Pro-urokinase: a study of its stability in plasma and of a mechanism for its selective fibrinolytic effect.
Highly purified pro-urokinase (pro-UK) or single-chain urokinase-type plasminogen activator (scu-PA) was treated with diisopropylfluorophosphate (1 mmol/L) to eliminate traces of two-chain UK activity. This preparation was found to retain a low activity against a chromogenic substrate (S2444), equivalent to 0.1% to 0.5% of the activity of its plasmin-activated derivative. Evidence is presented that the intrinsic activity of pro-UK (scu-PA) was sufficient to activate plasminogen on a fibrin plate or in buffer and was far more reactive against Lys-plasminogen than against Glu-plasminogen. The relative resistance of Glu-plasminogen to activation was overcome by the addition of lysine (25 mmol/L) to the reaction mixture. By contrast, in plasma, pro-UK (scu-PA) was stable and nonreactive for greater than 72 hours when incubated (37 degrees C). Pro-UK (scu-PA) did not form sodium dodecyl sulfate-stable inhibitor complexes, whereas complexation occurred rapidly with UK. Only at high concentrations of pro-UK (scu-PA) (greater than or equal to 250 IU/mL) did plasminogen activation in plasma occur. The relative inertness of pro-UK (scu-PA) in plasma, in contrast to its low-grade enzymatic activity in buffer, was attributed to the effect of inhibitors. The addition of EDTA or the removal of divalent cations by dialysis was associated with a lower threshold for nonspecific plasminogen activation by pro-UK (scu-PA) in plasma. Replacement of Ca++ but not other cations restored baseline conditions. In the presence of a clot, fibrin-selective plasminogen activation and clot lysis were triggered. Lysis was accompanied by less than 10% conversion of pro-UK (scu-PA) to two-chain UK, suggesting that the intrinsic activity of pro-UK (scu-PA) itself may have been responsible for fibrinolysis, although a contribution by the small amount of UK generated could not be excluded. Similarly, pro-UK (scu-PA) supported clot lysis for several days in the same plasma before the effect dissipated as a result of degradation to UK. When Glu-plasminogen in plasma was replaced by Lys-plasminogen, or when lysine was added to normal plasma, nonselective plasminogen activation and fibrinogenolysis occurred. It was concluded that under the experimental conditions, the fibrin specificity of pro-UK (scu-PA) can be explained by its selective activation of fibrin-bound plasminogen and is due to the latter's Lys-plasminogen-like conformation.(ABSTRACT TRUNCATED AT 400 WORDS)
Pannell
R
R
Gurewich
V
V
D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000097:blood*; D014568:Urokinase-Type Plasminogen Activator / Q000097:blood* / Q000378:metabolism
D009842:Oligopeptides; D010446:Peptide Fragments; C018998:lysyl-plasminogen; D007531:Isoflurophate; C028190:5-oxo-prolyl-glycyl-arginine-4-nitroanilide; D005337:Fibrin; D010958:Plasminogen; D004492:Edetic Acid; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium