PROUROKINASE - A STUDY OF ITS STABILITY IN PLASMA AND OF A MECHANISM FOR ITS SELECTIVE FIBRINOLYTIC EFFECT

  • 1 May 1986
    • journal article
    • research article
    • Vol. 67  (5) , 1215-1223
Abstract
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 > 72 hours when incubated (37.degree. 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) (.gtoreq. 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 < 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 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 of 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. The free plasminogen in plasma is spared due to its Glu-form as well as to the stability of pro-UK (scu-PA) in the presence of plasma inhibitors and Ca++. This mechanism appears not to require fibrin binding, and clot lysis is accompanied by little activation of pro-UK (scu-PA) to two-chain UK.