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Explants of fetal tissues were maintained in organ cultures for 2-3 weeks and the conditioned culture media analysed for the two main plasminogen activators, tissue activator and urokinase. A new immunoradiometric method was used for determining the tissue activator. The method is based on 125I-labelled antibodies to a tissue plasminogen activator which was purified from the culture medium of an established melanoma cell line. It detected tissue activator in a concentration of 1 microgram/1 or even less. Urokinase was measured with a RIA. Explants of kidney as well as thyroid gland and thymus released very substantial amounts of urokinase and smaller amounts of tissue activator. Urokinase was also detected in media conditioned by skin, spleen and pancreas. Aorta explants released only the tissue activator. The capacity of many tissues to release urokinase indicates that this is a more significant plasminogen activator in extrarenal organs that hitherto realized. The tissue activator is probably confined to vascular structures.

125I-labelled urokinase was incubated with plasma and plasminogen free plasma, and the incubation mixtures were analyzed by agarose gel electrophoresis. Autoradiography demonstrated that non-reacted urokinase remained around the slit and that complex-formation with inhibitors altered the migration and resulted in two bands, a major one and a minor one. Crossed immunoelectrophoresis combined with autoradiography showed that the major band contained a complex between alpha 2-antiplasmin and urokinase. The minor band contained a complex between alpha 2-macroglobulin and urokinase. Also di-isopropylfluorophosphate-inactivated urokinase was bound to alpha 2-macroglobulin but not to alpha 2-antiplasmin. Thus, an intact active site of urokinase is not necessary for complex formation with alpha 2-macroglobulin.

An inhibitor present in placenta and released in placental tissue culture forms specific complexes with each of two molecular forms of urokinase. Autoradiography demonstrated that the inhibitor shifted the electrophoretic position of 125I-labelled urokinase. It did not change the migration of diisopropyl-fluorophosphate-inactivated 125I-labelled urokinase, thereby indicating complex formation dependent on active serine site in urokinase. The inhibitor had a strong neutralizing effect on the plasminogen activators released from human ovarian carcinoma in tissue culture. The placental inhibitor might prove useful in inhibiting the fibrinolytic process necessary for proliferation of tumour vessels.