Kinetics of reciprocal pro-urokinase/plasminogen activation--stimulation by a template formed by the urokinase receptor bound to poly(D-lysine).
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.
Petersen
L C
LC
Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Gentofte, Denmark. lcp@novo.dk
D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D002863:Chromogenic Compounds / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009842:Oligopeptides / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism*; D011107:Polylysine / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D002863:Chromogenic Compounds; D004792:Enzyme Precursors; D005343:Fibrinolytic Agents; D009842:Oligopeptides; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D011107:Polylysine; C018788:valyl-leucyl-lysine 4-nitroanilide; D010958:Plasminogen; D007611:Aprotinin; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; C067551:saruplase