Plasminogen activator from human embryonic kidney cell cultures. Evidence for a proactivator

The nature of the trypsin-activatable plasminogen activator produced by kidney cell cultures (Bernik, M.B (1973), J. Clin. Invest. 52, 823-834) was investigated using human embryonic kidney (HEK) cell cultures in serum-free medium. Plaminogen activator activity ratios (trypsin-activated/ untreated controls) in HEK cell-conditioned media were maximal (up to 3) during the first week of culture and remained nearly constant at approximatley 2 for the next 3-5 weeks, while the total plasminogen activator titer increased in a nearly linear manner. Therefore, coincident with progressive cell degeneration and death, the ratios decreased to near unity due to "spontaneous" activation of the enzyme, which was inhibited in cell-free conditioned media by the pancreatic trypsin inhibitor Kunitz and benzamidine. Since the activator is not inhibited by the trypsin inhibitor, it is concluded that a protease other than the plasminogen activator is responsible for the activation. Increases in the plasminogen activator titers (about 2-fold) were similarly obtained by culturing the cells in medium containing low concentrations (0.05-0.10 mug/ml) of trypsin for up to about 6 weeks. The presence of the trypsin inhibitor in HEK cells cultures decreased the rate of activation, resulting in higher activity ratios (up to 6), and the total plasminogen activator activity was reduced only minimally (less than 20%), if at all, by the highest concentration of the trypsin inhibitor (100 mug/ml) tested. Affinity chromatography of conditioned media with activity ratios of 1.6--2 separated the plasminogen activator into an active fraction and a fraction which was activated a minimum of 200-fold by trypsin and contained no measurable activity prior to activation. Gel filtration of crude conditioned media or partially purified activator separated the plasminogen activator into two peaks; both were trypsin-activatable, and their relative proportions varied with the isolated conditions. The results indicate the occurrence of a proenzyme form of the plasminogen activator in the culture media.