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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Potential approaches to improve thrombolytic agents comprise the construction of mutants and variants of tissue-type plasminogen activator (tPA) or of single chain urokinase-type plasminogen activator (scuPA, pro-urokinase), of chimeric plasminogen activators and of conjugates of plasminogen activators with monoclonal antibodies. tPA mutants have been constructed with altered pharmacokinetic properties or altered functional properties, including binding to and stimulation by fibrin, resistance to plasmin and to protease inhibitors. Mutants of tPA described to date, obtained by deletion/substitution of functional domains or of single amino acids, have markedly reduced clearances, but usually also reduced specific thrombolytic potencies. Mutants of scuPA with improved thrombolytic potencies have thus far not been reported. Chimeric molecules containing functional domains of both tPA and scuPA have intact enzymatic properties of uPA and some fibrin affinity of tPA. Surprisingly, chimeras endowed with fibrin affinity usually have unaltered or reduced thrombolytic potencies. However, a chimera consisting of amino acids 87-274 of tPA and amino acids 138-411 of scuPA, with negligible fibrin affinity, has a 10-fold higher thrombolytic potency than scuPA in animal models of venous thrombosis, as a result of a delayed in vivo clearance and a relatively maintained specific thrombolytic activity. Plasminogen activators conjugated with antifibrin or antiplatelet monoclonal antibodies, either chemically or by recombinant DNA technology, are targeted to blood clots, resulting in a 5- to 10-fold increased thrombolytic potency. Thus, it is possible to develop plasminogen activators with improved thrombolytic potency. Whether such agents will be clinically useful remains to be established.
Ann N Y Acad Sci 1992 Dec 04
PMID:Remaining perspectives of mutant and chimeric plasminogen activators. 130 56

The properties of the cationic locus within the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) that are responsible for stabilization of its interaction with the carboxylate moiety of omega-amino acid ligands have been assessed by determination of the binding constants of several such ligands to a variety of r-[K2tPA] mutants obtained by oligonucleotide-directed mutagenesis. We have generated, expressed in Escherichia coli, and purified alanyl mutants of individual histidyl,lysyl, and arginyl residues of r-[K2tPA] and determined the dissociation constants of several omega-amino acids, viz., 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine (L-Lys), and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), to each of the r-[K2tPA] variants. We find that K33 plays the most significant role as a cationic partner of the complementary carboxylate group of these ligands. When K33 is altered to a variety of other amino acids, the K33R mutant best stabilizes binding of all of these ligands. However, the r-K33L and r-K33F variants selectively interact with 7-AHpA almost as strongly (ca. 2-fold reduction in binding strength) as wild-type r-[K2tPA]. Increased polarity (K33Q) or a negative charge (K33E) at this sequence position significantly destabilizes binding of omega-amino acids to the muteins. We also found that the r-K33E mutant and, to a lesser extent, the r-K33Q variant selectively interact with a new ligand, 1,6-diaminohexane. These observations show that the omega-amino acid binding site of wtr-[K2tPA] could be redesigned to provide a new binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 1992 Dec 01
PMID:The cationic locus on the recombinant kringle 2 domain of tissue-type plasminogen activator that stabilizes its interaction with omega-amino acids. 133 68

Primary cultures of peripheral lung lobes were grown in a highly supplemented medium. Human lung endothelial cells (HLE) were isolated from the mixed population by FACS. The cells proliferated rapidly and were serially cultivated for at least 16 passages. Both early and late passage cells were positive for the standard endothelial markers. Factor VIII related-antigen (Factor VIII R-Ag), angiotensin-converting enzyme, acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-1,3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL) uptake, and bound the lectin Ulex europaeus agglutinin (UEA). Prostaglandin E2 was the major cyclooxygenase product of HLE, in contrast to human umbilical vein endothelial cells (HUVE), which synthesized PGI2 in excess of PGE2. Factor VIII R-Ag exhibited a diffuse cytoplasmic as well as an extracellular fibrillar distribution in HLE, in contrast to a vesicular (Weibel-Palade body) cytoplasmic distribution in HUVE. The HUVE did demonstrate some extracellular fibrillar Factor VIII R-Ag as well. Urokinase was the predominant plasminogen activator (PA) secreted by HLE, whereas tissue PA was predominant in HUVE cultures. HLE formed tube-like structures within 2 h of plating on a Matrigel matrix whereas HUVE formed larger tube-like structures only after 1 or more days. The properties described here indicate that human lung microvessel endothelium can be isolated and continuously grown from small tissue segments and express a number of properties that differ from those of HUVE. These studies provide further support for the concept that endothelial cells from different sources can exhibit considerable heterogeneity relating to their phenotypic and biochemical properties.
Am J Respir Cell Mol Biol 1992 Dec
PMID:Isolation, cultivation, and partial characterization of microvascular endothelium derived from human lung. 133 46

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t-PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.
Blood 1992 Dec 01
PMID:Endocytosis and lysosomal delivery of tissue plasminogen activator-inhibitor 1 complexes in Hep G2 cells. 133 99

We have screened six human squamous carcinoma cell lines for their ability to invade connective tissue by using the experimentally modified chorioallantoic membrane of a chick embryo as an in vivo model of invasion. In confirmation of our earlier studies, all the invasive cell lines expressed high levels of surface-bound urokinase type plasminogen activator (uPA). However, some cell lines expressing this activity were not invasive, suggesting that surface uPA, although necessary, was not sufficient. Since in addition to fibronectin, that can be degraded by uPA or plasmin, chorioallantoic membrane connective tissue contains collagen, we examined the profile of collagenases secreted by the various cell lines in search for an activity that would coincide with the invasive phenotype. We found, using gelatin substrate gels, that type IV gelatinase was produced by all six cell types tested, three cell types produced the M(r) 92,000 gelatinase, and three a lower-molecular-weight activity, which we identified by immunoprecipitation with specific antibodies, and by a direct assay of activity, as interstitial collagenase. Only the latter cells were found to be highly invasive. We showed previously that continuous culture in vitro of one of the carcinoma cell lines, HEp3, led to a gradual extinction of their malignant phenotype. To confirm the correlation between invasion and the production of interstitial collagenase, we examined these two functions in cells freshly isolated from a HEp3 tumor and intermittently during passage in vitro. We found that, although the surface uPA activity was slightly diminished in the in vitro grown cultures, it was still within the range of values found in highly malignant cells, suggesting that it is not the reason for the decrease in invasiveness. In contrast, the reduction in interstitial collagenase closely followed the loss of the invasive phenotype; after 30 in vitro passages the cells were almost completely devoid of interstitial collagenase and unable to invade. The decrease in collagenase activity was not the result of an increased tissue inhibitor of metalloproteinases production.
Cancer Res 1992 Dec 15
PMID:Invasion of connective tissue by human carcinoma cell lines: requirement for urokinase, urokinase receptor, and interstitial collagenase. 133 82

We previously reported that extracellular matrix invasion by the prostate cancer cell lines, PC-3 and DU-145 was contingent on endogenous urokinase being bound to a specific cell surface receptor. The present study was undertaken to characterize the expression of both urokinase and its receptor in the non-invasive LNCaP and the invasive PC-3 and DU-145 prostate cells. Northern blotting indicated that the invasive PC-3 cells, which secreted 10 times more urokinase (680 ng/ml per 10(6) cells per 48 h) than DU-145 cells (63 ng/ml per 10(6) cells per 48 h), had the most abundant transcript for the plasminogen activator. This, at least, partly reflected a 3 fold amplification of the urokinase gene in the PC-3 cells. In contrast, urokinase-specific transcript could not be detected in the non-invasive LNCaP cells previously characterized as being negative for urokinase protein. Southern blotting indicated that this was not a consequence of deletion of the urokinase gene. Crosslinking of radiolabelled aminoterminal fragment of urokinase to the cell surface indicated the presence of a 51 kDa receptor in extracts of the invasive PC-3 and DU-145 cells but not in extracts of the non-invasive LNCaP cells. The amount of binding protein correlated well with binding capacities calculated by Scatchard analysis. In contrast, the steady state level of urokinase receptor transcript was a poor predictor of receptor display. PC-3 cells, which were equipped with 25,000 receptors per cell had 2.5 fold more steady state transcript than DU-145 cells which displayed 93,000 binding sites per cell.
Thromb Haemost 1992 Dec 07
PMID:Expression of urokinase and its receptor in invasive and non-invasive prostate cancer cell lines. 133 29

A mechanism for penetration of basement membranes by Escherichia coli is presented. The mechanism is based on the ability of the S fimbriae of meningitis-associated E. coli to bind to vascular endothelium and choroid plexuses in brain and to basement membranes. On the other hand, the S and the type 1 fimbriae of E. coli immobilize plasminogen and tissue-type plasminogen activator; this process generates proteolytic plasmin activity on the surface of fimbriate cells. Our hypothesis is that bacterium-bound plasma activity, directed to basement membranes through fimbrial binding, promotes bacterial penetration through basement membranes.
FEMS Microbiol Lett 1992 Dec 15
PMID:Penetration of fimbriate enteric bacteria through basement membranes: a hypothesis. 136 72

Cell clones derived from a human melanoma metastasis selected for different integrin profiles were examined in vitro for invasive potential and biological and biochemical features potentially related to this process. Clones which expressed high levels of integrins showed high invasive potential, extracellular matrix degradation, and adhesion to gelatin-coated substrates. A correlation was also found between invasiveness and intracellular and extracellular plasminogen activator activity. Heparanase and collagenase type IV activities were apparently unrelated to invasiveness. gamma-Glutamyl transferase (GGT) activity was high in highly invasive clones, whereas melanin content was high in slightly invasive clones. Heterogeneity was also observed in cellular parameters such as cell dimensions, growth features and DNA index. The intrinsic biological and biochemical heterogeneity of a cell population derived from a single metastasis may be responsible for the different behaviour of clones, regardless of their invasive potential. Since slightly invasive cells are more differentiated than highly invasive cells, malignancy and differentiation are inversely correlated in such human melanoma clones.
Melanoma Res 1992 Dec
PMID:Biological and enzymatic features of human melanoma clones with different invasive potential. 136 80

Forty-two strains of Neisseria meningitidis and 17 of Neisseria gonorrhoeae were tested for their ability to interact with 125I-labeled Glu-plasminogen. All strains tested reacted substantially with plasminogen, resulting in uptake values of 20%-48%. Scatchard analysis with selected N. meningitidis strains demonstrated a dual-phase receptor interaction, one more avid receptor with a Kd of 50 nM and 3000-6000 receptors per bacterium and a second receptor with a Kd of 200 nM and 10,000-20,000 receptors per bacterium. Plasminogen uptake could be completely eliminated by low concentrations of epsilon-aminocaproic acid, suggesting that the lysine binding sites on the plasminogen molecule are involved in the receptor-ligand interaction. The binding of plasminogen to the bacterial receptor facilitates the tissue-type plasminogen activator-mediated conversion to Glu-plasmin, which also modifies itself to the Lys form. Receptor-associated plasmin is enzymatically active, monitored as a breakdown of the chromogenic substrate S-2251, and retains its activity in the presence of naturally occurring inhibitors in plasma.
J Infect Dis 1992 Dec
PMID:Binding of plasminogen to Neisseria meningitidis and Neisseria gonorrhoeae and formation of surface-associated plasmin. 138 49

The pattern of expression of urokinase type plasminogen activator (PA) in granulocyte-macrophage-CSF transgenic mice and their normal littermates was studied using RNAse protection assays and a plasminogen-dependent fibrinolytic assay for PA. Urokinase type PA mRNA was expressed at a high level in transgenic peritoneal cells and at a lower level in transgenic eye tissue and spleen, but not in equivalent tissue from the normal mice. Enzymically active PA was detectable in protein extracts from peritoneal cells taken from transgenic mice of less than 8 wk of age (young mice) but not from normals. Paradoxically, extracts from transgenic mice of more than 12 wk of age (old mice) showed little detectable PA activity despite continuing transcription in some mice of this age. The production of PA by peritoneal cells may be responsible for the spontaneous i.p. bleeding which is a feature of the transgenic mice and production in other tissues may help explain the local pathologic changes.
J Immunol 1992 Dec 01
PMID:Plasminogen activator in granulocyte-macrophage-CSF transgenic mice. 143 Nov 38


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