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)

Recombinant variants of tissue-type plasminogen activator (t-PA) were constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Five variants were designed to improve the function of t-PA by mutagenesis in the kringle 1 (K1) domain. The amino acids were replaced with the corresponding residues present in the kringle 2 (K2) domain of native t-PA. The t-PA mutants expressed were as follows: variant E94V.D95G with point mutations in Glu94----Val and Asp95----Gly; variant N115P.S119M, Asn115----Pro and Ser119----Met; variant P125A.R129Q.R13OS, Pro125----Ala, Arg129----Gln and Arg130----Ser; variant G161R.K162R.-S165W, Gly161----Arg, Lys162----Arg and Ser165----Trp; and variant N115P, Asn115----Pro, respectively. The half-life following intravenous bolus injection in rabbits was prolonged in all variants except P125A.R130S. This was particularly true for N115P.S119M. The kinetic parameters for plasminogen activation were improved in t-PA G161R.K162R.S165W which showed a 0.6-fold decrease in Km, and a 1.8-fold increase in Vmax, thus promoting a 2.7-fold increase in kcat/Km compared to native t-PA. For a similar degree of thrombolysis in the rabbit jugular vein thrombosis model, the thrombolytic activity of G161R.K162R.S165W, at the dose tested, was four-fold greater than that of native t-PA. Thus, the substitution of the amino acids in the K1 domain with those corresponding in the K2 domain significantly enhanced the enzymatic activity of t-PA and improved the plasma survival.
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PMID:Characterization of human tissue-type plasminogen activator variants with amino acid mutations in the kringle 1 domain. 133 23

Tissue-type plasminogen activator (t-PA) induces synthesis of a rapidly acting inhibitor, plasminogen activator inhibitor type-1 (PAI-1) in human umbilical vein endothelial cells (HUVEC) in culture. In vivo, an analogous process may induce negative feedback on the fibrinolytic system. To define specific determinants in the t-PA molecule contributing to the induction, PAI-1 synthesis was characterized in 35S-methionine labeled HUVEC in response to several molecular variants of t-PA. Catalytically active variants devoid of several specific structural domains in the A-chain retained the capacity to form complexes with PAI-1 and to induce increased concentrations of total PAI-1 (free and complexed) in conditioned media without depleting PAI-1 from the extracellular matrix. Surprisingly, a mutant t-PA with markedly reduced catalytic activity reflecting replacement of the active site serine with threonine (S478T) formed complexes with PAI-1 and induced increased PAI-1 synthesis as well. However, in contrast to wild-type t-PA and A-chain variants, it did not release 35S-methionine labeled PAI-1 from the extracellular matrix. Thus, its effects appeared to reflect increased secretion exclusively. Our results suggest that induction of PAI-1 synthesis in HUVEC by t-PA depends on its protease domain but that an active site serine is not a requirement.
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PMID:Determinants of induction of increased synthesis of plasminogen activator inhibitor type-1 in human endothelial cells by t-PA. 162 Dec 43

The polarized secretions (apical/basal) of newly synthesized total protein and proteases from prostatic epithelial sheets of PA-III cells grown in dual compartment chambers were investigated at various cell densities and culture conditions. PA-III cells grown in a serum free defined medium (SFDM) form morphologically polarized monolayers of epithelial cells. These cells secreted their 35S-methionine labeled total protein in a predominantly apical direction (apical/basal ratio, 4-8 fold), with a lesser proportion of protein secreted apically at lower cell densities of the PA-III cell monolayer. PA-III cells grown in 5% fetal calf serum (FCS) are morphologically squamous, comparable to the anaplastic phenotype, and exhibited an inversion of polarized total protein secretion (apical/basal ratio, 0.4-0.9 fold), with an increased proportion of total protein secreted in a basal direction at lower cell densities. Since the culture of PA-III cells in FCS may approximate the anaplastic phenotype we investigated the polarized secretion of proteases from these cells at various cell densities, and compared them with the secretory pattern of protease secretion from polarized PA-III cells cultured in SFDM. At lower cell densities of the PA-III cells grown in FCS the polarity of protease secretion was inverted such that metalloproteinases, tissue type plasminogen activator, and a 72 kD gelatinase were secreted in a predominantly basal direction, as well as urokinase and a gelatinase of 26 kD that were secreted more or less equally into the apical and basal compartments of the chambers. On the other hand, for cultures of PA-III cells grown in SFDM the aforementioned proteases exhibited predominantly an apically directed polarity of secretion. These results suggest that the anaplastic phenotype characterized by a loss of polarized structure may also be characterized by a functional loss or inversion of polarized secretion. The consequences of such a loss or inversion of polarized secretion would be to increase the localized concentrations of proteases along the basal domain of cells thereby facilitating degradation of the basement membrane and interstitial tissue in vivo.
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PMID:Density dependent polarized secretion of a prostatic epithelial cell line. 173 75

Vascular endothelial cells undergo morphological and functional changes at sites of cell-mediated immune responses which may serve to promote the pathogenesis of inflammation. These changes, described as "endothelial cell activation" can be invoked by a variety of cytokines which include interleukin I (IL-1), tumor necrosis factor (TNF), and lipopolysaccharide (LPS). We report here on the regulation of the plasminogen activator (PA) proteolytic system by human recombinant TNF alpha in short term cultures (less than 4 passages) of human umbilical vein endothelial cells (HUVECs). TNF alpha treatment of HUVECs enhanced the production of 55 kDa urokinase (u) PA activity and uPA antigen by fourfold, in a concentration dependent manner (5-100 U/ml), following a 24 h treatment as determined by PA zymography and micro-ELISA assays, respectively. This response was specific for uPA since, no change in extracellular tissue type PA activity and tPA antigen levels were noted under analogous conditions. A similar 4-fold increase in the de novo synthesis of [35S]-methionine radiolabeled uPA was observed by immunoprecipitation following a 24 h TNF treatment. The induction of uPA by TNF was inhibited by actinomycin D and cycloheximide implying the necessity of RNA and protein synthesis, respectively. The effect of TNF could not be prevented by the addition of IL-1 neutralizing antibodies. Therefore, it is unlikely that TNF acts through the induction of IL-1 secretion. Time course studies using PA zymography indicate that within 8 h after TNF exposure, a 2-fold increase in uPA activity above untreated basal levels was observed. Upregulation of extracellular uPA production in HUVECs following TNF treatment suggests yet a new aspect of cellular and interstitial PA regulation in endothelium during inflammation and angiogenesis.
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PMID:Tumor necrosis factor induction of urokinase-type plasminogen activator in human endothelial cells. 180 6

Evidence has recently been presented that activated macrophages (M phi) express both urinary (u-PA) and tissue type (t-PA) plasminogen activator. Major cell products of M phi and polymorphonuclear neutrophils (PMN) are reactive oxidants of the HOCl/chloramine type. Since PMN and M phi are involved in inflammatory and fibrinolytic processes, we were interested in the interaction of u-PA, t-PA, and plasmin with oxidants of the leukocyte type. The enzymes were treated with chloramine-T, which at pH 8.5 is a selective oxidant for methionine residues. Oxidation by chloramine-T of t-PA abolishes about 40% of both stimulation susceptibility of t-PA by fibrinogen degradation products (FDP) and affinity of t-PA to FDP. However, the plasminogenolytic and amidolytic activity of unstimulated t-PA as well as the plasminogenolytic activity of u-PA and the amidolytic activity of plasmin are not impaired. Identification of the amino acid residues in the t-PA responsible for the interaction with fibrin might be of great importance in order to understand the mechanism of the clot- selectivity of t-PA. The present study gives evidence that fibrin specificity of t-PA partly depends on chloramine oxidizable amino acids, presumably methionine residues. Hence, experimental data on the interaction between t-PA and fibrin, using oxidized and labelled t-PA should be interpreted with caution. It may be suggested that oxidants of the leukocyte type might regulate t-PA activity and selectivity for fibrin.
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PMID:Effect of oxidants on proteases of the fibrinolytic system: possible role for methionine residues in the interaction between tissue type plasminogen activator and fibrin. 182 45

Several groups have demonstrated that radioiodinated tissue-type plasminogen activator (t-PA) binds to saturable sites on human umbilical vein endothelial cells (HUVECs) in culture (Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719; Beebe, D. P. (1987) Thromb. Res. 46, 241-254; Barnathan, E. S., Kuo, A., van der Keyl, H., McCrae, K. R., Larsen, G. L., and Cines, D. B. (1988) J. Biol. Chem. 263, 7792-7799). Here we report that most of the specific binding of 125I-t-PA to our HUVEC cultures is accounted for by binding to (i) plasminogen activator inhibitor type 1 (PAI-1), a t-PA inhibitor produced in abundance by HUVECs; and (ii) specific binding sites present on the plastic culture surface. The contribution of the sites on plastic can be eliminated by taking several precautions. Then, most or all of the specifically bound 125I-t-PA is present in a sodium dodecyl sulfate-stable 110-kDa 125I-t-PA.PAI-1 complex. Interestingly, a radioiodinated mutant form of t-PA, S478A, which is catalytically inactive and therefore unable to form the covalent complex with PAI-1, still binds to HUVECs. In fact, this ligand binds to HUVECs in 10-30-fold greater amounts than does wild-type 125I-t-PA (resulting in greater than 1 x 10(7) S478A 125I-t-PA molecules bound/cell at 12 nM ligand concentration). In contrast, diisopropyl fluorophosphate-treated t-PA binds to HUVECs in much smaller amounts than does wild-type t-PA. Several findings suggest that PAI-1 is a major binding site for S478A t-PA. The vast amount of binding observed with S478A t-PA, compared with wild-type t-PA, may be accounted for by an observed large scale release of wild-type 125I-t-PA.PAI-1 complexes from the solid phase (cells or extracellular matrix) into the culture medium. Immunoprecipitation experiments demonstrate that, in contrast to wild-type t-PA, S478A t-PA does not extract [35S]methionine-PAI antigen from metabolically labeled extracellular matrix. It is proposed that t-PA releases PAI-1 from the solid phase when it forms the irreversible covalent complex with the inhibitor, a process that does not occur with the catalytically inactive mutant form of t-PA.
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PMID:Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activator with human umbilical vein endothelial cell monolayers. 210 33

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.
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PMID:Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli. 211 12

Increased extracellular proteolysis because of unregulated activation of blood coagulation, complement, and fibrinolysis is observed in thrombosis, shock, and inflammation. In the present study, we have examined whether the plasma kallikrein-kinin system, the classical pathway of complement, and the fibrinolytic system could be inhibited by alpha 1-antitrypsin reactive site mutants. Wild-type alpha 1-antitrypsin contains a Met residue at P1 (position 358), the central position of the reactive center. It did not inhibit plasma kallikrein, beta-factor XIIa, plasmin, tissue-type plasminogen activator (t-PA), or urokinase. In contrast, these serine proteases were inhibited by alpha 1-antitrypsin Arg358. For the inhibition of C1s, a double mutant having Arg358 and a Pro----Ala mutation at P2 (position 357) was required. This double modification was made because C1-inhibitor, the natural inhibitor of C1s, has Arg and Ala residues at positions P1 and P2. Plasminogen activator inhibitor 1, the natural inhibitor of t-PA, also has Arg and Ala residues at positions P1 and P2. In a purified system, alpha 1-antitrypsin Ala357-Arg358 was 150-fold less efficient against C1s than C1-inhibitor and 27,000-fold less efficient against t-PA than plasminogen activator inhibitor-1. In plasma, 2.3 microM alpha 1-antitrypsin Ala357-Arg358 reduced by 65% the formation of a complex between kallikrein and C1-inhibitor following activation of the intrinsic pathway of blood coagulation by kaolin. Furthermore, after supplementation by 2.0 microM alpha 1-antitrypsin Ala357-Arg358, zymographic analysis showed that the majority of the free t-PA of normal plasma formed a bimolecular complex with the double mutant. In contrast, 3.4 microM alpha 1-antitrypsin Ala357-Arg358 did not prevent the activation of the classical pathway of complement observed when normal serum is supplemented with anti-C1-inhibitor F(ab')2 fragment. These results demonstrate that alpha 1-antitrypsin Ala357-Arg358 has therapeutic potential for disorders with unregulated activation of the intrinsic pathway of blood coagulation and the fibrinolytic system; however, the double mutant is not an efficient inhibitor for the classical pathway of complement.
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PMID:Reactivity of alpha 1-antitrypsin mutants against proteolytic enzymes of the kallikrein-kinin, complement, and fibrinolytic systems. 219 58

Urokinase activity is regulated by the specific endogenous plasminogen activator inhibitors type 1 (PAI-1) and type 2 (PAI-2). One of these inhibitors, PAI-1, has been directly implicated in connective tissue metabolism by virtue of its ability to bind extracellular matrix proteins. Because the normal lung is relatively rich in urokinase and abnormalities in urokinase activity have been associated with fibrotic lung diseases, we have explored the possibility of local production of PAI-1 and PAI-2 in human lung. Reverse transcription and subsequent amplification by the polymerase chain reaction of total lung RNA revealed PAI-1 mRNA in each of three normal samples and in two specimens from patients with the adult respiratory distress syndrome (ARDS). In situ hybridizations of lung biopsy specimens from a patient with ARDS with cRNA probes to PAI-1 and PAI-2 indicated that alveolar macrophages express PAI-1 mRNA during the acute injury phase. Subsequent reverse transcription and PCR amplification of normal human monocyte and alveolar macrophage mRNA revealed that neither cell type expressed mRNA for urokinase inhibitors. However, after 24 h stimulation with endotoxin in vitro, monocytes were strongly positive for PAI-2 but negative for PAI-1 mRNA whereas, under the same conditions, alveolar macrophages exhibited mRNA for both PAI-1 and PAI-2. Metabolic labeling of endotoxin-stimulated alveolar macrophages with 35S-methionine followed by immunoprecipitation with PAI-1 and PAI-2 antibodies revealed that macrophages synthesized both PAI-1 and PAI-2. As judged by immunoprecipitation and functional studies, PAI-2 was found to be the major intracellular PA inhibitor whereas PAI-1 was found to predominate outside the cell. Thus, mononuclear phagocytes exhibit a developmental potential for PAI-1 expression. The release of PAI-1 by stimulated macrophages, as observed in the setting of ARDS, may be one mechanism by which these cells promote connective tissue accumulation.
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PMID:Developmental expression of plasminogen activator inhibitor type 1 by human alveolar macrophages. Possible role in lung injury. 223 Jan 26

Calcitonin-induced changes in gene expression at the level of protein synthesis were examined in cultured porcine kidney cells (LLC-PK1) using two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides. Twelve intracellular polypeptides showed reproducible changes in the relative intensity of labeling. From that set of polypeptides ten showed an increase and two a decrease in the labeling intensity after calcitonin treatment. One of the induced polypeptides was identified as plasminogen activator and two others were shown to be related to cytokeratins. The labeling of two induced and well-separated polypeptides of 70K and 95K Mr was quantitated and correlated with an increase in secretion of plasminogen activator.
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PMID:Hormonal regulation of protein synthesis in cultured kidney cells. 240 39


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