INSIGHT INTO THE PROFIBRINOLYTIC ACTIVITY OF HEPARIN - EFFECTS ON THEACTIVATION OF PLASMINOGEN MEDIATED BY UROKINASE

The aim of this work was to clarify the role of urokinase-type plasmin ogen activator (uPA) on the profibrinolytic activity of heparin, chemi cally modified heparins [partially: N-desulfated (N-des), N-desulfated N-acetylated (N-des N-ac), O-desulfated (O-des), O/N-desulfated N-ace tylated (O/N-des N-ac)] and heparan sulfate. Binding competition assay s of plasminogen and uPA to heparin-sepharose demonstrated that hepari n bound to both enzymes. Moreover, in the presence of increasing amoun ts of heparin, plasminogen activation mediated by uPA occurred as a be ll-shaped curve, suggesting the formation of a ternary complex. In con trast, all chemically-modified heparins lacked this cofactor activity, although N-des and heparan sulfate partially retained the uPA binding capacity, and O-des partially bound to both plasminogen and uPA. Plas matic euglobulins from mice treated with heparin, as well as with modi fied heparins with uPA binding capacity, presented a 2-fold enhancemen t of 47 kDa lytic band, as assessed by zymographic analysis. Western b lotting analysis anti-uPA (47 kDa) showed that the enhanced uPA activi ty correlated with a true increase in uPA protein levels. These result s suggest that the profibrinolytic activity of heparin mediated by uPA could be caused by an increase in uPA protein levels rather than by a cofactor activity mediated by a formation of ternary complexes.