The main components in plasminogen activation include plasminogen, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), and plasminogen activator inhibitors-1 and -2 (PAI-1, PAI-2). These components are subject to extensive regulation and interactions with for example, pericellular adhesion molecules. Although uPA and tPA are quite similar in structure and have common inhibitors and physiological substrates, their physiological roles are distinct. Traditionally, the role of tPA has been in fibrinolysis and that of uPA in cell migration, especially in cancer cells. Recently several targets for tPA/plasmin have been found in neuronal tissues. The functional role of the PAIs is no longer simply to inhibit overexpressed plasminogen activators, and PAI-2 has an unidentified role in the regulation of cell death.
Pro-urokinase (pro-uPA) and activated uPA are confined to focal adhesions and cell-cell contacts. We studied the distribution of the uPA receptor (uPAR) on human fibroblasts (HES) and rhabdomyosarcoma (RD) cells by immunofluorescence and immunoelectron microscopy. Two monoclonal antibodies (MAb) utilized were against uPAR: MAb R4, which reacts with occupied and unoccupied uPAR, was concentrated at focal adhesions; MAb R3 reacting with unoccupied receptor stained cell surfaces diffusely. MAb R4 stained cell-cell contacts, tips of microspikes, and co-localized with vinculin. Of the matrix and integrin components tested, alpha v beta 3 integrin was found at focal adhesions but more centrally than uPAR. Since uPAR is anchored to the plasma membrane through a GPI lipid, we studied its mobility by antibody-induced clustering. This revealed that unoccupied uPAR was relatively mobile; MAb R3 redistributed it to clusters. In contrast, uPAR R4 and uPA antibodies at the focal contact sites remained mostly within focal contacts. Addition of exogenous uPA resulted in loss of R3 staining and increase of uPA in focal adhesions. These results suggest that occupancy of the receptor with uPA is associated with localization to cell contact sites and restricted lateral mobility.
We have examined the mononuclear cell fraction from 35 individuals, 18 with hematologic malignancies and 17 healthy controls for the presence of cell surface-associated plasminogen activator (PA) activity. PA activity was found on the cell surface of 10 out of 12 samples from patients with acute leukemia. In addition to active urokinase (uPA) found on the cell surface in four out of five acute myeloid leukemia patients, tissue-type PA activity was detected in the same samples (3 of 5). Two out of four samples from acute lymphoid leukemia displayed only uPA activity and three out of three samples from biphenotypic leukemia were also clearly uPA-positive. Plasmin activity was not detected in any of the samples. PA activity was not found on the surface of mononuclear cells from either patients with chronic lymphoid leukemia or healthy controls and, in this respect, the cell surface- bound uPA activity behaved as a marker for acute leukemia. The finding of PA activity on the cell surface in acute leukemia suggests that there may be continuous generation of plasmin with consequent consumption of plasma plasmin inhibitors.
Recently we have shown that heparin and related sulfated polyanions are low‐affinity ligands of the kringle domain in the amino‐terminal region (ATF) of human urokinase (u‐PA), and proposed that this may facilitate loading of u‐PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin‐nidogen) for u‐PA binding, and found that laminin‐nidogen is also a ligand of the u‐PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin‐nidogen showed that there are u‐PA binding sites in fragment E4 of laminin as well as in nidogen. The long‐arm terminal domain of laminin (fragment E3), which contains a heparin‐binding site, competed for binding of u‐PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u‐PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy‐terminal domain. Direct binding assays confirmed that u‐PA binds to nidogen through a site in the u‐PA ATF. We conclude that u‐PA binds to laminin‐nidogen by interactions involving the ATF region of u‐PA, the E4 domain of laminin and the rod or amino‐terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u‐PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.
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