Ross W. Stephens

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.

Production of an active urokinase by leukemia cells: a novel distinction from cell lines of solid tumors

A new screening test is described which enabled rapid determination of the proportion of single-chain and two-chain urokinase produced in the culture supernatants of 18 human cell lines. A clear distinction was found between two groups of cell lines: cells derived from ten solid tumors produced almost exclusively single-chain proenzyme, while the majority of the enzyme found in cultures of eight leukemia cell lines was in the active, two-chain form.

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.

REGULATION OF THE PERICELLULAR ACTIVATION OF PLASMINOGEN AND ITS ROLE IN TISSUE‐DESTRUCTIVE PROCESSES

While it is generally recognized today that cells use specific adhesion proteins such as fibronectin and laminin, both in cell adhesion and migration, it is equally clear that pericelldar proteolysis is critically involved in the invasion of malignant and certain normal cells through the extracellular matrices, both interstitial connective tissue matrices and basement membranes. There is interesting new evidence suggesting that a key component in pericellular proteolysis, i.e. the urokinase-receptor complex, may have a role also in both cell adhesion and cell migration. In addition there is direct evidence that adhesion proteins of pericellular matrix can provide a molecular link between the membrane receptor proteins, known as integrins, and plasminogen.

Tissue-Type Plasminogen Activator in Subretinal Fluid

The plasminogen activation system is quantitatively the major mechanism of extracellular proteolysis. To evaluate its role in retinal detachment, plasmin and plasminogen activators were measured in subretinal fluid (SRF) from 12 eyes of twelve patients. Plasmin was detected in 5 eyes (mean 6.26 micrograms/ml, SD = 3.7 micrograms/ml). Tissue-type plasminogen activator was present in 5 eyes (mean activity 0.33 IU/ml, SD = 0.24 IU/ml) but the activity did not associate with the plasmin activity in all of the SRF samples. Urokinase-type plasminogen activator was not detected in SRF. We conclude that the plasmin system has been activated in SRF in some eyes with retinal detachment with tissue-type plasminogen activator as the predominant activator. Plasmin in SRF may enhance dispersion of pigment epithelial cells into the subretinal space and the vitreous, a phenomenon seen frequently in eyes with retinal detachment.

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.

The Urokinase Plasminogen Activation System in Breast Cancer

Cancer invasion is a complex process in which degradation of the extracellular matrix plays a crucial role. This degradation is accomplished by the concerted action of several proteolytic enzyme systems, including generation of plasmin by the urokinase pathway of plasminogen activation, matrix metalloproteases and other extracellular proteases. Increased expression and secretion of urokinase plasminogen activator (uPA) strongly correlates with the malignant phenotype of many types of cells, and the central role of uPA in tumor invasion is now well established (1–3).