Jari Pöllänen

Directed Plasminogen Activation at the Surface of Normal and Malignant Cells

Publisher Summary This chapter discusses the concept of directed cell-surface plasminogen activation. Activation occurring on the solid phase of either the cell surface, in the case of the urokinase-type plasminogen activator (u-PA) pathway, or endothelial cells and the fibrin substrate of thrombi in the tissue-type plasminogen activator pathway (t-PA) has the advantage of escaping from the soluble plasmin inhibitors presented in great abundance in extracellular fluids. Receptor-bound pro-u-PA at focal contacts and the more uniformly distributed vitronectin-bound plasminogen activator inhibitor type 1 (PAI-1) on the pericellular substratum are the key determinants in the generation of directed cell-surface–bound plasmin activity. Given the generalized distribution pattern of plasminogen, it is clear that the u-PA receptor (u-PA-R) has a dominant role in directing proteolytic activity at the critical sites of contacts between cells and substratum. Receptor-bound active u-PA is clearly accessible to inhibition by PAI-1 and plasminogen activator inhibitor type 2 (PAI-2). The mechanism that targets u-PA receptors to the focal contact sites is poorly understood at present and needs further investigation as does the analysis of interactions between the components of the extracellular matrix and the proteolytic machinery.

Microplate immunocapture assay for plasminogen activators and their specific inhibitors

We report a convenient sensitive enzyme activity assay for urokinase and tissue-type plasminogen activators, based on a solid-phase microtitre plate method using readily available polyclonal antibodies. The sensitivities for urokinase (active and proenzyme) and tissue activator were better than 1 ng/ml. The specificity was very high, with no significant contribution of urokinase in tissue activator assays or vice versa. This method is particularly useful for the assay of urokinase proenzyme in samples containing inhibitors. We describe how this assay may also be used to measure specific inhibitors of plasminogen activators, making use of their rapid formation of stable complexes with solid-phase activator. Inhibitors may be assayed in samples containing proenzymes.

Cell Surface Plasminogen Activation

The assumption that the plasminogen activation system, through a breakdown of extracellular matrix proteins, plays a role in invasiveness and destruction of normal tissue during growth of malignant tumors is supported by a variety of findings. These include a close correlation between transformation of cells with oncogenic viruses and synthesis of urokinase-type plasminogen activator (u-PA), the finding that u-PA is involved in tissue destruction in many nonmalig-nant conditions, and the immunohistochemical localization of u-PA in invading areas of tumors (for reviews, see Dan0 et al. 1985; Saksela 1985). Further support for this hypothesis has come from studies with anticatalytic antibodies to u-PA in model systems for invasion and metastasis. Such antibodies were found to decrease metastasis to the lung from a human u-PA-producing tumor, HEp-3, transplanted onto the chorioallantoic membrane of chicken embryos (Ossowski and Reich 1983; Ossowski 1988), penetration of amniotic membranes by B16 melanoma cells (Mignatti et al. 1986), basement membrane invasion by several human and murine cell lines of neoplastic origin (Reich et al. 1988), and formation of lung metastasis after intravenous injection of B16 melanoma cells in mice (Hearing et al. 1988). In some of these studies (Mignatti et al. 1986; Reich et al. 1988), a plasmin-catalyzed activation of procollagenases (see Tryggvason et al. 1987) appeared to be a crucial part of the effect of plasminogen activation.