Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.

Plasminogen activators, tissue degradation, and cancer.

The 23S twisted circular form of ColE1 DNA has been isolated from Escherichia coli as a tightly associated DNA-protein complex with a sedimentation coefficient of approximately 24S. Treatment of this complex with pronase, trypsin, sodium dodecyl sulfate, Sarkosyl, or heat results in a conversion to a slower sedimenting form of 17S or 18S, as determined by centrifugation in neutral sucrose gradients. These treatments do not alter the sedimentation properties of noncomplexes supercoiled ColE1 DNA even in the presence of the ColE1-protein complex. Electron microscopic analyses indicate that the decrease in sedimentation rate of the ColE1-protein complex after treatment with these various agents is largely owing to an induced transition of ColE1 DNA from the supercoiled to the open circular state.

https://www.pnas.org/content/pnas/62/4/1159.full.pdf

SUPERCOILED CIRCULAR DNA-PROTEIN COMPLEX IN Escherichia coli: PURIFICATION AND INDUCED CONVERSION TO AN OPEN CIRCULAR DNA FORM

On the regulation and control of fibrinolysis. Edward Kowalski Memorial Lecture.

The secretion of plasminogen activators has been implicated in the controlled extracellular proteolysis that accompanies cell migration and tissue remodeling. We found that the human plasminogen activator urokinase (Uk) (Mr 55,000 form) binds rapidly, specifically, and with high affinity to fresh human blood monocytes and to cells of the monocyte line U937. Upon binding Mr 55,000 Uk was observed to confer high plasminogen activator activity to the cells. Binding of the enzyme did not require a functional catalytic site (located on the B chain of the protein) but did require the noncatalytic A chain of Mr 55,000 Uk, since Mr 33,000 Uk did not bind. These results demonstrate the presence of a membrane receptor for Uk on monocytes and show a hitherto unknown function for the A chain of Uk: binding of secreted enzyme to its receptor results in Uk acting as a membrane protease. This localizes plasminogen activation near the cell surface, an optimal site to facilitate cell migration.

https://rupress.org/jcb/article-pdf/100/1/86/1051168/86.pdf

A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase.

The Regulation of Rat Liver Xanthine Oxidase CONVERSION IN VITRO OF THE ENZYME ACTIVITY FROM DEHYDROGENASE (TYPE D) TO OXIDASE (TYPE O)

The plasminogen activator content of surgically removed lung cancers, as well as that of adjacent normal lung tissue has been determined quantitatively. Optimum conditions for the quantitative extraction have been worked out using a modification of the technique described by Nagy et al. (Int. J. Cancer, 19: 614–620, 1977) which utilizes a buffered solution of the non-ionic detergent Triton X-100. Plasminogen activator was significantly elevated in the tumors (2.5- to 4.3-fold over the normal lung, depending on sample selection and other criteria), although individual variations between tumors were extremely large. No significant correlation was found between the histopathological character of the tumors and the activator content, or between invasiveness and activator content. Using rabbit antibody formed against purified urokinase as an inhibitor of activator action, it was found that lung tumors contain a urokinase-like enzyme as the predominant plasminogen activator, while the activator content of the adjacent normal lung tissue consists of some urokinase-like enzyme, but mostly of an enzyme which is not inhibited by the antibody. The urokinase-like activator has been purified approximately 20,000-fold from lung tumors by the combination of two affinity chromatographic procedures, and was compared with purified urokinase with a molecular weight of 55,000 on all criteria used, the lung tumor activator was identical to urokinase.

https://cancerres.aacrjournals.org/content/canres/40/3/841.full.pdf

Content and characterization of plasminogen activators in human lung tumors and normal lung tissue.

The plasminogen activator activity of human breast tumors was determined quantitatively in extracts of 33 primary tumors, 11 lymph node metastases, and 11 normal tissues. Activities varied more widely in the neoplastic tissues than in the normal ones. The mean activator content of the tumors (primary and metastasis) was significantly higher ( p < 0.01) than that of normal tissues: 4.4-fold on a g wet tissue basis and 2.1-fold when the activities were referred to the protein content of the extracts. Primary and metastatic tumors were not significantly different from one another in their plasminogen activator content. In the six cases where tumors and normal tissue pairs from the same patients could be compared, the mean of the individual tumor:normal ratios was 10.0 (wet weight) or 3.6 (extract protein). In addition to the activator content, the molecular type of the enzymes was also determined, using goat antibody formed against human urinary urokinase as an inhibitor of activator activity. On the average, 80% of the activity of the tumor extracts could be inhibited by the antibody; extracts of normal breast tissue contained only 62% urokinase-like activator. The activators were also characterized by sodium dodecyl sulfate gel electrophoresis using two different methods for the zymographic visualization of activator bands. The fraction of urokinase-like enzyme determined by the fibrinogen copolymerization method agreed well with the results obtained by immunoinhibition. No correlation was found between activator activity or activator composition, on the one hand, and the degree of differentiation of the tumors, the fraction of positive lymph nodes found, or the amount of estrogen-binding protein (or their molecular size) in the cytosol of the tumors, on the other. This apparent lack of correlation may reflect variations in local physiological conditions which necessitate the presence of widely differing amounts of activator in order to ensure a critical amount of the product of the reaction catalyzed by this enzyme, i.e. , plasmin.

Plasminogen Activator Activity and Composition in Human Breast Cancer

The secretion of plasminogen activator by explants of 92 human malignant tumors was studied in short-term organ culture. Where possible, adjacent normal tissue of the presumed origin of the tumors was also studied. The study included adenocarcinomas of the lung, colon, prostate, breast, and stomach and different types of sarcomas. In addition to the measurement of secretion rates, all tissues were quantitatively extracted to determine the amount of cell-bound enzyme. Both culture fluids and extracts were analyzed with respect to the type of plasminogen activator they contained by immunoinhibition with goat immunoglobulin G formed against purified human urinary urokinase sodium dodecyl sulfate:gel electrophoresis followed by zymography. The study yielded the following conclusions: ( a ) the measurement of plasminogen activator secretion rates gives a much sharper differentiation between malignant and normal tissues than does the amount of extractable enzyme; ( b ) the enzymes secreted in short-term organ culture are, in the great majority of the cases studied, of the urokinase type, even when a large fraction of the activator contained in the tissue is of the vascular type; ( c ) the secretion rates of metastatic tumors of the colon are much lower than those of the primary ones; ( d ) immunoperoxidase staining of tissue sections reveals that urokinase is localized predominantly in the tumor cells. The low secretion rates of metastatic tumors, probably a reflection of this property in the original cell that gave rise to the metastatic focus, could be of advantage to circulating cancer cells. Such cells would not dissolve the microthrombus thought to be essential for the arrest of cancer cells in the capillaries of target organs.

https://cancerres.aacrjournals.org/content/canres/43/11/5517.full.pdf

Plasminogen Activator Secretion of Human Tumors in Short-Term Organ Culture, Including a Comparison of Primary and Metastatic Colon Tumors

Plasminogen activator content was determined quantitatively in extracts of 23 pairs of surgically removed colon tumors and adjacent normal mucosa specimens. The activator content averaged 4.4 times higher in the tumor samples than in the corresponding normal tissue. Polyps removed with the adenocarcinomas gave values intermediate between those for tumors and those for the normal mucosae. The enzyme content of the group of tumors that showed invasive propagation or metastatic spread was significantly higher (P < 0.05) than was the enzyme content of the group not manifesting these conditions. Activator activity of the tumor extracts was completely inhibited by rabbit antibody formed against human urokinse. The activity of the normal mucosae was variably inhibited, suggesting the presence of several kinds of activator in normal tissues. The activator from the normal tissues could be separated into a completely refractory fraction and a completely inhibitable fraction by means of an affinity column made of Sepharose-linked, rabbit antiurokinase antibody. The activator, eluted from this column with 1 M acetic acid in 0.5 M NaCl (pH 2.2), was highly purified and had an isoelectric point of 8.6, as does authentic urokinase. The details of the isoelectric profile of the two, however, differed. The data are discussed in relation to earlier studies on the fibrinolytic system in colon cancer.

Plasminogen activator content of human colon tumors and normal mucosae: separation of enzymes and partial purification.

When streptokinase-activated, urokinase-activated, or spontaneously activated human plasmin is mixed with increasing amounts of human antiplasmin, caseinolytic effect decreases much more rapidly th...

Gabor Markus

Papers

Content and characterization of plasminogen activators in human lung tumors and normal lung tissue.

Plasminogen Activator Activity and Composition in Human Breast Cancer

Plasminogen Activator Secretion of Human Tumors in Short-Term Organ Culture, Including a Comparison of Primary and Metastatic Colon Tumors

Plasminogen activator content of human colon tumors and normal mucosae: separation of enzymes and partial purification.

Plasmin-antiplasmin complex as a reservoir of fibrinolytic enzyme