Effect of purified, soluble urokinase receptor on the plasminogen-prourokinase activation system

AbstractThe extracellular proteolytic pathway mediated by the urokinase plasminogen activator (uPA) is a cascade system, initiated by activation of the zymogen, pro-uPA. Pro-uPA as well as uPA binds to the cellular uPA receptor (uPAR) which has a central function in cell-dependent acceleration of the cascade system. This role of uPAR is generally assumed to be a positioning effect since uPAR-expressing cells exclusively stimulate the activation of cell surface-bound plasminogen (Ellis et al. (1993) Methods Enzymol. 223, 223–233). However, it was recently reported that a recombinant, soluble uPAR (suPAR) was capable of accelerating plasminogen activation in solution (Higazi et al. (1995) J. Biol. Chem. 270, 17375–17380). In this work we show that suPAR as such has no accelerative role. In contrast, the progress of the activation reactions in a soluble system with pro-uPA and plasminogen was found to be attenuated by suPAR. This delay of the activation system was shown to include a partial inhibition of the plasmin-mediated activation of pro-uPA as well as of the uPA-mediated activation of plasminogen

uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion

The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor–associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions

The pro-urokinase plasminogen-activation system in the presence of serpin-type inhibitors and the urokinase receptor:rescue of activity through reciprocal pro-enzyme activation

The reciprocal pro-enzyme activation system of plasmin, urokinase-type plasminogen activator (uPA) and their respective zymogens is a potent mechanism in the generation of extracellular proteolytic activity. Plasminogen activator inhibitor type 1 (PAI-1) acts as a negative regulator. This system is complicated by a poorly understood intrinsic reactivity of the uPA pro-enzyme (pro-uPA) before proteolytic activation, directed against both plasminogen and PAI-1. We have studied the integrated activation mechanism under the repression of PAI-1 in a purified system. A covalent reaction between pro-uPA and PAI-1 was positively demonstrated but the reaction of PAI-1 with two-chain uPA was found to be at least 1000-fold faster. However, in spite of this very fast inhibition, two-chain uPA still became the dominant plasminogen activator when plasminogen was incubated with pro-uPA and PAI-1. The activity pattern observed under these conditions revealed an initial lag phase, followed by a continuous generation of minute amounts of active two-chain uPA, this uPA having a short lifetime before inhibition but still succeeding to generate new plasmin activity, thus preventing a complete inactivation of the feedback system. This property of the activation system was retained even in the simultaneous presence of PAI-1 and alpha(2)-antiplasmin. Addition of soluble uPA receptor to the system did not change the role of pro-uPA and the same pattern was observed when pro-uPA was bound to the uPA receptor on U937 cells. The present mechanism maintains the system at standby level and may be triggered to increased activity without the need for an external initiating event