Valyl-leucyl-lysyl-4-aminomethylcoumarin

Urokinase can form a tripartite complex binding urokinase receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1), a component of the extracellular matrix (ECM). The components of the tripartite complex are modulated throughout the in vitro myogenic differentiation process. A series of experiments aimed at elucidating the role of the urokinase tripartite complex in the fusion of human myogenic cells were performed in vitro. Myogenic cell fusion was associated with increased cell-associated urokinase-type plasminogen activator (uPA) activity, cell-associated uPAR, and uPAR occupancy. Incubation of cultures with either uPA anticatalytic antibodies, or the amino-terminal fragment of uPA (ATF), which inhibits competitively uPA binding to its receptor, or anti-PAI-1 antibodies, which inhibit uPA binding to PAI-1, resulted in a 30 to 47% decrease in fusion. Incubation of cultures with the plasmin inhibitor aprotinin did not affect fusion. Decreased fusion rates induced by interfering with uPAR/uPA/PAI-1 interactions were not associated with significant changes in mRNA levels of both the myogenic regulatory factor myogenin and its inhibitor of DNA binding, Id. Incubation of cultures with purified uPA resulted in a decrease in fusion, likely due to a competitive inhibition of PAI-1 binding of endogenous uPA. We conclude that muscle cell fusion largely depends on interactions between the members of the urokinase complex (uPAR/uPA/PAI-1), but does not require proteolytic activation of plasmin. Since the intrinsic muscle cell differentiation program appears poorly affected by the state of integrity of the urokinase complex, and since cell migration is a prerequisite for muscle cell fusion in vitro, it is likely that the urokinase system is instrumental in fusion through its connection with the cell migration process. Our results suggest that the urokinase tripartite complex may be involved in cell migration in a non conventional way, playing the role of an adhesion system bridging cell membrane to ECM.

The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.

The two zymogens, plasminogen and pro-urokinase plasminogen activator (pro-uPA), constitute a system of reciprocal activation, since plasmin, generated by uPA-catalysed plasminogen activation, can activate pro-uPA to uPA. Two such zymogens, when mixed, will undergo autocatalytic, reciprocal activation resulting in generation of proteolytic activity. As an example of reciprocal zymogen activation, the plasminogen/pro-uPA system was analysed in terms of a kinetic model which describes the progression in activated enzymes. This model gave a detailed description of the progress curves in plasmin and uPA. It accounted for the effects of varying the concentration of the zymogens, and also for the effects of plasmin substrates and inhibitors in the reaction mixture. The model assumes non-significant zymogen activity. It did not, however, exclude that a very low initial proteolytic activity, accounting for maximally 0.01% of that obtained when pro-uPA is fully activated, could be attributed to a genuine pro-uPA activity. Binding of the uPA receptor (uPAR) to pro-uPA/uPA might affect separate steps of the reciprocal activation reaction, or it might induce a significant pro-uPA activity. To distinguish between these possibilities the effect of a recombinant soluble (residues 1-277) form of uPAR, uPAR-(1-277)-peptide, on reciprocal pro-uPA/plasminogen activation was studied. uPAR-(1-277)-peptide attenuated reciprocal zymogen activation, and the results suggested that this was due to a decreased accessibility of the pro-uPA/uPAR-(1-277)-peptide complex to activation by plasmin. The uPAR-(1-277)-peptide in the presence of poly(D-lysine) caused a 20-fold enhancement of reciprocal zymogen activation. Kinetic analysis of separate activation steps revealed that this was due to a threefold stimulation of plasminogen activation by uPA/uPAR-(1-277)-peptide combined with a sixfold stimulation of plasmin's activation of pro-uPA/uPAR-(1-277)-peptide. The results suggested that poly(D-lysine) provided a template for a catalytically favourable interaction between plasminogen/plasmin and the uPAR-(1-277)-peptide complex with pro-uPA/uPA. There was no indication of a significant uPAR-(1-277)-peptide-induced enhancement of pro-uPA activity.