Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. 2. An alkaline (pH10.5) extraction of cytokinase from rabbit kidney lysosome-microsome fraction, followed by chromatography on DEAE-cellulose at pH7.6 with stepwise or linear increase in concentration of phosphate buffer, gave an 86-fold purification of the enzyme. The purified material was non-proteolytic against casein and heated fibrin and was freeze-dried without significant loss of activity or solubility. 3. Cytokinase is a protein with E(0.1%) (1cm.)=0.87 at 280mmu, and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S(20,w) 2.9-3.1s and molecular weight 50000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH8 and pH9, and is maximally active and stable at pH8.5. It is inactivated by heat at 78 degrees . 4. Cytokinase and human urokinase have the same K(m) value and are inhibited in a partially competitive manner by in-aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. 5. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.
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PMID:Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase. 564 83

Fibrinolytic inhibitor was prepared from human aortas and some of its biochemical properties were investigated. The fibrinolytic inhibitor suppressed urokinase activity, but did not inhibit plasmin activity when assayed by fibrin plate method and synthetic fluorogenic substrate method. The urokinase inhibitor was a glycoprotein and migrated similar to alpha-globulin upon fibrin-agar electrophoresis. The molecular weight determined by gel filtration was approximately 98,000. The urokinase inhibitor was immunologically different from other known plasma protease inhibitors, such as alpha 2-plasmin inhibitor, alpha 2-macroglobulin, and alpha 2-antitrypsin. The interaction of urokinase with the inhibitor was dose-dependent. Progressive inactivation of urokinase occurred by increasing time of incubation with the inhibitor at 37 degrees C, and over 90% inhibition of urokinase required 30 min of incubation. The inhibitor of plasminogen activator in human aorta may be noteworthy in relation to thrombogenesis and atherogenesis.
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PMID:Inhibitor of plasminogen activator in human arterial wall. II. Biochemical characterization. 623 47

The function of fibrinolysis is to dissolve fibrin clots. The agent of fibrinolysis is plasmin, a glycoprotein with gram molecular weight (GMW) of 90,000. Under natural conditions, plasminogen is converted to plasmin by tissue plasminogen activator (TPA). Activation occurs on the fibrin surface, thus confining proteolytic activity to the appropriate site. Tissue plasminogen activator, produced by monoclonal methods, has recently been made available for limited therapeutic use. Currently streptokinase and urokinase are widely used therapeutically to activate plasminogen. These agents cause plasmin to be formed which is free in the circulation as well as bound to fibrin, resulting in proteolysis of circulating plasminogen and clotting factors. Fibrinolytic therapy has proven to be more beneficial than anticoagulation alone for deep vein thrombi and for pulmonary emboli. During therapy, laboratory studies demonstrate reduced concentrations of plasminogen, fibrinogen, and of alpha-2 plasmin inhibitor, and prolongation of activated partial thromboplastin time and thrombin time. Laboratory findings must be correlated with the clinical course. Demonstration of circulating plasmin-antiplasmin complex may be a useful indicator of active fibrinolysis.
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PMID:Fibrinolysis--a review. 623 87

Cultured bovine aortic endothelial cells are associated with an unusually stable fibrinolytic inhibitor (Loskutoff, D.J., van Mourik, J.A., Erickson, L.A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2956-2960). This inhibitor was purified to apparent homogeneity from medium conditioned by these cells by a combination of concanavalin A affinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a single-chain glycoprotein of apparent Mr 50,000 +/- 2,500 and isoelectric point of 4.5-5.0, and inhibits the ability of both urokinase and tissue-type plasminogen activator to cleave and active plasminogen. This inhibition of plasminogen activator activity is associated with the formation of an enzyme-inhibitor complex which can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified inhibitor retains full activity after incubation in the presence of 0.1% sodium dodecyl sulfate, or at pH 2.7, two treatments which rapidly destroy the activity of protease nexin, another cellular inhibitor of fibrinolysis. The inhibitor purified from cloned endothelial cells cultured in the presence of L-[3,4,5-3H]leucine represented 2.5-12% of the total radiolabeled protein released by the cells in a 24-h period. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a protein which inhibits plasminogen activators and is distinct from protease nexin. It is a major endothelial cell product, and, as such, probably plays an important role in regulating the fibrinolytic system of these cells.
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PMID:Purification of an inhibitor of plasminogen activator (antiactivator) synthesized by endothelial cells. 643 6

In the MCF7 human breast cancer cell line, estradiol stimulates the synthesis of a 52 K secretory glycoprotein and has been reported to increase the plasminogen activator (PA) activity in the culture medium. Since one PA isozyme has a molecular weight close to 52 000 daltons under denaturing conditions, we asked whether the 52 K protein was a PA. The PA activity released in serum-free conditioned medium was evaluated by the increase in [125I]casein digestion observed in the presence of plasminogen. The 52 K protein was estimated by analysing the released proteins on SDS-polyacrylamide gel electrophoresis. When the conditioned medium was chromatographed on concanavalin A-Sepharose, the 52 K protein was retained on the gel, but not the PA. The two proteins also appeared different on the basis of their competing efficiency in a radioimmunoassay developed to quantify the 52 K protein. An antiserum against human urokinase failed to immunoprecipitate the 52 K protein. Under our culture conditions estradiol increased 52 K, but not PA, production. These results clearly indicate that the estradiol-regulated 52 K protein is not a plasminogen activator.
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PMID:The estrogen-regulated 52 K protein adn plasminogen activators released by MCF7 cells are different. 653 16

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.
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PMID:Localization of vitronectin binding domain in plasminogen activator inhibitor-1. 751 53

Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepatocyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete significantly increased quantities of urokinase, an enzyme associated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These findings suggest that scatter factor may act as a paracrine mediator in pathologic angiogenesis associated with human inflammatory disease.
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PMID:Scatter factor induces blood vessel formation in vivo. 768 Apr 81

The deficiency of platelet function is the main defect of the hemostatic mechanism during cardiopulmonary bypass, which greatly exacerbates the postoperative bleeding complications. In this study, we assessed, from basic and clinical perspectives, the mechanism of relieving platelet damage by means of aprotinin. In vitro research confirmed that the addition of urokinase (40 U/ml) to platelet-rich plasma and the addition of plasmin (0.3 U/ml) to washed platelets made ristocetin-induced agglutination decline to 31.6% and 38.5% of control values, respectively. The extent of decline was positively correlated with the concentration of urokinase and plasmin. In addition, the platelet membrane glycoprotein Ib decreased to 76.4% of control value. With the addition of urokinase or plasmin to aprotinin-pretreated platelet-rich plasma or washed platelets, the changes in agglutination are not statistically significant and the decrement in glycoprotein Ib is much less marked. Further in vivo research revealed that cardiopulmonary bypass caused a decrease in plasma alpha 2-antiplasmin, indicating the fibrinolytic system activation. Meanwhile, ristocetin-induced agglutination decreased to 39.6% and platelet glycoprotein Ib decreased to 50% of preoperative values. However, with the administration of aprotinin, plasma alpha 2-antiplasmin during cardiopulmonary bypass did not change; platelet agglutination was improved, platelet glycoprotein Ib was preserved, and this consequently resulted in 46% lower blood loss after the operation. The results showed that fibrinolysis impaired platelet function, and this effect may be associated with the hydrolysis of glycoprotein Ib. Fibrinolytic activation occurred during cardiopulmonary bypass and contributed to postoperative platelet dysfunction to a great extent. Aprotinin may inhibit fibrinolysis during cardiopulmonary bypass and thus relieve the platelet damage and improve the postoperative hemostatic mechanism.
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PMID:Mechanism of the preserving effect of aprotinin on platelet function and its use in cardiac surgery. 768 94

The urokinase plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol-linked glycoprotein, plays a central role in the regulation of pericellular proteolysis and participates in events leading to cell activation. Here, we demonstrate that uPAR, on a human melanoma cell line, is localized in caveolae, flask-shaped microinvaginations of the plasma membrane found in a variety of cell types. Indirect immunofluorescence with anti-uPAR antibodies on the melanoma cells showed a punctated staining pattern that accumulated to stretches along sides of cell-cell contact and membrane ruffles. uPAR colocalized with caveolin, a characteristic protein in the coat of caveolae, as demonstrated by double staining with specific antibodies. Further, uPAR could be directly localized in caveolae by in vivo immunoelectron microscopy. Both uPAR and its ligand, uPA, were present in caveolae enriched low density Triton X-100 insoluble complexes, as shown by immunoblotting. From such complexes, caveolin could be coprecipitated with uPAR-specific antibodies suggesting a close spatial association between uPAR and caveolin that might have implications for the signal transduction mediated by uPAR. Further, functional studies indicated that the localization of uPAR and its ligand in caveolae enhances pericellular plasminogen activation, since treatment of the cells with drugs that interfere with the structural integrity of caveolae, such as nystatin, markedly reduced cell surface plasmin generation. Thus, caveolae promote efficient cell surface plasminogen activation by clustering uPAR, uPA, and possibly other protease receptors in one membrane compartment.
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PMID:The urokinase-type plasminogen activator receptor, a GPI-linked protein, is localized in caveolae. 772 38

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg561-Val562. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn161. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.
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PMID:A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. 773 Mar 29


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