Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human endothelial cells (EC) assemble plasmin-generating proteins on their surface. We have previously identified an EC membrane protein (Mr approximately 40,000) which specifically binds tissue plasminogen activator (t-PA) but not urokinase (Hajjar, K.A., and Hamel, N. M. (1990) J. Biol. Chem. 265, 2908-2916). In the present study, t-PA receptor protein (t-PA-R) was purified to apparent homogeneity from a detergent extract of human placental tissue by diisopropyl fluorophosphate-t-PA affinity chromatography and preparative gel electrophoresis. In a solid phase binding assay wells coated with t-PA-R bound both 125I-t-PA and 125I-Lys-plasminogen (PLG), but not 125I-urokinase in a specific, reversible, and noncompetitive fashion. Binding of 125I-Lys-PLG, but not 125I-t-PA, to t-PA-R was 80% inhibited by a 20-100-fold molar excess of the PLG-like lipoprotein(a), or by the lysine analog, epsilon-aminocaproic acid (50 mM). A polyclonal anti-t-PA-R antibody inhibited 66 and 79% of the specific 125I-t-PA and 125I-Lys-PLG binding, respectively, to EC monolayers. Biosynthetically labeled 40-kDa protein coprecipitated with t-PA- or Lys-PLG-Sepharose beads, but not with unconjugated Sepharose. In a functional assay, t-PA associated with immobilized t-PA-R generated 6.4 times more plasmin than an equivalent amount of t-PA in the fluid phase. These results suggest that t-PA-R can bind both t-PA and Lys-PLG in a manner that mimics the EC surface. This protein may play a role in modulating plasmin generation on cell surfaces.
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PMID:The endothelial cell tissue plasminogen activator receptor. Specific interaction with plasminogen. 165 83

The effect of Escherichia coli strains isolated from blood and cerebrospinal fluid of septic infants on plasminogen activation was studied. These strains typically carry a filamentous surface protein, S fimbria, that has formerly been shown to bind to endothelial cells and interact with plasminogen. The bacteria effectively promoted plasminogen activation by tissue plasminogen activator (t-PA) which was inhibited by epsilon-aminocaproic acid. A recombinant strain expressing S fimbriae accelerated t-PA-catalyzed plasminogen activation to a similar extent as did the wild-type strains whereas the nonfimbriate recipient strain had no effect. After incubation with t-PA and plasminogen, the S-fimbriate strain displayed bacterium-bound plasmin activity whereas the nonfimbriate strain did not. Bacterium-associated plasmin generation was also observed with a strain expressing mutagenized S fimbriae that lack the cell-binding subunit SfaS but not with a strain lacking the major subunit SfaA. Both t-PA and plasminogen bound to purified S fimbriae in a lysine-dependent manner and purified S fimbriae accelerated t-PA-catalyzed plasminogen activation. The results indicate that E. coli S fimbriae form a complex with t-PA and plasminogen which enhances the rate of plasminogen activation and generates bacterium-bound plasmin. This may promote bacterial invasion and persistence in tissues and contribute to the systemic activation of fibrinolysis in septicaemia.
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PMID:Enhancement of tissue plasminogen activator-catalyzed plasminogen activation by Escherichia coli S fimbriae associated with neonatal septicaemia and meningitis. 167 56

The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.
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PMID:The mechanism of activation of plasminogen at the fibrin surface by tissue-type plasminogen activator in a plasma milieu in vitro. Role of alpha 2-antiplasmin. 169 17

Untreated Staphylococcus aureus cells, strain Cowan I, specifically bound 125I-Glu-plasminogen. The binding was inhibited by both unlabeled Glu-plasminogen and Glu-plasmin. The Lys form of plasminogen, which lacks the 8-kDa amino-terminal activation peptide, was approximately 100-fold more effective than the Glu form in competing with the binding of 125I-labeled Glu-plasminogen. This suggests an increase in binding affinity upon removal of the activation peptide. Fibronectin, fibrinogen and IgG, plasma components known to bind to the staphylococcal surface, did not significantly interfere with the binding. The competing activity in plasma was abolished by specifically absorbing plasminogen from the plasma sample. L-Lysine and a fragment of plasminogen containing three of the first five protein attachment domains present in the molecule (kringle structures) also competed with plasminogen for binding suggesting that the lysine-binding sites of plasminogen were involved in its interaction with staphylococci. Scatchard analysis revealed high- and low-affinity binding sites. Kd and the number of high-affinity binding sites were 1.7 nM and 780 binding sites/bacterial cell, respectively. 125I-Glu-plasminogen bound to staphylococcal surface was converted to plasmin by tissue-type plasminogen activator. The conversion took place also in the presence of plasma. If the conversion was carried out in the absence of low-molecular-mass plasmin inhibitors such as aprotinin, the bound Glu-plasmin was further converted to Lys-plasmin. The surface-bound plasmin was enzymically active, as judged by digestion of the synthetic substrate, S-2251. The plasminogen conversion shown by the present experiments not only leads to the surface-bound plasmin but seems to considerably increase the affinity of plasmin for its binding site. This may represent a physiologically relevant method for a bacterial cell to retain surface-bound active plasmin which is also protected from its soluble plasma inhibitors. This novel mechanism for staphylococci to adopt surface-bound proteolytic activity, without the interference of plasma components, may have some role in the tissue penetration and invasion of microbes during infection.
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PMID:Binding and activation of plasminogen at the surface of Staphylococcus aureus. Increase in affinity after conversion to the Lys form of the ligand. 170 Nov 46

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.
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PMID:Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion. 171 33

Thrombolytic effects of tissue-type plasminogen activator (t-PA) are limited by in vivo platelet activation and dynamic coronary vasoconstriction. To examine if the concurrent administration of a fibrin(ogen)-degradation product, pentapeptide 6A (Ala-Arg-Pro-Ala-Lys) with t-PA would improve the thrombolytic effects of t-PA, dogs with electrically induced coronary thrombus were given t-PA alone or with peptide 6A. In dogs given t-PA alone (0.75 mg/kg over 20 min), coronary blood flow was restored in 69% of animals (9 of 13 dogs), with a mean time to reflow of 21 +/- 10 min and duration of reflow of 35 +/- 18 min. Reocclusion occurred in 77% of dogs (7 of 9 dogs). With concurrent administration of peptide 6A (200 mumol), coronary venous 6-keto-PGF1 alpha concentrations increased from 221 +/- 71 to 422 +/- 161 pg/ml (p less than 0.05), but not with t-PA given alone. Coronary blood flow was restored in 7 of 11 dogs (reperfusion rate 64%), with mean time to reflow of 17 +/- 7 min and duration of reflow of 35 +/- 15 min. The coronary reocclusion rate was 86%. All these indices of thrombolysis were similar to those in dogs given t-PA alone. In ex vivo experiments, we also demonstrated release of endothelium-derived relaxing factor from canine coronary artery rings in response to peptide 6A. To further examine the role of prostacyclin (PGI2) in thrombolytic response to t-PA, eight other dogs were given t-PA with a PGI2 analog iloprost (100 ng/kg/min for 40 min).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Combined thrombolytic effects of tissue-plasminogen activator and a fibrinogen-degradation product peptide 6A or iloprost. 171 84

A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296-G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296-G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296-G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA-related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt-PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296-G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1. 173 87

The activation kinetics of single chain urinary-type plasminogen activator (scu-PA) by plasmin have been studied in detail. Nonstandard Michaelis-Menten kinetics were observed. To explain our results, we propose a model in which plasmin can exist in two conformations of lower activity (kcat/Km = 1.4 x 10(6) M-1 s-1) or higher activity (kcat/Km = 16.7 x 10(6) M-1 s-1) depending on whether a lysine binding site is occupied or free, respectively. These kinetic studies demonstrate that scu-PA interacts at this binding site (KD approximately 30 nM) and so is able to act as both a substrate and effector in this reaction. Binding was also demonstrated between scu-PA and Glu- or Lys-plasminogen at a high affinity site (KD approximately 65 nM), sensitive to the presence of lysine analogs. This suggests that scu-PA may be almost completely bound to plasminogen in plasma under normal physiological conditions and provides a possible explanation for the fibrin specificity of this activator, as discussed.
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PMID:Kinetics of plasmin activation of single chain urinary-type plasminogen activator (scu-PA) and demonstration of a high affinity interaction between scu-PA and plasminogen. 173 May 85

The pathogenesis of Hailey-Hailey disease and Darier's disease was investigated using immunocytological and explant-tissue-culture techniques. There was breakdown of the intercellular adhesions between keratinocytes in explants from clinically uninvolved skin of patients with Hailey-Hailey disease or Darier's disease. The major desmosomal components were present in the cultures and were expressed in a punctate peripheral pattern at cell-cell contact sites, but there was diffuse staining of acantholytic cells. Plasminogen, which is expressed by basal keratinocytes in normal skin, was detected in association with suprabasal acantholytic cells in skin biopsies from these diseases. Plasminogen was reversibly displaced from the cells by 6-aminohexanoic acid, suggesting that binding is mediated by a reaction with the lysine receptor on the plasminogen molecule. Plasminogen was also detected in separating cells in explant cultures and there was cytoplasmic expression of the plasminogen activator urokinase by these cells. These abnormalities are not unique to either disease and do not account for the phenotypic differences between Darier's disease and Hailey-Hailey disease, but plasmin generation may have a role in perpetuating cell separation.
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PMID:Cell adhesion in Hailey-Hailey disease and Darier's disease: immunocytological and explant-tissue-culture studies. 175 48

We investigated production of plasminogen activator (PA) and cartilage degradation induced by injection of recombinant human interleukin-1 (rhIL-1 alpha) in rabbit knees. Rabbits were injected intra-articularly (i.a.) with 100 ng rhIL-1 alpha and necropsied at 0, 3, 6, 18 and 54 h and synovial lavage and articular cartilage were collected. PA activity in the joint lavage was measured using Z-Lys-thiobenzyl ester as a substrate. Cartilage degradation was assessed by quantitating sulfated glycosaminoglycan (S-GAG) to hydroxyproline (Hyp) and appearance of keratan sulfate (KS) in synovial lavage by and ELISA. The PA activity in the lavage of IL-1 injected knees at 3, 6, and 18 h was elevated 8 to 10 fold compared to vehicle controls. At 54 h the activity declined to approximately one third of that seen at the earlier time points. KS in the joint lavage was highest at 18 h, suggesting proteoglycan degradation. The maximal loss of cartilage proteoglycan (S-GAG/Hyp) occurred by 54 h. These observations demonstrate that i.a. injection of IL-1 stimulated the production of PA activity within the rabbit joint. Since elevation of PA activity is followed by cartilage degradation, we investigated effect of anti-inflammatory agents on PA activity and cartilage degradation in this model. We found that triamcinolone, indomethacin and dexamethasone were able to suppress PA activity but not the cartilage degradation. These observations suggest that in this model of cartilage degradation suppression of PA is not sufficient to inhibit cartilage degradation.
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PMID:Elevation of synovial plasminogen activator activity after injection of interleukin-1 alpha into rabbit knee joint. 179 35


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