Vascular smooth muscle cells potentiate cell surface plasminogen activation by both upa and tpa-dependent mechanisms

Experiments in both normal and transgemc animals indicate that plasminogen activators play a role in the response of the vessel wall to injury, presumably by mediating the degradation of extracellular matrix (ECM) by vascular smooth muscle cells (VSMC) which is necessary to facilitate their migration and proliferation. We have therefore investigated the ability of human VSMC in culture to assemble specific plasminogen activating systems on their cell surface. uPA bound to a single class of binding sites on VSMC with high affinity (Kd = 2 nM), and this binding could be competed by monoclonal antibodies to the uPA receptor (uPAR). Binding of pro-uPA to these cells resulted in a large potentiation of plasmin generation which was dependent on the cellular binding of plasminogen, consistent with our previous observations on leukocytes and various cells of neoplastic origin. Disruption of caveolae on these cells with cholesterol binding agents had no effect on uPAR-mediated plasminogen activation. tPA was also found to bind to VSMC, using a functional assay to ensure that binding to PAI-1 was not being measured. Analysis of the binding isotherms obtained revealed 2 classes of binding site with Kds of 20 and 600 nM. Neither inactivated uPA or plasminogen could compete this binding. Enzyme kinetic analysis demonstrated that although both of these sites mediated plasminogen activation, the large enhancement of tPA activity observed (greater than 50-fold) was solely due to tPA binding to the higher affinity site and required the cellular binding of plasminogen. The tPA domain deletion mutant K2P was found not to interact with the higher affinity binding site. Comparison of the two specific plasminogen activator binding sites revealed that at saturation the binding of tPA led to an approx. 10-fold greater plasmin generation than uPA. This rose to approx. 25-fold after thrombin stimulation of the cells, which increased tPA but not uPA binding. These data demonstrate the occurrence of a novel specific tPA receptor on human VSMC which may be important for the regulation of plasminogen activation and ECM degradation by these cells in various vascular pathologies.