SPECIFIC BINDING OF URINARY-TYPE PLASMINOGEN-ACTIVATOR (U-PA) TO VITRONECTIN AND ITS ROLE IN MEDIATING U-PA-DEPENDENT ADHESION OF U937 CELLS

The present paper described interactions of urinary-type plasminogen a ctivator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as ad hesion studies with uPA-saturated U937 monocytic cells. Kinetic analys es showed that fibronectin and laminin were moderately effective at de creasing activation of plasminogen by u-PA (3-4-fold decrease in k(cat .)/K-m), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in k(cat.)/K-m). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds pr edominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (K-d,K-app. of 97 nM). Limited proteolysis of vitronec tin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49 000), and binding was shown t o occur through the N-terminal fragment of u-PA, N-terminal sequence a nalysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the hepari n-binding region of the vitronectin molecule. Plasminogen activator in hibitor type I did not compete with u-PA for binding to vitronectin, s uggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasmino gen, suggesting these molecules share a common binding site on vitrone ctin. In addition to in vitro studies, experiments were performed to a ssess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cell s to vitronectin was inhibited 66% by excess vitronectin, suggesting t hat direct binding of u-PA to vitronectin is the mechanism by which u- PA-dependent adhesion of U937 cells to vitronectin is mediated.