells

Abstract

The fibrinolytic system of rabbit aortic endothelial cells (RAECs) isolated from male New Zealand White rabbits was investigated. The components of the fibrinolytic system in the conditioned media (CM) were analysed by SDS-PAGE followed by fibrin autography and reverse fibrin autography. The major lytic zones appeared at 54 and 70 KD and minor lytic zones could be detected between 100 and 120 KD. A plasminogen activator inhibitor was localised at 50 KD. Addition of amiloride abolished the lytic zones at 54 KD and 100 KD. There was a time dependent increase in plasminogen activator and inhibitor activities as revealed by an assay involving plasminogen and chromogenic plasmin substrate. A plateau was reached after 16–20 hours. When mRNA obtained from RAECs cultured for 16 hours in serum free medium was analysed for the presence of specific messages of tissue type plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA) and plasminogen activator inhibitor 1 (PAI-1) using human probes, weak specific binding could be seen for both t-PA and u-PA while the PAI-1 probe gave a strong specific signal at 3.4 Kb and a weak signal at 2.3 Kb. Both thrombin (1U/ml) and endotoxin (100 ng/ml) increased the release of PAI-1 activity into CM significantly, while there was no significant change in PAs. The increase of PAI-1 was reflected by increased PAI-1 mRNA levels. The data suggests that rabbit ECs can be used to investigate endothelial cell mediated fibrinolysis and supplement results obtained in investigations employing the rabbit as an in vivo animal model to study the fibrinolytic system.