Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PLASMINOGEN”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,189 records · Page 66Linked to original sources
PubMed1986

Plasminogen interacts with human platelets through two distinct mechanisms.

Glu-plasminogen, the native form of plasminogen, interacts in a specific and saturable manner with unstimulated human platelets, and the binding is enhanced fivefold by thrombin stimulation (Miles and Plow, 1985. J. Biol. Chem. 260:4303). This study characterizes the nature of the Glu-plasminogen binding sites by analyzing platelets deficient in selected proteins and functions. Platelets from patients with afibrinogenemia, Gray platelet syndrome, and the Cam Variant of thrombasthenia, a form of thrombasthenia with near normal levels of glycoprotein IIb/IIIa (GPIIb/IIIa), showed minimal augmentation of plasminogen binding to thrombin-stimulated platelets but normal binding to unstimulated platelets. This selective deficiency indicates that two distinct mechanisms are involved in the interaction of plasminogen with platelets. These abnormal platelets share a deficiency in fibrinogen. Surface expression of platelet fibrinogen, however, was not sufficient for enhanced plasminogen binding to stimulated platelets, and experiments with alpha-thrombin and gamma-thrombin indicated that fibrin formation on the platelet surface is necessary for the augmented plasminogen binding. Unstimulated and stimulated thrombasthenic platelets deficient in GPIIb/IIIa bound markedly reduced levels of plasminogen, which suggests a role for GPIIb/IIIa in plasminogen binding to unstimulated platelets. Treatment of platelets to dissociate the heterodimeric complex of GPIIb/IIIa did not significantly perturb plasminogen binding to unstimulated platelets, but the complex may be necessary for thrombin-stimulated plasminogen binding via its interaction with platelet fibrin.

Blood Platelets↗
PubMed2001

Immunologic and hematologic properties of antibodies to prothrombin and plasminogen in a mouse model.

Antibodies to prothrombin have been associated with venous and arterial thrombosis, and they cross-react with a structurally closely related protein plasminogen. We immunised 16 mice with human prothrombin and 15 mice with human plasminogen. Mice immunised with prothrombin developed cross-reactive antibodies to plasminogen (12/16), beta2-glycoprotein I (4/16), tissue-type plasminogen activator (6/16) and cardiolipin (11/16). Mice immunised with plasminogen developed cross-reactive antibodies to prothrombin (8/15), tissue-type plasminogen activator (2/12) and cardiolipin (5/12). Functional effects of antibodies were examined. Immunisation with prothrombin induced lupus anticoagulant activity in 9/14 mice. In mice immunised with plasminogen, radial fibrinolysis was inhibited in 8/10 and plasminogen activation in the chromogenic assay was inhibited in 9/11. No cross-functionality was observed. In conclusion, antibodies to prothrombin and plasminogen cross-react in vivo. Antibodies to prothrombin and plasminogen have different functional profiles, immunisation with prothrombin leads to prolonged blood clotting time, and immunisation with plasminogen induces antibodies interfering with fibrinolysis.

Animals↗
PubMed1983

Gonadotropins regulate plasminogen activator production by rat granulosa cells.

The role of FSH in the regulation of plasminogen activator production was studied in granulosa cells obtained from 23- to 25-day-old female rats. Cells cultured without FSH secreted a negligible amount of plasminogen activator. Purified ovine, rat, and human FSH produced dose-dependent increases in plasminogen activator production. This FSH effect was mimicked by analogs of cAMP, prostaglandin E2, and choleratoxin. Purified ovine LH and ovine PRL had no effect on plasminogen activator production by these immature granulosa cells. However, when these granulosa cells were treated in vitro or in vivo with FSH and then exposed to LH or PRL, the cells responded to LH in a dose-dependent manner with increased plasminogen activator production. These cells remained unresponsive to PRL. Similarly to FSH, in vitro pretreatment of the cells with choleratoxin or analogs of cAMP also induced responsiveness to LH with increased plasminogen activator production. This responsiveness to both FSH and LH with increased plasminogen activator production was also observed in granulosa cells obtained from rat preovulatory follicles. These studies demonstrate that: 1) FSH but not LH regulates plasminogen activator production by immature granulosa cells from preantral follicles; 2) Pretreatment of the undifferentiated granulosa cells with FSH, choleratoxin, or cAMP induced granulosa cell responsiveness to LH with increased plasminogen activator production; and 3) Granulosa cells obtained from preovulatory follicles respond to both FSH and LH with increased plasminogen activator secretion. These results suggest that with the LH surge at ovulation, plasminogen activator production in follicles is increased and may be important in follicular rupture.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

Apolipoprotein(a): structure-function relationship at the lysine-binding site and plasminogen activator cleavage site.

Apolipoprotein(a) [apo(a)] is the distinctive glycoprotein of lipoprotein Lp(a), which is disulfide linked to the apo B100 of a low density lipoprotein particle. Apo(a) possesses a high degree of sequence homology with plasminogen, the precursor of plasmin, a fibrinolytic and pericellular proteolytic enzyme. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogen-like kringle 4 and single copies of kringle 5, and the protease region including the backbone positions for the catalytic triad (Ser, His, Asp). A lysine-binding site that is similar to that of plasminogen kringle 4 is present in apo(a) kringle IV type 10. These kringle motifs share some amino acid residues (Asp55, Asp57, Phe64, Tyr62, Trp72, Arg71) that are key components of their lysine-binding site. The spatial conformation and the function of this site in plasminogen kringle 4 and in apo(a) kringle IV-10 seem to be identical as indicated by (i) the ability of apo(a) to compete with plasminogen for binding to fibrin, and (ii) the neutralisation of the lysine-binding function of these kringles by a monoclonal antibody that recognises key components of the lysine-binding site. In contrast, the lysine-binding site of plasminogen kringle 1 contains a Tyr residue at positions 64 and 72 and is not recognised by this antibody. Plasminogen bound to fibrin is specifically recognised and cleaved by the tissue-type plasminogen activator at Arg561-Val562, and is thereby transformed into plasmin. A Ser-Ile substitution at the activation cleavage site is present in apo(a). Reinstallation of the Arg-Val peptide bond does not ensure cleavage of apo(a) by plasminogen activators. These data suggest that the stringent specificity of tissue-type plasminogen activator for plasminogen requires molecular interactions with structures located remotely from the activation disulfide loop. These structures ensure second site interactions that are most probably absent in apo(a).

Apolipoproteins A↗
PubMed2006

Nitration of human plasminogen by RAW 264.7 macrophages reduces streptokinase-induced plasmin activity.

Despite many years of study, clinical trials of new drugs to prevent thrombosis have often been disappointing. Part of the problem lies in our incomplete understanding of the regulation of plasminogen activation and/or inhibition in vivo. We have previously shown that in vitro nitration of plasminogen in plasma by peroxynitrite resulted in decreased plasmin activity. We hypothesized that macrophages may be agents of plasminogen nitration and designed this study to prove this hypothesis. We first better characterized our previous observations using purified plasminogen instead of whole plasma, studied the time and concentration dependence of these reactions, and co-incubated plasminogen with macrophages, as well as with non-inflammatory cells as controls, to assess nitration and impaired activity. When plasminogen (10 micromol/L) is incubated in the presence of SIN-1 (0.01-2 mmol/L), plasmin activity (generated by streptokinase) is reduced in a time- and concentration-dependent fashion. We performed experiments incubating human plasminogen in the presence of murine RAW264.7 macrophages, allowing for free diffusion of reactive oxygen species, while preventing the action of proteases. In this way we show that incubation of plasminogen with macrophages also decreases plasmin activity, while increasing nitration of the molecule, an effect that is already apparent after 2 h and reaches a plateau of 60% inhibition after 24 h of incubation. This effect appears specific for macrophages, since 31EG4 murine mammary cells used in parallel and under the same conditions failed to produce any deleterious changes in plasminogen. Our data on quick functional inactivation of plasminogen by nitration, mediated by macrophages, adds a new pathophysiological dimension to our previous work showing plasminogen as a target for peroxynitrite damage. Nitrosative stress may be implicated in impaired fibrinolysis. New therapeutic approaches for nitrosative stress in atherosclerosis and diabetes should limit the formation of superoxides and peroxynitrite.

Animals↗
PubMed2000

Ethanol-induced up-regulation of candidate plasminogen receptor annexin II in cultured human endothelial cells.

INTRODUCTION: Epidemiological studies indicate that moderate alcohol consumption reduces the risk for coronary heart disease and that this cardioprotective benefit may be mediated, in part, by increased fibrinolysis. Endothelial cells (ECs) synthesize plasminogen activators, tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), receptors for plasminogen activators, and a receptor for plasminogen, annexin II (Ann-II). These receptors localize and facilitate receptor-bound plasminogen activator-mediated conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface, which results in the regulated expression of surface-localized EC fibrinolytic activity. Ethanol is a systemic factor that affects these components, which increases EC fibrinolysis and hence reduces the risk for thrombosis, coronary heart disease, and myocardial infarction (MI). METHODS: This study was carried out to determine whether low ethanol (0.1% v/v) increased plasminogen receptor, Ann-II antigen (western blot), messenger ribonucleic acid (mRNA) (reverse transcription polymerase chain reaction; RT-PCR) expression, activity (ligand binding/Scatchard analysis), and hence fibrinolysis (plasmin generation) in cultured human ECs. RESULTS: Plasminogen receptor activity increased approximately 2-fold (2.5 vs. 5.6 x 10(6) sites/cell), as evidenced by increased 125I-labeled Glu-plasminogen ligand binding/Scatchard analysis. In addition, western blot analyses indicated an increase in Ann-II antigen, and mRNA levels increased approximately 2-fold (RT-PCR). This increase in Ann-II expression was concomitant with approximately 2- to 3-fold sustained increase (approximately 24 hr) in surface-localized EC fibrinolytic activity. Nuclear transcription run-on assays showed an approximately 5- to 6-fold increase in new 32P-labeled Ann-II mRNA levels, compared with controls (no ethanol). CONCLUSIONS: These results demonstrated that low ethanol increased Ann-II antigen/mRNA levels and up-regulated Ann-II gene expression at the transcriptional level. The results further identify and define the contribution and role of the plasminogen receptor, Ann-II, in the ethanol-induced mechanism of increased EC fibrinolysis that may underlie and contribute, in part, to the cardioprotective benefit associated with moderate alcohol consumption.

Annexin A2↗
PubMed2002

Plasminogen has a broad extrahepatic distribution.

Plasmin is the major enzyme that dissolves fibrin in the vasculature and the predominant source of its zymogen, plasminogen, is liver. However, plasmin has a broad substrate spectrum and, if present in other tissues, may perform additional functions. We tested the hypothesis that plasminogen is expressed broadly extrahepatically. A sensitive and specific isotopic quantitative RT-PCR assay was developed to detect plasminogen mRNA from total RNA isolated from C57BL/6J mice tissues. Plasminogen mRNA was detected in adrenal, kidney, brain, testis, heart, lung, uterus, spleen, thymus and gut. Of these tissues, adrenal had the highest plasminogen mRNA content. In situ hybridization was utilized to localize plasminogen mRNA expressing cell types. Besides hepatocytes, positive cells were identified in both adrenal and kidney medullae and cortexes. Plasminogen mRNA expression was detected in cerebral, hippocampal and cerebellar neurons. Plasminogen mRNA was detected in cells in early stages of spermatogenesis in testis, present in the cortex and medulla of the thymus and in splenic white and red pulps. Our results suggest that the plasminogen gene is expressed broadly in extrahepatic tissues. Thus, tissues separated by local anatomic barriers as well as tissues accessible to circulating plasminogen have the capacity to provide local sources of plasminogen.

Animals↗
PubMed1977

Activation of human plasminogen by equimolar levels of streptokinase.

Native Glu-human plasminogen (Mr approximately 92,000 with NH2-terminal glutamic acid) is able to combine directly with streptokinase in an equivalent molar ratio, to yield a stoichiometric complex. The plasminogen moiety in the complex then undergoes streptokinase-induced conformational changes. As a result of such, an active center develops in the plasminogen moiety of the complex. This proteolytically active complex then activates plasminogen in the complex to plasmin and at least two peptide bonds are cleaved in the process. The data presented in this paper reveal that initially an internal peptide bond of plasminogen (in the complex) is cleaved to yield a two-chain, disulfide-linked plasmin molecule. The heavy chain (Mr approximately 67,000 with NH2-terminal glutamic acid) of this plasmin molecule has an identical NH2-terminal amico acid as the native plasminogen. The light chain (Mr approximately 25,000 with NH2-terminal valine) of plasmin is known to be derived from the COOH-terminal portion of the parent plasminogen molecule. A second peptide is then cleaved from the NH2-terminal end of the heavy chain of plasmin producing a proteolytically modified heavy chain (Mr =60.000 with NH2-terminal lysine). This cleavage of the NH2-terminal peptide from the heavy chain of plasmin is shown to be mediated by the dissociated free plasmin present in the activation mixture. Plasmin in the streptokinase-plasmin complex is unable to cleave this NH2-terminal peptide. This same NH2-terminal peptide can also be cleaved from native Glu-plasminogen or from the Glu-plasminogen-streptokinase complex by free plasmin and not by a complex of streptokinase-plasmin. From these studies we conclude (a) in the streptokinase-plasminogen complex, the NH2-terminal peptide need not be released prior to the cleavage of the essential Arg-Val peptide bond which leads to the formation of a two chain plasmin molecule and (b) that this peptide is cleaved from the native plasminogen or from the heavy chain of the initially formed plasmin in the streptokinase complex by free plasmin and not by the plasmin associated with streptokinase. In agreement with this, plasmin associated with streptokinase was unable to cleave the NH2-terminal peptide from the isolated native heavy chain possessing glutamic acid as the NH2-terminal amino acid; whereas free plasmin readily cleaved this peptide from the same isolated Glu-heavy chain.

Amino Acids↗
PubMed1988

Plasminogen interactions with platelets in plasma.

In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5'-diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg-Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen-platelet binding, nor does antithrombospondin antibody. epsilon-Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP-activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.

Adenosine Diphosphate↗
PubMed1993

Fibrinolysis in patients with acute ischaemic heart disease. With particular reference to systemic effects of tissue-type plasminogen activator treatment on fibrinolysis, coagulation and complement pathways.

The plasminogen activator systems in the blood, the coagulation system, and the complement pathways are reviewed. The review describes the role of the vascular intima in activation of coagulation and fibrinolysis and the interrelations between the complement system and haemostatic mechanisms. Physiological activation of fibrinolysis may be triggered by and limited to fibrin because of a special affinity of plasminogen and plasminogen activators. The binding of plasminogen to fibrin is regulated by histidine-rich glycoprotein, and the primary physiological inhibitor of generated plasmin is alpha 2-antiplasmin and especially the plasminogen-binding form of this immediate plasmin inhibitor. Plasminogen activator inhibitors in the blood, that is, notably plasminogen activator inhibitor type 1 (PAI-1), bind circulating tissue-type plasminogen activator (t-PA). However, local fibrinolysis in vivo mediated by t-PA may be independent of complex formation between plasminogen activator inhibitors and t-PA in the fluid phase. Circulating plasminogen activator inhibitors might regulate fibrinolysis by increasing the clearance of t-PA from the blood. The urokinase-type and factor XII-dependent fibrinolytic proactivator system can be activated following t-PA-mediated generation of plasmin, and could thus serve as an amplification system of t-PA-induced fibrinolysis. It is claimed that the as yet uncharacterized proactivator is essential for optimal generation of plasminogen activator activity by the factor XII-dependent fibrinolytic system. The normal antithrombotic condition of the vascular intima probably results from lack of tissue factor activity and the presence of significant antithrombotic components comprising, among others, antithrombin III and the protein C-protein S system. A number of pathophysiologic stimuli, notably mediators of the acute phase response such as the cytokines interleukin-1 and tumour necrosis factor-alpha (cachectin), have the potential to induce the vascular endothelium to express procoagulant activity. Vascular endothelium promoting coagulant activity releases increased amounts of t-PA antigen and PAI-1 antigen into the circulation, and elevated levels in the blood of both may be regarded as a marker of a generalized procoagulant condition involving the vascular endothelium. In a prospective study in patients with unstable angina pectoris, patients in whom disease progresses and acute myocardial infarction develops, have increased amounts of t-PA antigen and PAI-1 antigen in the blood. This suggests that the procoagulant potential and atherosclerotic process of the vascular intima is more pronounced in the risk group.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute Disease↗
PubMed1998

Modulation of the plasminogen activator/plasmin system in rat liver regenerating by recruitment of oval cells.

The proteolytic cascade involving plasminogen activators and plasmin appears to have an important function in tissue regeneration. We have investigated the expression and cellular localization of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) as well as plasminogen activation in rat liver regeneration by recruitment of progenitor (oval) cells. Using a model in which surgical partial hepatectomy is combined with feeding of 2-acetylaminofluorene (2-AAF) to induce liver regeneration by proliferation and differentiation of oval cells, expression of uPA, uPAR, and PAI-1 was detected by immunohistochemistry mainly in the duct-like formations of expanding oval cells. Plasminogen activation, as assessed by direct zymography on frozen liver sections, was located over the expanding oval cell populations but not over mature hepatocytes. Plasminogen activation was not detected in control liver. Expression of uPA, uPAR, and PAI-1, as assessed by immunohistochemical and Northern blot analyses, was also observed, when cells located in and in close proximity to the bile epithelial structures were activated to enter DNA-synthesis in response to 2-AAF, and after in vivo infusion of various growth factors. Given the physiologic function of plasminogen activation in fibrinolysis, and plasminogen activators in activation of latent growth factors, the selective expression of the plasminogen activator/plasmin proteolytic cascade in oval cells expanding during liver regeneration in response to the combination of 2-AAF and partial hepatectomy, may confer a proliferative advantage to these cell populations in an extracellular matrix containing both fibrin and latent growth factors.

2-Acetylaminofluorene↗
PubMed1997

Denatured proteins as cofactors for plasminogen activation.

Activation of covalently intact plasminogen by tissue-type plasminogen activator (tPA) is facilitated by a majority of proteins subjected to denaturing conditions. Except for heat-denatured apoferritin, the denatured proteins examined require partial proteolysis by plasmin for cofactor activity. The same proteins in their native state are resistant to proteolysis with plasmin and develop no activity. Denatured preparations of apoferritin, antithrombin, alpha1-protease inhibitor, alpha2-macroglobulin, and albumin also accelerate des(1-77)-plasminogen activation by tPA. The rate enhancements are comparable with that of the fibrin(ogen) fragments on a w/w basis. The cofactor activities are inhibited by 6-aminohexanoate and inactivated by pepsin. Analysis of heat-denatured apoferritin and albumin preparations by ultracentrifugation and gel chromatography indicates that cofactor is associated predominately with aggregates, which have binding capacity for both tPA and zymogen. Heat-denatured albumin pretreated with plasmin decreases K(M) and increases k(cat) for both intact plasminogen and des(1-77)-plasminogen activation by tPA, yielding catalytic efficiencies in excess of 8 x 10(3) M(-1) s(-1) and 2 x 10(4) M(-1) s(-1), respectively. Because of enhanced plasmin-catalyzed proteolysis of plasminogen to des(1-77)-plasminogen, activation by urokinase-type plasminogen activator is also facilitated by denatured proteins; activation of des(1-77)-plasminogen is not affected. It is concluded that denatured proteins serve as both cofactors and substrates in the fibrinolytic system, and that enhancement of plasminogen activation by denatured proteins is mechanistically indistinguishable from that observed with fibrin.

Animals↗
PubMed1981

Expression of the catalytic activity of plasminogen activator under physiologic conditions.

We have investigated the factors governing the plasminogen-dependent fibrinolysis catalyzed by the serine proteinase, plasminogen activator (EC 3.4.21.-), under physiologic conditions. We found that live rabbit fibroblasts digested much less fibrin than predicted by cell-free assay of the secreted plasminogen activator. The reduced catalytic activity of plasminogen activator expressed by cells growing on fibrin was regulated by the salt concentration of culture medium. The plasminogen activators of cells from several mammalian species were inhibited by physiologic salt concentrations (0.15 M NaCl) in cell-free assays. CaCl2 and KCl, but not D-glucose, were also effective inhibitors. The catalytic activity of purified human urokinase and of plasmin was unaffected by increased ionic strength. Plasminogen activators secreted both spontaneously and in response to stimulation by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, were inhibited by 0.15 M NaCl. Physiologic salt concentration appeared to function by interacting with plasminogen activator, or plasminogen, and a third component, possibly a reversible inhibitor. One consequence of this regulation of plasminogen activator under physiologic conditions is the limitation of plasminogen-dependent fibrin degradation by living cells.

Animals↗
PubMed1986

The active and the inactive plasminogen activator inhibitor from human endothelial cell conditioned medium are immunologically and functionally related to each other.

In human endothelial cell conditioned medium a fast-acting inhibitor of tissue-type plasminogen activator and urokinase has been detected. Moreover, an inactive inhibitor of these plasminogen activators is present, that can be activated by denaturing agents such as sodium dodecyl sulphate (SDS). The mutual relationship between these inhibitors was studied. The fast-acting plasminogen activator inhibitor from human endothelial cell conditioned medium was purified in a complex with tissue-type plasminogen activator by immune adsorption, using an immobilized anti-tissue-type plasminogen activator antibody. With the complex as an antigen, specific antibodies were raised against this inhibitor in rabbits. The antiserum immunoreacted with both the inactive and the fast-acting plasminogen activator inhibitor. Endothelial cell conditioned medium (containing the inactive plasminogen activator inhibitor) was treated with SDS and the inhibitory activity that emerged was purified. The SDS-generated product formed complexes with tissue-type plasminogen activator with the same molecular mass as those formed with the fast-acting inhibitor. Moreover, the inhibitory activity generated by SDS treatment showed the same kinetic behaviour with tissue-type plasminogen activator as did the fast-acting inhibitor. These data show that the fast-acting and the inactive plasminogen activator inhibitor are immunologically and functionally related to each other, and probably represent different molecular forms of the same protein.

Antibody Specificity↗
PubMed2001

Oxygen modulates the release of urokinase and plasminogen activator inhibitor-1 by retinal pigment epithelial cells.

The aims of this study were to examine the effect of oxygen, in the presence or absence of exogenous growth factors, on the release of plasminogen activators and plasminogen activator inhibitor-1 by cultured human retinal pigment epithelial cells. Antigen and activity levels of urokinase, tissue plasminogen activator and plasminogen activator inhibitor were measured in conditioned media after cells were exposed to three different oxygen environments: hypoxia, normoxia and hyperoxia. Overall proteolytic balance was determined by zymography. The effects of exogenous basic fibroblast growth factor and transforming growth factor-beta were also examined. it was found that retinal pigment epithelial cells released urokinase, tissue plasminogen activator and plasminogen activator inhibitor in measurable quantities. After 48 h, urokinase levels were highest at normoxia, reaching 7.2ng/10(6) cells (+/-2.0 SEM), whereas plasminogen activator inhibitor 1 levels were highest at hyperoxia, reaching 67.5ng/10(6) cells (+/-3.7 SEM). Tissue plasminogen activator levels were minimal (<0.5ng/10(6) cells) and unaffected by both oxygen and growth factors. Overall proteolytic activity was also greatest at normoxia. Fibroblast growth factor stimulated urokinase production dose-dependently, but plasminogen activator inhibitor only minimally. Transforming growth factor-beta stimulated plasminogen activator inhibitor production dose-dependently but urokinase only at higher concentrations. These results suggest that both oxygen tension and growth factors may interact to modulate the proteolytic properties of the human retinal pigment epithelium.

Cells, Cultured↗
PubMed1989

Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants.

Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.

Cell Line↗
PubMed1994

Comparison of plasminogen binding and activation on extracellular matrices produced by vascular smooth muscle and endothelial cells.

Plasminogen is the zymogen form of the serine proteinase plasmin. Although plasmin functions primarily as a fibrinolytic enzyme, recent evidence from numerous laboratories indicates that plasmin is also active in extracellular-matrix (ECM) proteolysis. The role of plasmin in ECM degradation suggests that activation of plasminogen may be regulated by interaction with components of the ECM. In the current study, we have investigated binding and kinetic interactions between plasminogen, plasminogen activators and ECM synthesized by either vascular smooth muscle cells (SMCECM) or endothelial cells (ECECM). We report binding of plasminogen, tissue-type plasminogen activator (t-PA) and urinary-type plasminogen activator (u-PA) to intact SMCECM with concentrations of ligand yielding half-maximal binding (B50) of 34, 5 and 15 nM, respectively. ECECM bound only plasminogen and t-PA, with B50 values of 32 nM and 10 nM, respectively. The initial rate of t-PA-catalyzed plasminogen activation was enhanced 41-fold in the presence of SMCECM and 27-fold on ECECM. In contrast, u-PA-catalyzed activation on SMCECM and ECECM was increased only 1.5-fold or 3-fold, respectively. These data suggest that the ECM may provide an alternative surface for assembly and regulation of plasminogen activation.

Amino Acid Sequence↗
PubMed1997

Plasminogen activation by pro-urokinase in complex with its receptor--dependence on a tripeptide (Spectrozyme plasmin).

The intrinsic activity of single-chain pro-urinary-type plasminogen activator (pro-uPA) and whether its receptor (uPAR) potentiates this activity remains controversial. In this report, the pro-uPA/uPAR-(1-281)-peptide complex in solution is shown to have equivalent plasminogen-activator activity to that of active two-chain uPA (tc-uPA). However, the activity of the complex was dependent on a synthetic tripeptide, Spectrozyme plasmin (Spl, H-D-2-aminohexanoic acid(Ahx)-hexatyrosyl-lysine-p-nitroanilide), which can also be used as a chromogenic substrate for plasmin. Furthermore, this activity could be completely suppressed by commonly used carrier proteins and detergents. The pro-uPA/uPAR-(1-281)-peptide complex at 1 nM displayed similar activity to that of tc-uPA for either [Glu1]plasminogen or [Lys77]plasminogen in chromogenic assays with Spl present as the plasmin substrate. When assayed with another plasmin substrate, S2251, the pro-uPA/uPAR-(1-281)-peptide complex was unable to activate plasminogen. The pro-uPA/uPAR-(1-281)-peptide complex and tc-uPA also showed a similar extent of plasminogen activation as measured by SDS/PAGE, when incubated with plasminogen and Spl in the presence of 100 micro M aprotinin, and plasminogen activation by pro-uPA alone was also stimulated in the presence of Spl in this assay. Activation of plasminogen by the pro-uPA/uPAR-(1-281)-peptide strictly required the presence of Spl, and pro-uPA remained in single-chain form during these assays. This activity of the pro-uPA/uPAR-(1-281)-peptide complex but not that of tc-uPA was completely inhibited by human serum albumin, bovine serum albumin, Tween-80, Triton X-100, and Pluronic-F68. Taken together, the data indicates that uPAR-(1-281)-peptide itself is not sufficient to augment pro-uPA activity and the presence of an effector molecule (e.g. Spl) is required to elicit the full plasminogen-activator activity of the pro-uPA/uPAR-(1-281)-peptide complex. It remains to be seen whether there is a physiological counterpart to this phenomenon.

Enzyme Activation↗