Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. 2. Primary astrocytes were cultured under standard conditions and prelabeled with [(14)C]DHA or with [(3)H]AA. Adenosine 5'-triphosphate (ATP) (20 micro M applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca(2+)-independent phospholipase A(2) (iPLA(2)), since it is blocked by the selective iPLA(2) inhibitor 4-bromoenol lactone (BEL, 5 micro M) and is not affected either by removal of Ca(2+) from extracellular medium or by suppression of intracellular Ca(2+) release through PLC inhibitor (U73122, 5 micro M). 3. AA release, on the other hand, which is stimulated by ATP, is attributed to Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)). AA release is abolished by U73122 and, by removal of extracellular Ca(2+), is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 micro M), a general inhibitor of intracellular PLA(2) s. 4. Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA(2) and 40 kDa protein reactive to iPLA(2) antibodies. 5. The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 micro M), and by the protein kinase A (PKA) activator dibutyryl-cAMP (1 mM). In contrast, release of AA is not affected by forskolin or dibutyryl-cAMP, but is almost completely blocked by 2,3-dideoxyadenosine (20 micro M) and inhibited by 34% by H89 (10 micro M), inhibitors of adenylyl cyclase and PKA, respectively. 6. Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. 7. Different sensitivities of iPLA(2) and cPLA(2) to Ca(2+) and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.
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PMID:Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipase A2 and is differently regulated by cyclic AMP and Ca2+. 1283 76

Bothropstoxin-II (Bthtx-II), an Asp-49 phospholipase A(2) (D-PLA(2)) isolated from Bothrops jararacussu snake venom is able to induce platelet aggregation in a concentration-dependent manner. This effect was not due to the release of ADP from platelets since the aggregation was not suppressed by ADP scavenger systems. PMSF and PPACK were unable to inhibit Bthtx-II-induced platelet aggregation. Thus, a thrombin-like proaggregating activity of Bthtx-II can be excluded as its mechanism of action. On the other hand, indomethacin at low concentrations inhibited more markedly the ATP-release reaction than the aggregation induced by Bthtx-II, indicating that generation of cyclooxigenase products is not the most important event for the platelet aggregation reaction. It was also found that staurosporine and genistein suppressed both platelet aggregation and ATP-release reactions, but not the platelet shape-change induced by Bthtx-II. Substances that either directly activates adenylyl cyclase enzyme (forskolin and PGE(1)) or cell-permeant increasing agents (dibutyril-cAMP) inhibited in a concentration-dependent fashion, the platelet aggregation effects induced by the protein. It is concluded that Bthtx-II induces platelet aggregation and secretion through multiple signal transduction pathways.
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PMID:Signal transduction pathways involved in the platelet aggregation induced by a D-49 phospholipase A2 isolated from Bothrops jararacussu snake venom. 1555 84

Increased mast cell numbers and mast cell activation represent one of the prevalent etiologic theories for interstitial cystitis, an inflammatory condition in the bladder. This study was designed primarily to determine whether increased mast cell tryptase in the bladder wall may play a role in activating bladder endothelial cell phospholipase A(2) (PLA(2)), leading to increased inflammatory phospholipid metabolite accumulation, which may propagate the inflammatory process. We stimulated human bladder microvascular endothelial cells with thrombin or tryptase and measured the activation of PLA(2) and the production of multiple membrane phospholipid-derived inflammatory mediators. Thrombin and tryptase stimulation resulted in activation of a Ca(2+)-independent PLA(2), leading to increased release of arachidonic acid and prostacyclin and increased production of platelet-activating factor. These responses were blocked completely by pretreatment of human bladder microvascular endothelial cells with the Ca(2+)-independent PLA(2)-selective inhibitor bromoenol lactone. The combination of increased prostacyclin and platelet-activating factor in the bladder circulation may result in vasodilation and increased polymorphonuclear leukocyte adherence to the endothelium and may facilitate recruitment of polymorphonuclear leukocytes to the bladder wall of patients with interstitial cystitis.
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PMID:Protease-activated receptor stimulation activates a Ca2+-independent phospholipase A2 in bladder microvascular endothelial cells. 1556 75

Thrombin stimulation of isolated rabbit ventricular myocytes activates a membrane-associated, Ca(2+)-independent PLA(2) (iPLA(2)) that selectively hydrolyzes plasmalogen phospholipids and results in increased production of arachidonic acid and lysoplasmenylcholine. To determine whether MAPK regulates myocardial iPLA(2) activity, we isolated ventricular myocytes from rabbit heart by collagenase digestion and pretreated them with MAPK inhibitors before stimulating them with thrombin. Pretreatment with PD-98059 to inhibit p42/44 MAPK or SB-203580 to inhibit p38 MAPK had no significant effect on thrombin-stimulated, membrane-associated iPLA(2) activity. Thrombin stimulation resulted in significant increases in both p42/44 and p38 MAPK activity after 2 min. Pretreatment with the iPLA(2)-selective inhibitor bromoenol lactone completely inhibited thrombin-stimulated MAPK activity, suggesting that activation of MAPKs was dependent on iPLA(2) activation. Ventricular myocyte MAPK activity was increased by incubation of the myocytes with lysoplasmenylcholine, a metabolite produced by iPLA(2)-catalyzed membrane plasmalogen phospholipid hydrolysis. Altogether, these data suggest that activation of MAPKs occurs downstream of and is dependent on iPLA(2) activation in thrombin-stimulated rabbit ventricular myocytes.
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PMID:Activation of MAPKs in thrombin-stimulated ventricular myocytes is dependent on Ca2+-independent PLA2. 1633 69

Heterotrimeric GTP-binding (G) proteins transduce hormone-induced signals to their effector enzymes, which include several phospholipases. In particular, the G(o)/G(i) and G(q) protein families have been shown to couple signaling to phospholipase A(2) (PLA(2)), phospholipase C, and phospholipase D, while the G(12)/G(13) family has been linked to the activation of small GTPases of the Rho family, and hence, to phospholipase D activation. Here, we demonstrate that in CHO cells, the G(12)/G(13) family is also able to activate cPLA(2)alpha, through the activation of RhoA and, subsequently, ERK1/2. Hormone-induced arachidonic acid release increased as a consequence of Galpha(13) overexpression, and was inhibited through inhibition of Galpha(13) signaling. The Galpha(13)-mediated cPLA(2)alpha activation was inhibited by pharmacological blockade of ERK1/2 with either U0126 or PD98059, and by RhoA inactivation with C3 toxin or a dominant-negative RhoA (N19RhoA), and was stimulated by the serine-threonine phosphatase inhibitor calyculin A. Our data thus identify a pathway of cPLA(2)alpha regulation that is initiated by thrombin and purinergic receptor activation, and that signals through Galpha(13), RhoA and ERK1/2, with the involvement of a calyculin-sensitive phosphatase.
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PMID:Galpha13 mediates activation of the cytosolic phospholipase A2alpha through fine regulation of ERK phosphorylation. 1680 23

In the present study, phospholipase A(2) (PLA(2))-catalyzed hydrolysis of platelet membrane phospholipids was investigated by measuring PLA(2) activity, phospholipid hydrolysis, arachidonic acid release and choline lysophospholipid production in thrombin-stimulated human platelets. Thrombin-stimulated platelets demonstrated selective hydrolysis of arachidonylated plasmenylcholine and plasmenylethanolamine, with little change in diacyl phospholipids. Accelerated plasmalogen hydrolysis was accompanied by increased arachidonic acid and thromboxane B(2) release and increased lysoplasmenylcholine production. Thrombin stimulation caused an increase in PLA(2) activity measured in the cytosolic fraction with plasmenylcholine only; no increase in activity was measured with phosphatidylcholine. No change in membrane-associated PLA(2) activity was observed with either substrate tested. Pretreatment with the Ca(2+)-independent PLA(2)-selective inhibitor, bromoenol lactone, inhibited completely any thrombin-stimulated phospholipid hydrolysis. Thus, thrombin stimulation of human platelets activates a cytosolic PLA(2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids.
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PMID:Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids in thrombin-stimulated human platelets. 1705 38

Atherosclerotic plaque formation is a dynamic process involving repeated injury and inflammation of the endothelium. We have demonstrated previously that thrombin and tryptase stimulation of human coronary artery endothelial cells (HCAEC) leads to increased phospholipase A(2) (PLA(2)) activity and generation of membrane phospholipid derived inflammatory metabolites, including eicosanoids and platelet activating factor. Thus, our hypothesis is that selective PLA(2) inhibitors have therapeutic potential as anti-inflammatory agents. Stimulation of confluent HCAEC monolayers with thrombin or tryptase resulted in a concentration and time-dependent increase in both prostaglandin E(2) (PGE(2)) and prostacyclin (PGI(2)) production. Pretreatment with PX-18 to inhibit secretory PLA(2) or BEL to inhibit calcium-independent PLA(2) prior to thrombin or tryptase stimulation resulted in a significant inhibition of both PGI(2) and PGE(2) release. However, pretreatment with methyl arachidonyl fluorophosphonate (MAFP), a widely used inhibitor of cytosolic PLA(2) isoforms, resulted in a significant potentiation of both thrombin and tryptase stimulated PGI(2) and PGE(2) release as a consequence of increased free arachidonic acid production. We conclude that the use of selective PLA(2) inhibitors may be of therapeutic benefit in the development and progression of atherosclerosis, however, the development of such an agent requires rigorous screening.
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PMID:Prostaglandin production in human coronary artery endothelial cells is modulated differentially by selective phospholipase A(2) inhibitors. 1737 79

We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.
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PMID:The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity. 1745 99

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A(2) ([Lys(49)]PLA(2)), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as alpha-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for alpha-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.
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PMID:A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag. 1820 18

To advance our knowledge on the snake venom composition and transcripts expressed in venom gland at the molecular level, we constructed a cDNA library from the venom gland of Agkistrodon piscivorus leucostoma for the generation of expressed sequence tags (ESTs) database. From the randomly sequenced 2112 independent clones, we have obtained ESTs for 1309 (62%) cDNAs, which showed significant deduced amino acid sequence similarity (scores >80) to previously characterized proteins in National Center for Biotechnology Information (NCBI) database. Ribosomal proteins make up 47 clones (2%) and the remaining 756 (36%) cDNAs represent either unknown identity or show BLASTX sequence identity scores of <80 with known GenBank accessions. The most highly expressed gene encoding phospholipase A(2) (PLA(2)) accounting for 35% of A. p. leucostoma venom gland cDNAs was identified and further confirmed by crude venom applied to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and protein sequencing. A total of 180 representative genes were obtained from the sequence assemblies and deposited to EST database. Clones showing sequence identity to disintegrins, thrombin-like enzymes, hemorrhagic toxins, fibrinogen clotting inhibitors and plasminogen activators were also identified in our EST database. These data can be used to develop a research program that will help us identify genes encoding proteins that are of medical importance or proteins involved in the mechanisms of the toxin venom.
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PMID:Complementary DNA sequencing and identification of mRNAs from the venomous gland of Agkistrodon piscivorus leucostoma. 1850 63


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