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)

The tetra-alanine substitution variant KHRR 296-299 AAAA of tissue-type plasminogen activator (t-PA) was previously shown to have enhanced fibrin specificity and enhanced activity in the presence of fibrin compared with the wild-type form of the molecule. The structural requirements for these alterations in enzymatic activity were investigated by constructing several amino acid substitution variants at each of the positions from 296 to 299 and evaluating their activities under a variety of conditions. Effects on plasminogen activator activity were common among the point mutants at positions 296-299; nearly all had a phenotype similar to the KHRR 296-299 AAAA variant. The greatest effects on enzymatic function were found with multiple substitution variants, but some single charge reversals and proline substitutions had substantial effects. The enhanced fibrin specificity of KHRR 296-299 AAAA t-PA results in less fibrinogenolysis than seen with wild-type t-PA. Approximately four times greater concentration of KHRR 296-299 AAAA compared with wild-type t-PA was required to consume 50% of the fibrinogen in human plasma.
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PMID:Involvement of residues 296-299 in the enzymatic activity of tissue-type plasminogen activator. 140 47

Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of the serine proteases tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). To systematically investigate the roles of the reactive center P1 and P1' residues in PAI-1 function, saturation mutagenesis was utilized to construct a library of PAI-1 variants. Examination of 177 unique recombinant proteins indicated that a basic residue was required at P1 for significant inhibitory activity toward uPA, whereas all substitutions except proline were tolerated at P1'. P1Lys variants exhibited lower inhibition rate constants and greater sensitivity to P1' substitutions than P1Arg variants. Alterations at either P1 or P1' generally had a larger effect on the inhibition of tPA. A number of variants that were relatively specific for either uPA or tPA were identified. P1Lys-P1'Ala reacted 40-fold more rapidly with uPA than tPA, whereas P1Lys-P1'Trp showed a 6.5-fold preference for tPA. P1-P1' variants containing additional mutations near the reactive center demonstrated only minor changes in activity, suggesting that specific amino acids in this region do not contribute significantly to PAI-1 function. These findings have important implications for the role of reactive center residues in determining serine protease inhibitor (serpin) function and target specificity.
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PMID:Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center. 155 96

rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahern et al. (J Biol Chem 1990; 265:5540-5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse). The specific fibrinolytic activities on fibrin plates were 160,000 +/- 17,000, 210,000 +/- 88,000 and 460,000 +/- 72,000 IU/mg (mean +/- SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k2/Km) in the absence of fibrin were comparable (1.1 to 1.7 x 10(-3) microM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 +/- 0.14 micrograms/ml rt-PA P47G, K49N, 0.36 +/- 0.01 micrograms/ml rt-PA and 0.17 +/- 0.01 micrograms/ml alteplase, respectively (mean +/- SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemical, thrombolytic and pharmacokinetic properties of rt-PA P47G, K49N, a substitution variant of human tissue-type plasminogen activator. 163 93

The effect of pretreatment of metastatic B16 melanoma cells with 10(-6) M all trans-retinoic acid resulted in a significant inhibition of lung colonization following injection of 10(5) cells into the tail vein of syngeneic C57BL mice. Adhesion of melanoma cells to vascular endothelial cell monolayers, and subendothelial extracellular matrix was also inhibited by pretreatment with retinoic acid, as was tumour cell aggregation following seeding of pretreated cells on to 0.5% agar. Release of 35SO4 from radiolabelled subendothelial extracellular matrix by melanoma cells was essentially unaltered by retinoic acid pretreatment, as was the release of radiolabel from [3H]proline-labelled matrix, while plasminogen activator activity was enhanced in retinoic-acid-treated cells. These observed changes in adhesive properties may be responsible, at least in part, for the retinoic-acid-induced inhibition of lung colonization.
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PMID:Retinoic acid-induced inhibition of metastatic melanoma cell lung colonization and adhesion to endothelium and subendothelial extracellular matrix. 173 48

The present study determined whether inhibitors of angiotensin converting enzyme (ACE) can ameliorate radiation-induced pulmonary endothelial dysfunction and pulmonary fibrosis in rats sacrificed 2 months after a range of single doses of 60Co gamma rays to the right hemithorax. Four indices of pulmonary endothelial function were monitored: right lung ACE and plasminogen activator (PLA) activity, and prostacyclin (PGI2) and thromboxane (TXA2) production. Hydroxyproline (HP) content served as an index of pulmonary fibrosis. Rats consumed either control powdered chow or feed containing one of five modifying agents continuously after irradiation. The modifiers included three ACE inhibitors: Captopril, CL242817, and CGS13945, respectively, a thiol, a thioacetate, and a nonthiol compound. All of the ACE inhibitors are analogues of proline. Two additional modifiers were tested: penicillamine, a thiol with no ACE inhibitory activity; and pentoxifylline, a vasodilator that is neither a thiol nor an ACE inhibitor. Radiation produced a dose-dependent decrease in lung ACE and PLA activity, and an increase in PGI2 and TXA2 production and in HP content. All ACE inhibitors attenuated the radiation-induced suppression in lung ACE and PLA activity. All thiol or thioacetate compounds ameliorated the radiation-induced increase in PGI2, TXA2, and HP. The two agents that were both thiols and ACE inhibitors (Captopril and CL242817) spared all of the radiation reactions, while the compound that was neither a thiol nor an ACE inhibitor (pentoxifylline) spared none of the reactions. These data suggest a novel application for ACE inhibitors in general, and for Captopril in particular, as modifiers of radiation pneumotoxicity.
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PMID:Radiation pneumotoxicity in rats: modification by inhibitors of angiotensin converting enzyme. 173 1

Inhibition of in vitro platelet aggregation and release of contents of platelet granules is necessary in order to assess accurately platelet activation in vivo. This can be accomplished by using a variety of inhibitors added to blood collection containers. An additive mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD) provides a practical alternative to a mixture of acid citrate dextrose (ACD), acetylsalicylic acid (aspirin), and prostaglandin E1 (PGE1) because of the stability problems associated with PGE1. Inhibition of in vitro fibrinolysis is essential for the accurate measurement of fibrin degradation products (FDP). This can be accomplished by using a mixture of thrombin, soybean trypsin, or aprotinin into which blood is collected. However, in patients receiving heparin, the fibrinolysis inhibitor mixture is ineffective unless it is supplemented with reptilase. With increasing use of recombinant tissue-type plasminogen activator therapy (rt-PA), an inhibitor such as D-phenylalanine-proline-arginine-chloromethylketone (PPACK) used as a blood collection additive is superior to a conventional protease inhibitor, such as aprotinin.
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PMID:Inhibition of in vitro platelet aggregation and release and fibrinolysis. 250 43

A number of physiological responses and adjustments occur at high altitude to compensate for hypoxia. We hypothesized that interference with one component of the normal compensatory process, the sympathetic nervous system, would hinder altitude acclimatization and thereby exacerbate acute mountain sickness (AMS) and compromise well-being. Twelve young males (21.2 +/- 0.4 years) received either 80 mg propranolol (PRO; n = 6) or placebo (PLA; n = 6), t.i.d. at sea level (SL) and during the first 15 d of a 19-d residence at 4,300 m (HA). Individuals were randomly assigned to each group. The Environmental Symptoms Questionnaire (ESQ) was administered at SL and twice daily (AM and PM) during the entire altitude exposure in order to assess AMS symptoms and subjective feelings of well-being. Supine heart rate (HR) was determined at rest twice at SL and four times at HA. HR in the PLA group increased 40% over SL values (57 +/- 3 to 80 +/- 4 beats/min) by day 7 at HA (p less than 0.01). HR in the PRO group did not increase above SL values during medication at HA. By 4 d after the medication administration was terminated, HR in the PRO group had increased and did not differ from the PLA group. Throughout the entire altitude exposure, ESQ scores for the PRO group were lower than or similar to the PLA group. Furthermore, cessation of PRO treatment did not result in a change in well-being. These findings suggested that interference with the normal acclimatization process by beta-adrenergic blockade did not exacerbate AMS or reduce feelings of well-being.
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PMID:Effects of propranolol on acute mountain sickness (AMS) and well-being at 4,300 meters of altitude. 276 52

Monolayers of bovine microvascular endothelial cells (BMECs) grown on connective tissue derived from human amniotic membrane were used to examine the transendothelial migration of human neutrophils in vitro. Neutrophils placed above these cultures migrated in response to a chemotactic gradient generated by placing 10(-7) M-formyl-methionyl-leucyl-phenyl-alanine (fMLP) below the cultures. Under these conditions, an average of 29 +/- 12% of the total population of neutrophils migrated beneath the endothelium after 1 or 2 h of incubation. Neutrophil migration in the absence of fMLP or in the presence of equal concentrations of fMLP above and below the cultures was less than 8% of the response to a 10(-7) M-fMLP gradient. Migration was a rapid event. Neutrophils began adhering to the apical surface of the endothelium within 2 min following exposure to an fMLP gradient; Ca2+ was required for this initial adhesion. Within 10 min, the majority of neutrophils associated with the BMEC-amnion cultures had migrated beneath the endothelial monolayer. Ultrastructural studies revealed that the initial adhesion between migrating neutrophils and endothelium was characterized by close contact between the two types of cell in focal areas. This close association was maintained as the neutrophils traversed the clefts between endothelial cells. Following their migration across the endothelium, neutrophils often were observed lying between the endothelium and its basement membrane. With time, the neutrophils penetrated the basement membrane and moved into the underlying amniotic connective tissue. To test the role of neutrophil proteinases in breaching endothelial and subendothelial barriers, migration was allowed to proceed in the presence of a variety of proteinase inhibitors, including p-nitrophenyl p'-guanidinobenzoate, soybean trypsin inhibitor, 6-aminocaproic acid, alpha 1-proteinase inhibitor, leupeptin, antipain and methoxysuccinyl alanine-alanine-proline-valine chloromethyl ketone. None of these had a significant effect on the number of neutrophils that migrated or the depth to which they penetrated the amniotic tissue as compared with controls. In contrast, pepstatin and chymostatin reduced migration in response to fMLP to 7% and 52% of control values, respectively. However, these two inhibitors did not affect migration in response to another chemoattractant, leukotriene B4. Migration was neither enhanced nor inhibited by the following treatments: (1) removal of plasminogen from the calf serum used in the assay medium and addition of polyclonal antibody to plasminogen; (2) addition of monoclonal or polyclonal antibody to plasminogen activator.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Migration of neutrophils across monolayers of cultured microvascular endothelial cells. An in vitro model of leucocyte extravasation. 296 75

Pulmonary injury induced by the plant alkaloid monocrotaline is partially prevented by the angiotensin-converting enzyme (ACE) inhibitor captopril. CL242817 [(S-[R*,S*])-1-([3-acetylthio]-3-benzoyl-2-methyl-propionyl)- L-proline] is a new orally active ACE inhibitor under evaluation as an antihypertensive agent. To determine whether CL242817 also can modify monocrotaline-induced pulmonary injury, male rats were divided into four groups: control; CL242817 (60 mg/kg/day, po); monocrotaline (2.4 mg/kg/day, po); or monocrotaline plus CL242817, and were sacrificed after 6 weeks of continuous treatment. Rats receiving monocrotaline alone exhibited occlusive medial thickening of the pulmonary arteries, cardiomegaly, and right ventricular hypertrophy. Electron micrographs of monocrotaline-treated lung revealed degeneration of both endothelial and Type I epithelial cells, as well as marked interstitial hypercellularity and fibrosis. Hydroxyproline (collagen) content of monocrotaline-treated lung also increased significantly, confirming the fibrosis observed in the electron micrographs. These structural changes were accompanied by decreased lung ACE and plasminogen activator (PLA) activities, indicative of pulmonary endothelial dysfunction. Concomitant CL242817 treatment ameliorated all anatomic manifestations of monocrotaline injury, particularly the right ventricular hypertrophy, pulmonary arterial occlusion, epithelial degeneration, and interstitial fibrosis. CL242817 also significantly prevented the monocrotaline-induced increase in lung hydroxyproline content. In contrast, concomitant CL242817 did not significantly influence the suppressed lung ACE and PLA activities in monocrotaline-treated rats. CL242817 alone produced retarded weight gain, decreased heart weight relative to body weight, decreased lung hydroxyproline content and ACE activity, and increased serum ACE activity and plasma AII concentration. Thus CL242817 resembles captopril, both in its ability to ameliorate monocrotaline-induced pulmonary injury in rats, and in many of its side effects.
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PMID:Monocrotaline-induced cardiopulmonary damage in rats: amelioration by the angiotensin-converting enzyme inhibitor CL242817. 301 47

Tissue-type plasminogen activator (tPA) cDNA derived from human uterine mRNA was inserted into different yeast expression vectors. All such expression plasmids carried a yeast acid phosphatase (PHO5) promoter, a 2-micron plasmid replication origin, transcription termination signals, and a selectable TRP1 gene. Plasmid pYBDT-10 contained the entire tPA coding region ("pre-pro-tPA"), pYBDT-10-PRO contained a sequence encoding the putative pro-tPA precusor, and pYBDT-6 contained only a mature tPA cDNA fused precisely in frame to the sequence encoding the entire signal peptide of acid phosphatase. All constructions directed the synthesis of single-chain tPA proteins that were readily precipitated with a specific antibody directed against human uterine tPA. Electrophoretic mobilities were approximately the same as those of the Bowes melanoma single-chain tPA and a 68-kD protein marker. Treatment of immunoprecipitates with endoglycosidase H resulted in increased electrophoretic mobilities, suggesting that these yeast products are glycosylated. Despite the use of either human or yeast signal sequences, however, tPA produced in yeast was not secreted into the culture medium, but rather was found only in cells following disruption with glass beads. Although this cellular tPA exhibited fibrinolytic activity, most of the activity was associated with large cellular debris.
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PMID:Expression of active human uterine tissue plasminogen activator in yeast. 393 15


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