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PubMed1992

Multiple members of the plasminogen-apolipoprotein(a) gene family associated with thrombosis.

Plasminogen and apolipoprotein(a) [apo(a)] are closely related plasma proteins that are associated with hereditary thrombophilia. Low plasminogen levels are found in some patients who developed venous thrombosis, while a population with high plasma concentrations of apo(a) have a higher incidence of arterial thrombosis. Two different genes coding for human apo(a) have been isolated and characterized in order to study and compare these genes with four other closely related genes in the plasminogen-apo(a) gene family. These include the gene coding for plasminogen, two unique plasminogen-related genes, and a gene coding for hepatocyte growth factor. Nucleotide sequence analysis of these genes revealed that the exons and their boundaries of the genes for plasminogen and apo(a), and the plasminogen-related genes, differ only 1-5% in sequence. The types of exon/intron junctions and positions of introns in the molecules are also exactly identical, suggesting that these genes have evolved from an ancestral plasminogen gene via duplication and exon shuffling. By utilizing these results, gene-specific probes have been designed for the analysis of each of the genes in this gene family. The plasminogen and two apo(a) genes were all localized to chromosome 6 by employing the gene-specific primers and genomic DNAs from human-hamster cell hybrids. These data also make it possible to characterize the apo(a) and plasminogen genes in individuals by in vitro amplification.

Amino Acid Sequence↗
PubMed1988

The course and prerequisites of Lys-plasminogen formation during fibrinolysis.

Plasmin-catalyzed modification of the native plasma zymogen Glu1-plasminogen to its more reactive Lys78 form has been shown to be enhanced in the presence of fibrin. The aim of the present work has been to characterize the influence of fibrinopeptide release, fibrin polymerization, and plasmin cleavage of fibrin on the rate of Lys78-plasminogen formation. 125I-Labeled Glu1- to Lys78-plasminogen conversion was catalyzed by performed Lys78-plasmin, or by plasmin generated during plasminogen activation with tissue plasminogen activator or urokinase. The two forms of plasminogen were quantitated following separation by polyacrylamide gel electrophoresis in acetic acid/urea. Plasmin generated by plasminogen activator was monitored by a fixed-time amidolytic assay. The rate of Lys78-plasminogen formation was correlated, in separate experiments, to the simultaneous, plasmin-catalyzed cleavage of 125I-labeled fibrinogen or fibrin to fragments X, Y, and D. The radiolabeled components were quantitated after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results show that the formation of both bathroxobin-catalyzed des-A-fibrin and thrombin-catalyzed des-AB-fibrin leads to marked stimulation of Lys78-plasminogen formation, whereas inhibition of fibrin polymerization, with Gly-Pro-Arg-Pro, abolishes the stimulatory effect. The rate of Lys78-plasminogen formation varies markedly in the course of fibrinolysis. The apparent second-order rate constant of the reaction undergoes a transient increase upon transformation of fibrin to des-A(B) fragment X polymer and decreases about 10-fold to the level observed during fibrinogenolysis upon further degradation to soluble fragments Y and D.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrin↗
PubMed1984

The AH-site of plasminogen and two C-terminal fragments. A weak lysine-binding site preferring ligands not carrying a free carboxylate function.

Glu-plasminogen [native plasminogen (Glu-1-Asn-790)], Lys-plasminogen [plasmin-cleaved fragment of plasminogen (Lys-77-Asn-790)] and miniplasminogen [fragment of plasminogen (Val-440-Asn-790)] were all found to interact specifically with immobilized 6-aminohexyl ligands. The interactions apparently are mediated by a single weak lysine-binding site, termed the AH-site, as seen from the patterns of inhibition obtained from frontal-quantitative-affinity-chromatography experiments with 6-aminohexanoic acid and alpha-N-acetyl-L-lysine methyl ester as competing ligands. The AH-site, in contrast with the strong lysine-binding site of Glu-plasminogen and Lys-plasminogen, may prefer ligands not carrying a free carboxylate function and therefore may interact with lysine side chains of proteins. In Glu-plasminogen the AH-site is present, but is apparently only partially free to react. It is suggested that it participates in an intramolecular complex and that an equilibrium state between two Glu-plasminogen forms exists. It is further suggested that binding of the plasminogens to fibrin is mainly determined by the AH-site.

Aminocaproates↗
PubMed1992

Binding of human plasminogen to basement-membrane (type IV) collagen.

Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix.

Basement Membrane↗
PubMed2002

Inhibition of human MDA-MB-231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen.

Matrix metalloproteinase (MMP)-3 inhibited human MDA-MB-231 breast cancer cell invasion through reconstituted basement membrane in vitro. Inhibition of invasion was dependent upon plasminogen and MMP-3 activation, was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2 and was associated with: rapid MMP-3-mediated plasminogen degradation to microplasminogen and angiostatin-like fragments; the removal of single-chain urokinase plasminogen activator from MDA-MB-231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t-PA) and membrane-mediated plasminogen activation; and reduced laminin-degrading capacity. Purified human plasminogen lysine binding site-1 (kringles 1-3) exhibited a similar capacity to inhibit MDA-MB-231 invasion, impair t-PA and cell membrane-mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP-3 can inhibit breast tumour cell invasion in vitro by a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP-3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP-3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.

Angiostatins↗
PubMed2001

Regulation of plasminogen activator inhibitor 1 expression by interaction of epidermal growth factor with progestin during decidualization of human endometrial stromal cells.

OBJECTIVE: During human pregnancy implantation, trophoblasts invade maternal blood vessels in a process that risks hemorrhage. Previous studies have demonstrated enhanced expression of type 1 plasminogen activator inhibitor, the primary inhibitor of fibrinolysis, during progestin-induced decidualization of estradiol-primed human endometrial stromal cells in vivo and in vitro. Decidual cell-expressed plasminogen activator inhibitor 1 is appropriately positioned to avert implantational hemorrhage. Because of the absence of estrogen or progesterone response elements from the plasminogen activator inhibitor 1 gene promoter, I posited that epidermal growth factor mediates these steroid effects and that expression of epidermal growth factor receptor in human endometrial stromal cells is under ovarian steroid control. STUDY DESIGN: Confluent human endometrial stromal cells were exposed to vehicle control or to either estradiol (10(-8) mol/L) or medroxyprogesterone acetate (10(-7) mol/L), or both, with or without growth factors. After 40 hours the cultures were analyzed for plasminogen activator inhibitor 1 protein and messenger ribonucleic acid expressions. Immunostaining for epidermal growth factor receptor was carried out in sections of cycling and gestational endometrial tissues. RESULTS: In the absence of steroids, epidermal growth factor did not alter plasminogen activator inhibitor 1 expression. In the absence of epidermal growth factor, estradiol and medroxyprogesterone acetate enhanced human endometrial stromal cell-secreted plasminogen activator inhibitor 1 protein levels 8-fold (n = 12; P <.001), whereas estradiol alone had no effect. Marked synergistic increases in plasminogen activator inhibitor 1 levels were elicited when epidermal growth factor was added with estradiol and medroxyprogesterone acetate (n = 12; 65-fold; P <.0001). Both transforming growth factor alpha and epidermal growth factor, which act through epidermal growth factor receptor, increased steady-state plasminogen activator inhibitor 1 messenger ribonucleic acid levels several-fold when added with estradiol and medroxyprogesterone acetate. In contrast, transforming growth factor beta, which does not activate epidermal growth factor receptor, did not elevate plasminogen activator inhibitor 1 messenger ribonucleic acid or protein levels whether added alone or with estradiol and medroxyprogesterone acetate. In correspondence with these in vitro observations, immunostaining for epidermal growth factor receptor was increased in human endometrial stromal cells undergoing decidualization in sections of secretory phase and first-trimester endometrial tissue. CONCLUSIONS: Taken together, these in vitro and in vivo results indicate that both epidermal growth factor and progesterone receptors are required for maximal plasminogen activator inhibitor 1 expression by human endometrial stromal cells.

Blotting, Northern↗
PubMed1995

Cytokines and plasminogen activator inhibitor-1 in posttrauma disseminated intravascular coagulation: relationship to multiple organ dysfunction syndrome.

OBJECTIVES: a) To investigate the relationships between tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), plasminogen activator inhibitor-1, and disseminated intravascular coagulation (DIC); b) to determine the influence of DIC on the mortality rate, adult respiratory distress syndrome (ARDS), and multiple organ dysfunction syndrome; and c) to find a useful prognostic index for outcome. DESIGN: Prospective, case-control study. SETTING: General intensive care unit (tertiary care center) in a city hospital serving a population of 1.5 million people. PATIENTS: Fifty-eight trauma patients; 22 of the patients with DIC and 36 of the patients without DIC. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: TNF-alpha, IL-1 beta, plasminogen activator inhibitor-1 activity, and plasminogen activator inhibitor-1 antigen concentration were measured on the day of the injury, and on days 1, 3, and 5 after admission. The results of these measurements, demographic data, severity of illness score, mortality rate in the intensive care unit and frequencies of ARDS, multiple organ dysfunction syndrome, and sepsis were compared according to the occurrence of DIC. DIC patients were classified into subgroups of survivors and nonsurvivors, and the changes in plasminogen activator inhibitor-1 between subgroups were studied. The Acute Physiology and Chronic Health Evaluation II scores, the Injury Severity Scores, and the frequency of ARDS and multiple organ dysfunction syndrome were higher in the DIC patients. The mortality rate of the DIC patients was higher than the rate of the non-DIC patients (59.0% vs. 13.8%; p = .0009). TNF-alpha and IL-1 beta concentrations increased more in the DIC patients than in the non-DIC patients. Plasminogen activator inhibitor-1 activity and plasminogen activator inhibitor-1 antigen concentrations in the DIC patients, especially those values in the nonsurvivors, continued to be markedly high up to day 5 of admission. The most favorable prognostic value of plasminogen activator inhibitor-1 for the prediction of death in all of the trauma patients and the DIC patients was determined on days 3 and 5, respectively. No significant correlation was noted between the two cytokines and plasminogen activator inhibitor-1. CONCLUSIONS: In the patients with trauma, DIC is a predictor of ARDS, multiple organ dysfunction syndrome, and death. TNF-alpha and IL-1 beta might be one of the causes of DIC, while plasminogen activator inhibitor-1 may be one of the aggravating factors of ARDS and multiple organ dysfunction syndrome. Plasminogen activator inhibitor-1 is a good predictor of death for posttrauma DIC patients.

APACHE↗
PubMed1996

Direct interactions of plasminogen activators with human aortic and pulmonary artery endothelial cells in vitro: implications for thrombolytic therapy.

Direct interactions of plasminogen activators with arterial endothelial cells are important in the pathogenesis of vascular complications associated with thrombolytic therapy. We investigated the direct effects of various plasminogen activators on human aortic and pulmonary artery endothelial cell functions in vitro. The effects of plasminogen activators on endothelial cells were not caused by generation of plasmin, as shown by the absence of plasminogen and alpha(2)-plasmin inhibitor-plasmin complex both before and after addition of plasminogen activators to endothelial cells. High concentrations of plasminogen activators increased the permeability of aortic endothelial cells to albumin. Alteplase (50 x 10(3) IU/ml), a recombinant tissue-type plasminogen activator (t-PA), increased prostaglandin I(2) (PGI(2)) production by aortic endothelial cells from 175.5 +/- 13.8 to 870.8 +/- 131.0 pg/mg cellular protein during a 2-h incubation; other plasminogen activators increased PGI(2) production to a lesser extent. Alteplase (100 x 10(3) IU/ml) also increased PGI(2) production from 152.0 +/- 16.2 to 1,080 +/- 95.1 pg/mg cellular protein in human pulmonary artery endothelial cells. High concentrations of urokinases decreased the amount of endothelin-1 in the medium of aortic or pulmonary artery endothelial cells by as much as 93%; part of this decrease was attributable to degradation of endothelin-l by urokinases. Other plasminogen activators either had no effect on or slightly increased the production of endothelin-1. These changes in the function of human arterial endothelial cells induced by plasminogen activators may affect regional vascular tone, endothelial permeability, and platelet aggregability, all of which are important in the efficacy of thrombolysis and in the pathogenesis of such vascular complications as rethrombosis and hemorrhage.

Aorta↗
PubMed1983

Molecular size of secreted and cell-associated plasminogen activators from cultured epidermal cells.

The molecular sizes of secreted and cell-associated plasminogen activators from four cultured cell types were determined using an SDS-PAGE technique in which plasminogen and casein were included during polymerization of the polyacrylamide gel. The major bands of plasminogen activators secreted by human neonatal epidermal cells, human adult epidermal cells and transformed human squamous cells migrated the same distance as the high molecular weight band of authentic urokinase, indicating that the apparent molecular weight of these plasminogen activators was approximately 55,000 daltons. Plasminogen activator extracted from normal adult human epidermis also migrated with this major band of plasminogen activator, and a minor higher molecular weight band was also detected. In contrast, plasminogen activators secreted by transformed mouse squamous cells migrated between the high molecular weight band (approximately 55K) and the low molecular weight band of urokinase (approximately 32K), indicating that plasminogen activators of mouse epidermal cells differ from those of human epidermal cells. The mobility of the major bands of plasminogen activators detected in cell lysates of the four cell types was identical to that of secreted plasminogen activators.

Adult↗
PubMed1995

The role of an enolase-related molecule in plasminogen binding to cells.

The alpha isoform of enolase is a candidate plasminogen receptor on U937 monocytoid cells [Miles, L. A., Dahlberg, C. L., Plescia, J., Felez, J., Kato, K. & Plow, E. F. (1991) Biochemistry 30, 1682-1691]. In the present study, an enolase-related molecule was detected on the surfaces of peripheral blood monocytes and neutrophils by fluorescence-activated cell sorting. A mRNA transcript encoding a unique membrane form of an enolase-related molecule was not detected by Northern-blotting and primer-extension analyses, consistent with the cell-surface protein being authentic alpha-enolase. Both the alpha and beta isoforms of purified enolase, bound plasminogen with an affinity similar to that of the cell surface. Moreover, immunopurified alpha-enolase enhanced plasminogen activation by tissue plasminogen activator and blocked the binding of plasminogen to alpha 2-antiplasmin, mimicking functions arising from the association of plasminogen with cells. The interaction of the enolase isoforms with plasminogen was dependent upon recognition of the C-terminal lysyl residue of the enolases by the lysine-binding sites of plasminogen, as the interaction was blocked by (a) peptides with C-terminal lysine residues and (b) an antibody to the C-terminal aspect of enolase. A monoclonal antibody was developed, characterized and utilized to quantify the enolase molecules present on the surface of U937 cells. A substantial number of molecules, 1.8 x 10(6)/cell, was present, accounting for approximately 10% of the plasminogen-binding capacity of these cells. These studies clearly establish the role of enolase as a cell-surface plasminogen-binding site with profibrinolytic functions.

Amino Acid Sequence↗
PubMed1995

Structural domains of streptokinase involved in the interaction with plasminogen.

Two fragments of recombinant streptokinase, comprising amino acids Val143-Lys293 (17-kDa rSK) or Val143-Lys386 (26-kDa rSK), were cloned and expressed in Escherichia coli, purified to homogeneity and their interactions with plasmin(ogen) were evaluated. Both 17-kDa rSK and 26-kDa rSK bound to plasminogen with a 1:1 stoichiometry and with affinity constants of 3.0 x 10(8) M-1 and 12 x 10(8) M-1, respectively, as compared to 6.3 x 10(8) M-1 for the binding of intact recombinant streptokinase to plasminogen. Binding of 17-kDa rSK to plasminogen-Sepharose was displaced by addition of increasing concentrations of recombinant streptokinase, whereas bound recombinant streptokinase was not displayed by 17-kDa rSK. In equimolar mixtures of plasminogen and 26-kDa rSK, the appearance of amidolytic activity as monitored with a chromogenic substrate, was significantly delayed compared to the equimolar mixture with recombinant streptokinase (60% of the maximal activity after 30 min, compared to maximum activity within < or = 2 min). In contrast, no amidolytic activity was generated in equimolar mixtures of plasminogen and 17-kDa rSK. Plasminogen was rapidly activated by catalytic amounts (1:100 molar ratio) of recombinant streptokinase (60-70% within 10-15 min), whereas only 4% of the plasminogen was activated within 60 min with 26-kDa rSK, and no plasmin was generated with 17-kDa rSK. Complexes of plasmin with 17-kDa rSK or with 26-kDa rSK were very rapidly inhibited by alpha 2-antiplasmin (apparent second-order inhibition rate constant of approximately 2 x 10(7) M-1 s-1), whereas the complex with recombinant streptokinase was resistant to inhibition. With 26-kDa rSK, inhibition by alpha 2-antiplasmin resulted in dissociation of the complexes and recycling of functionally active 26-kDa rSK to other plasminogen molecules; 17-kDa rSK, in contrast, remained associated with the plasmin-alpha 2-antiplasmin complex. These findings suggest that different regions of the streptokinase molecule are involved in binding to plasminogen, in active-site exposure, and in impairment of the inhibition of plasmin by alpha 2-antiplasmin. Thus, the 17-kDa region spanning Val143-Lys293 in streptokinase mediates its binding to plasminogen but does not induce activation. Furthermore, this region does not interfere with the inhibition of the complex with plasmin by alpha 2-antiplasmin.

Amino Acid Sequence↗
PubMed1994

Binding of vitronectin and plasminogen to Helicobacter pylori.

We have studied how some extracellular matrix proteins, fibronectin, fibrinogen, collagen type I and type IV, plasminogen and vitronectin bind to Helicobacter pylori. Radiolabelled vitronectin and plasminogen bound to the haemagglutinating H. pylori strain 17874 at a high level (53% and 32%, respectively), type IV collagen showed an intermediate level of binding (16%), while binding by 125I-labelled fibrinogen, fibronectin and collagen type I remained at a low level (5-7%). Both 125I-vitronectin and plasminogen showed a dose-dependent binding to cells of H. pylori 17874. Plasminogen binding by this strain was specific since the binding was inhibited by nonlabelled plasminogen, but not by highly glycosylated glycoproteins such as fetuin and orosomucoid or by a variety of monosaccharides. We have previously shown that 125I-vitronectin shows a specific and saturable binding to H. pylori 17874, and that sialic acid-rich glycoproteins such as fetuin and orosomucoid drastically reduced binding. We now report that a simultaneous incubation of 125I-vitronectin and 125I-plasminogen with cells of H. pylori 17874 showed a total binding approximately similar to the level of binding when either 125I-plasminogen, or 125I-vitronectin only were incubated with the bacterial cells. Nonlabelled vitronectin inhibited the binding of 125I-plasminogen by H. pylori, but nonlabelled plasminogen had no effect on the binding of 125I-vitronectin. Our findings suggest that there are different but probably closely localized binding sites for vitronectin and plasminogen on H. pylori 17874.

Collagen↗
PubMed1984

Modulation of cell-associated plasminogen activator activity by cocultivation of a stem cell and its tumorigenic descendant.

The effect of the presence of one cell type on the plasminogen activator activity of another cell type was studied. The cell types, AC and D, were isolated from a rat neuroblastoma (I. Imada and N. Sueoka, Dev. Biol. 66:97-108, 1978). AC cells are stem cells capable of multipotential differentiation in vitro and have little or no cell-associated plasminogen activator activity. D cells are tumorigenic and have high levels of cell-associated plasminogen activator activity. When AC cells were cocultivated with D cells, the plasminogen activator activity of the D cells was dramatically inhibited. The presence of as few as 1,250 AC cells inhibited 70% of the plasminogen activator activity of 20,000 D cells, as determined by a highly quantitative assay. The amount of inhibition by AC cells was proportional to the number of AC cells present. At increasing numbers of AC cells and a constant number of D cells, the Vmax for the activation of plasminogen proportionately decreased and the Km remained constant, implying that AC cells did not alter the structure or concentration of plasminogen. Inhibition was not mediated by a soluble inhibitor secreted by AC cells. Rather, attachment of AC cells adjacent to D cells, i.e., cell-to-cell contact, seemed to be required for inhibition. The substratum-attached material of AC cells, that which remained on the microwell surface after removal of AC cells with EDTA, inhibited D cell plasminogen activator activity. If plasminogen activator activity is involved in metastasis, then regulation of the plasminogen activator activity of one cell type by another cell type may be involved in determining which cells in a tumor can metastasize and where secondary tumors can arise.

Animals↗
PubMed1988

Production of plasminogen activator by alveolar macrophages in normal subjects and patients with interstitial lung disease.

Increased production of the serum protease plasminogen activator is associated with tissue damage. The in vitro production of plasminogen activator by alveolar macrophages obtained by bronchoalveolar lavage was studied in 22 normal subjects and 28 patients with interstitial lung disease to determine whether plasminogen activator is produced by normal alveolar macrophages and whether this is increased in patients with interstitial lung disease. Plasminogen activator activity, measured with an iodine-125 labelled fibrin release assay, was found to be dependent on time, effector cell numbers, and plasminogen concentration. Plasminogen activator production by alveolar macrophages from 14 normal non-smokers and eight normal smokers was similar and the mean value was 0.78 (SEM 0.16) urokinase (UK) units x 10(-8)/cell/hour. Alveolar macrophages from the seven patients with cryptogenic fibrosing alveolitis and six patients with histiocytosis-X produced more plasminogen activator (1.89 (0.25) and 4.54 (1.3) x 10(-8) UK units/cell/hour respectively) than macrophages from normal subjects (p less than 0.05), whereas those from 15 patients with sarcoidosis did not (1.09 (0.2) x 10(-8) UK units/cell/hour). Exposure of normal alveolar macrophages to immune complexes enhanced plasminogen activator production to 2.07 (0.27) x 10(-8) UK units/cell/hour, whereas exposure to products of activated T cells and to purified gamma interferon reduced plasminogen activator production (to 0.38 (0.11) and 0.62 (0.11) x 10(-8) UK units/cell/hour respectively). These studies show that plasminogen activator is produced by normal human alveolar macrophages and that its production is increased in patients with cryptogenic fibrosing alveolitis and histiocytosis-X.

Antigen-Antibody Complex↗
PubMed1983

Plasminogen activators in tissues of the immature and estrogen-stimulated rat uterus and in uterine luminal fluid: characterization and properties.

We have characterized the molecular properties of the plasminogen activators in different cell types comprising the immature and the estrogen-stimulated rat uterus and in rat uterine luminal fluid. There were two plasminogen activators in the immature (day 20) rat uterus with apparent molecular weights, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of 70,000 and 46,000. Both plasminogen activators were present in epithelial and in stromal plus myometrial cell fractions of the immature uterus, and after stimulation by 17 beta-estradiol, no new plasminogen activators were detected in either cell fraction. The Michaelis constants (Km) for the activation of dog plasminogen by extracts from epithelial cells and from stromal plus myometrial cells obtained from either immature or 17 beta-estradiol-stimulated uteri were similar (approximately 11 microM). The maximal velocity (Vmax), normalized to protein concentration, increased 2.5-fold in the stromal plus myometrial cell fraction and 6.5-fold in the epithelial cell fraction, upon hormone stimulation (2 micrograms 17 beta-estradiol/day X rat for 3 days). The greatest concentration of plasminogen activator activity was found in the luminal fluid from estrogen-stimulated uteri, where the Vmax per mg protein was more than 10-fold greater than that in the cell fractions from estrogen-stimulated uteri. The plasminogen activator activity of luminal fluid was inhibited by diisopropyl fluorophosphate and rho-nitrophenyl rho-guanidinobenzoate, was not inhibited by human alpha-1-proteinase inhibitor and human antithrombin III, and was inhibited by high, but not low, concentrations of soybean trypsin inhibitor and bovine pancreatic trypsin inhibitor. These studies indicate that the plasminogen activators in different cell types comprising the uterus are similar and show that the estrogen enhancement of uterine plasminogen activator activity is the result of an increase in Vmax. The presence, upon hormone stimulation, of an apparent concentration gradient of increasing plasminogen activator activity through the uterus from myometrium to epithelium to luminal fluid may be a reflection of the dynamic role of this protease in the physiology of the uterus.

Animals↗
PubMed1987

Preclinical pharmacological evaluation of anisoylated plasminogen streptokinase activator complex.

An ideal thrombolytic (or fibrinolytic) agent is one which would generate the formation of plasmin only where it is required, i.e. bound to fibrin within the thrombus. However, the capacity of even the newer thrombolytic agents to achieve localised plasmin generation within the thrombus is relative and depends on the concentration of the agent administered. For all available activators, the concentration required for effective clinical thrombolysis is also capable of converting plasminogen to plasmin within the circulation (plasminaemia). Since the action of plasmin is not specific to fibrin, plasminaemia results in dissolution not only of fibrin but also of several other clotting factors. For example, plasmin can degrade fibrinogen and cause impaired haemostasis. The plasminogen activators which are available, or have been developed to date, include streptokinase, urokinase, pro-urokinase, anisoylated plasminogen-streptokinase activator complex (APSAC) and tissue plasminogen activator (t-PA). All of these agents have the same biochemical mechanism of action, cleaving an arginine-valine bond in the plasminogen molecule to form plasmin, but they differ with regard to other important properties. The first property to be considered is clot specificity; the ability to dissolve fibrin as opposed to fibrinogen, and also to dissolve the clot as opposed to a haemostatic plug. Unfortunately, fibrin specificity does not equate entirely with thrombus specificity, and all currently developed plasminogen activators, by dissolving fibrin, will induce the destruction of haemostatic extravascular plugs as well as intravascular thrombi. Thus, no agent is thrombus-specific in this respect. The degree of fibrinogenolysis does vary between plasminogen activators. Those which have the least effect on haemostasis or clotting capability would seem, at first, to be preferable. However, a short term reduction in fibrinogen could also be beneficial, since it may reduce the incidence of early reocclusion and, by reducing blood viscosity, improve microcirculation to the infarct zone. The intrinsic efficiency of the plasminogen activators is a second important property. In vitro, under conditions pertaining to the circulation, urokinase is about 10 times more efficient than t-PA at converting glu-plasminogen to plasmin (on the basis of the Vmax to Km ratio), while streptokinase-plasmin is 20 times more efficient. The efficiency of these activators is increased in the presence of fibrin and lys-plasminogen, 1800-fold for t-PA, 8-fold for urokinase and 180-fold for streptokinase-plasmin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Plasminogen binding and cancer: promises and pitfalls.

The urokinase plasminogen activation system is a key modulator of the tissue remodeling processes required for tumor cell invasion and metastasis. Malignant progression is characterised by inappropriately high cell surface levels of receptor- bound active urokinase. This enhances the rate of plasminogen activation resulting in markedly increased levels of cell surface plasmin. The repercussions of this are significant and include the activation of growth factors and signaling pathways, and the degradation of extracellular matrices, either directly or indirectly, via the activation of matrix metalloproteinases. Native, circulating plasminogen binds in a lysine- and/or carbohydrate-dependent manner to tumor and endothelial cells with low affinity but high capacity and a heterogeneous group of plasminogen receptors have been identified. This heterogeneity underscores the complexity of the mechanisms responsible for the regulation of cell-surface plasminogen binding. This review summarizes the literature on known plasminogen receptor candidates and shows that they can be subdivided into three classes based on their mode of interaction with plasminogen. We also aim to emphasize the notion that in the tumor environment the known intrinsic functional relationship between plasminogen conformation and activation is essentially connected to cellular binding. This allows plasminogen to be co-localised in an activation-susceptible form with the enhanced uPA levels seen in malignancy and together furnishes tumor cells with elevated tissue remodeling capacity. In addition, some of the pitfalls and strategies encountered when conducting plasminogen receptor experiments are also addressed.

Animals↗
PubMed1991

Partial purification and characterization of native plasminogen activators from bovine milk.

At least four native plasminogen activators were detected in bovine milk, and two partially purified plasminogen activators were characterized. The plasminogen activators were dissociated from casein proteins by treatments with sulfuric acid and dimethylformamide. The plasminogen activators in the resulting fractions were partially purified with size exclusion, affinity, or metal chelate chromatographic techniques. Molecular weights of the two partially purified plasminogen activators were 47.2 and 30.5 kDa by gel electrophoresis. Size exclusion chromatography gave a molecular weight of 43.2 kDa for the first plasminogen activator. The isoelectric points of the two plasminogen activators were in the pH range 6.2 to 6.7. Because activity was not enhanced by the presence of fibrinogen fragments in a plasminogen activator assay mixture and decreased when human anti-urokinase Ig were added, at least some bovine milk native plasminogen activators appear to be urokinase-type plasminogen activators.

Animals↗