Binding of tpa to human vascular smooth muscle cells evidence for a novel, specific receptor

Experiments in both normal and transgenic 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 by vascular smooth muscle cells (VSMC) which is necessary to facilitate their migration and proliferation. As these cells express both uPA and tPA in vivo, we have studied whether they can assemble a tPA-catalysed plasminogen activation system on their surface in addition to the established uPA/uPAR system. tPA was found to bind to human VSMC, using a functional assay i.e. by plasminogen activation. Analysis of the binding isotherms revealed 2 classes of binding site with Kjs of 25 and 660 nM. Enzyme kinetic analysis demonstrated that although both of these sites mediated plasminogen activation, the large enhancement of tPA activity observed (> 100-fold) involved the higher affinity site and required the cellular binding of plasminogen. The binding of tPA was not competed for either by antibodies to annexin II, proposed as an endothdial tPA binding site, or by ligands competing the binding of tPA to a variety of other molecules such as LRP, mannosc receptor or glycosaminoglycam. The specificity of tPA binding was demonstrated, as Ser47s>Ala tPA competed tPA binding by >85% By contrast, PhePro-Arg-chloromethyl ketone (PPACK)-inactivated tPA did not compete the binding of active tPA to VSMC, although it was effective at competing tPA binding to fibrin. As the domain deletion mutant K2P did not bind with high affinity, these data implicate both the serine protease and N-terminal domains of tPA in the binding interaction. Preliminary affinity chromatography experiments have detected a tPAbinding protein of approx. 350 kD in detergent extracts of these cells. Comparison with the uPAR system reveals that tPA binding has the potential to mediate 10-25-fold greater plasmin generation on these cells. 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.