Fibrin activatable urokinase (FA-UK): A latent form of UK found in urine related to a complex with an inhibitor/fibrin-interacting cofactor

Abstract

In order to investigate the binding of pro-urokinase (pro-UK) in urine to fibrin/Celite, the property which led to its discovery, the effect of fibrin on the plasminogen activator activity of urine was studied. The plasminogen activator activity in urine was found to be consistently about 2-fold higher when measured by fibrin plate assay than by amidolytic substrate (S-2444), when normalized against the UK reference standard. When the amidolytic activity measurement was preceded by incubation of urine with soluble fibrin, a 2-fold increase in amidolytic activity was also found. Fibrin similarly increased plasmin generation in urine enriched with Glu- or Lys-plasminogen as determined by synthetic substrate S-2251. The observed promoting effect was common to several forms of soluble fibrin and was dose dependent, whereas fibrinogen had little effect. The promoting effect of fibrin was not expressed in the presence of pro-UK or two-chain UK (TC-UK) in buffer and therefore was attributed to another constituent of urine. Since the activity was inhibited by antibodies to UK but not to t-PA, it was called fibrin activatable UK (FA-UK). Gel filtration (Sephacryl-200) of urine revealed FA-UK activity in fractions eluting at a molecular weight of ∼ 100K. A 100K band of activity was also consistently seen when concentrated urine was subjected to zymography. Treatment of concentrated urine with hydroxylamine (1 M) eradicated both these activities and was associated with an increase in baseline amidolytic activity in the urine sample indicative of the release of UK from an inhibitor complex. Moreover, passage of urine over insolubilized monoclonal antibody against UK-inhibitor (PAI-3) complexes resulted in loss of FA-UK activity and of the 100K band on the zymogram suggesting that the complex responsible for FA-UK was related to a PAI-3 complex. Since the FA-UK activity appeared to bind to fibrin/Celite, attempts were made to investigate complexation with pro-UK. Unfortunately, due to the instability of pro-UK in urine, no reliable data were obtained. It was concluded that PAI-3 may serve as a fibrin-interacting co-factor of UK, and therefore may play a role in fibrinolysis.