An ELISA was set up using polyvinylchloride microtiter plates coated with rabbit anti-UK IgG's and affino-purified goat anti-UK IgG's as second antibody. Detection occurred with rabbit anti-goat IgG antibodies conjugated with alkaline phosphatase. The assay is specific for urokinase (UK) with a detection limit of 100 pg/ml sample. Tissue-type plasminogen activator, up to concentrations of 100 ng/ml, does not interfere. The assay measures the antigen of the inactive zymogen pro-UK, the active enzyme UK and the UK-inhibitor complex with equal efficiency and gives the total UK antigen present, irrespective of its molecular form. Culture media of fibroblasts, endothelial- and kidney cells showed, despite the absence of active UK, antigen levels of 1.2, 23 and 65 ng/ml, respectively. In human plasma the UK concentration was found to be 3.5 +/- 1.4 ng/ml (mean +/- SD, n = 54). The inter- and intra-assay variations were 20% and 6%, respectively.
The specific fibrinolytic properties of both high molecular weight (55 kd) and low molecular weight (30 kd) pro-urokinase from a monkey kidney cell culture were evaluated in a plasma clot lysis system and compared with those of human urokinase. The system was composed of a radiolabelled plasma clot immersed in plasma containing the fibrinolytic agent. On unit base, 55 kd pro-urokinase was approximately 1.5 times more effective in lysing the clot than 30 kd pro-urokinase and equally effective as urokinase. In contrast to urokinase, both pro-urokinase forms induced clot lysis without degrading fibrinogen in the surrounding plasma. However, a considerable activation of the fibrinolytic system in the plasma occurred as a large amount of alpha 2-antiplasmin was consumed, indicating that pro-urokinase was not fully fibrin-specific. Quenching antibodies against tissue-type plasminogen activator (t-PA) added to the plasma clot lysis system retarded but did not prevent pro-urokinase-induced clot lysis. This indicated that not only was t-PA in plasma involved in the activation of pro-urokinase (probably via plasmin), but that an additional mechanism also existed.
Endothelial cells were isolated from arteries and veins obtained from elderly people at autopsy and propagated for 37 to 69 population doublings. The cells secreted tissue-type plasminogen activator (t-PA) and PA inhibitor-1, and, after subculturing, urokinase-type PA (u-PA) antigen. The following differences between endothelial cells from adult arteries and veins were observed: 1) The cells had the potential to be propagated as a healthy monolayer. The diameter of aortic endothelial cells increased after 8 to 19 population doublings, while a homogeneous population of small diameter vena cava cells was retained for 35 population doublings. 2) The amount of secreted t-PA varied. Vena cava cells produced four times more t-PA than aorta cells, and 20-fold more than umbilical artery or vein endothelial cells. The t-PA mRNA content of vena cava cells did not exceed that of aorta cells, but was fourfold greater than that of umbilical cord endothelial cells. 3) The release of u-PA antigen varied. No u-PA antigen was detectable in conditioned medium of primary cultures of human aorta and vena cava endothelial cells or of early passage vena cava cells. After prolonged subculturing, vena cava cells started to secrete u-PA. Endothelial cells from aorta and other adult arteries, however, started secreting u-PA after one to four passages, parallel to the occurrence of enlarged endothelial cells. u-PA was present as a u-PA/inhibitor complex and as a single-chain u-PA. These differences may be developmentally related to their artery or vein origin or may reflect differences acquired during the "life history" of these blood vessels in vivo. Our data suggest that the release of u-PA antigen by human macrovascular endothelial cells can be used as an indicator of cell senescence.
