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Abstract. Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serumcontaining medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation.

The urokinase-type plasminogen activator (uPA) binds to a specific cell-surface receptor, uPAR. On several cell types uPAR is present both in the full-length form and a cleaved form, uPAR(2+3), which is devoid of binding activity. The formation of uPAR(2+3) on cultured U937 cells is either directly or indirectly mediated by uPA itself. In a soluble system, uPA can cleave purified uPAR, but the low efficiency of this reaction has raised doubts as to whether uPA is directly responsible for uPAR cleavage on the cells. We now report that uPA-catalyzed cleavage of uPAR on the cell surface is strongly favored relative to the reaction in solution. The time course of uPA-catalyzed cleavage of cell-bound uPAR was studied using U937 cells stimulated with phorbol 12-myristate 13-acetate. Only 30 min was required for 10 nM uPA to cleave 50% of the cell-bound uPAR. This uPA-catalyzed cleavage reaction was inhibited by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cellbound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2 + 3), purified from U937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occusred at two positions in the hinge region connecting domain 1 and 2, between Arg83-Ah84 and Arg89-Ser90, respectively. The uPA-catalyzed cleavage of uPAR is a new negativefeedback regulation mechanism for cell-surface plasminogen activation. We propose that this mechanism plays a physiological role at specific sites with high local concentrations of uPA, thus adding another step to the complex regulation of this cascade reaction.

We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro-u-PA and plasminogcn with LJ937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated rz51]-u-PA to U937 cells, is directed against the u-PA binding NH,-terminal domain of u-PAR. a well-defined fragment formed by limited chymotrypsin digestion of pm-ified u-PAR. demonstrating the functional independence of the U-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system.

The receptor for urokinase-type plasminogen activator (uPAR) is an integral membrane protein that specifically binds urokinase-type plasminogen activator (uPA) and plays a crucial role in cell surface plasmin generation. We have previously found that transforming growth factor-beta, type 1 (TGF-beta 1), increases uPAR gene transcription in the human lung carcinoma cell line A549 and now report that also epidermal growth factor (EGF) and the tumour promoter phorbol 12-myristate 13-acetate (PMA) cause increased uPAR transcription and that PMA and TGF-beta 1 in addition increase the stability of uPAR mRNA, while EGF has no effect on this parameter. All three compounds also increase the uPAR protein level, as measured by cell-binding experiments with radiolabelled ligand. The increase in uPAR protein level was however considerably lower with all three compounds than the increase in mRNA level, suggesting that they also exert a translational or post-translational control. Accompanying the increase in the number of uPAR molecules there was a proportional decrease in their ligand-binding affinity, the mechanism of which is unknown. Platelet-derived growth factor, basic fibroblast growth factor and cyclic AMP analogues did not induce any change in the uPAR mRNA level in A549 cells. Previous studies have shown that expression of uPA and its type-1 inhibitor is regulated by a variety of cytokines in a cell-specific manner. The present study indicates that cytokines in addition influence cell surface plasminogen activation by regulating uPAR expression.