Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids. A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities. Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis. The same qualitative variations are observed during thrombin-induced aggregation. At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates. On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action. The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed.
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PMID:Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets. Possible involvement of a platelet lipase. 21 64

We provide evidence that the mechanism for arachidonate release from stimulated human platelets involves two enzymes: a phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) and a diglyceride lipase. After incubation of platelets with thrombin for 15 seconds, 1.2 nmol of 1-stearoyl-2-arachidonoyl diglyceride per 10(9) platelets, was isolated. Arachidonate was released from this substrate by the action of diglyceride lipase located in the particulate fraction of platelets. The enzyme has a pH optimum of 7.0, is stimulated by calcium ions and reduced glutathione, and liberates 31 nmol of fatty acid per min per mg of platelet particulate protein. The diglyceride lipase has sufficient activity to account for the 5-10 nmol of arachidonate released per 10(9) platelets upon thrombin stimulation. That only arachidonate is released upon thrombin stimulation may be explained by the fact that the diglyceride substrate in platelets contains only arachidonate in the 2 position. The lipase activity found in platelet membranes can also hydrolyze the 1-position fatty acid. Stearate is not released when intact platelets are stimulated with thrombin, and the fate of this fatty acid remains to be elucidated.
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PMID:Diglyceride lipase: a pathway for arachidonate release from human platelets. 29 Sep 99

Injection of bacterial lipopolysaccharide into pregnant mice resulted in fibrinogen accumulation, thrombosis and haemorrhage in the placental tissue and foetal death. Depletion of circulating fibrinogen by a thrombin-like enzyme from the venom of Malayan pit viper, Arvin, prevents foetal death. Foetal protection was also obtained by treating the mothers with a preparation of phospholipase C from Bacillus cereus known to inactivate tissue thromboplastin. It is suggested the lipopolysaccharide causes foetal death by inducing thrombosis as a consequence of activation of placental thromboplastin.
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PMID:Protection of pregnant mice with phospholipase C and with Arvin against foetal death induced by bacterial lipopolysaccharide. 44 21

The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C. A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity. The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.
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PMID:The effect of phospholipase C on human blood platelets. 81 57

Rat blood platelets were treated with phospholipase C in vitro or phospholipase C was injected i.v. to rats. In both cases its effect on the functions of the platelets in vivo has been studied. No change was found in primary bleeding time or in platelet survival. Treatment with phospholipase C gave a moderate reduction of ADP-induced platelet aggregation in the pulmonary circulation whereas the aggregation induced by thrombin was unchanged. Iv. injection of phospholipase C caused a rapid, very moderate and transient increase of 51Cr-activity in the lungs without concomitant overt respiratory distress. A moderate increase in 51Cr-activity was noted in liver and kidney 24 and 48 h after injection of phospholipase C. This may be caused by a slightly increased leakage of 51Cr-labelled material from the platelets during exposure to phospholipase C.
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PMID:The effect of phospholipase C on rat blood platelets in vivo. 84 96

Endothelial cells can produce contracting factors; endothelin, a 21-amino acid peptide, is one of the most potent of these factors, which can control local vascular tone. The peptide is formed from its precursor, big endothelin, via the activity of the endothelin converting enzyme. The basal production of the peptide is stimulated by epinephrine, angiotensin II, arginine vasopressin, transforming growth factor beta, thrombin, interleukin-1 and the calcium ionophore A23187. In vascular smooth muscle cells, endothelin binds to its specific receptor (ETA-receptor and possibly ETB-receptor) which activate phospholipase C and lead to the formation of inositol trisphosphate, diacylglycerol and increased intracellular calcium levels. In certain blood vessels, the endothelin receptor is linked to voltage-operated calcium channels via a Gi-protein. This linkage may explain why calcium antagonists inhibit endothelin-induced contractions in certain, but not other blood vessels. In large conduit arteries, such as the human internal mammary artery, endothelin-induced contractions are primarily mediated by release of intracellular calcium and hence, calcium antagonists do not markedly affect the response. In contrast, in the human forearm circulation, calcium antagonists of different classes do prevent endothelin-induced contractions. Similarly, in mesenteric resistance arteries of the rat, calcium antagonists can reverse endothelin-induced contraction suggesting that calcium antagonists are particularly potent in inhibiting endothelin-induced contraction in resistance arteries, where peripheral vascular resistance and hence, blood pressure is regulated.
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PMID:Endothelin-induced vasoconstriction and calcium antagonists. 128 11

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin-induced aggregation. These data indicate that EDRF blocks thrombin-induced platelet aggregation by inhibiting the activation of PIP2-specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.
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PMID:Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C. 130 23

Thrombin is thought to stimulate responsive cells by cleaving cell-surface receptors coupled to intracellular second-messenger-generating enzymes via G-proteins. In order to understand this process better, we have examined the regulation of adenylate cyclase by thrombin in the megakaryoblastic HEL cell line and compared it with platelets. A notable difference was found. In HEL-cell membrane preparations, thrombin inhibited cyclic AMP (cAMP) formation by a pertussis-toxin-sensitive mechanism comparable with that observed in platelets. In contrast, when added to intact HEL cells, thrombin activated adenylate cyclase and caused an increase in cAMP formation synergistic with that produced by forskolin and prostaglandin I2. This increase, which was not seen with platelets, was accompanied by an increase in cAMP metabolism by phosphodiesterase. Like other responses to thrombin, the increase in cAMP formation required proteolytically active thrombin and was subject to homologous desensitization. An equivalent response could be evoked by the addition of a polypeptide, derived from the N-terminus of the thrombin receptor, that has been shown to activate the receptor. The effects of thrombin could not, however, be reproduced by the addition of phorbol ester and the Ca2+ ionophore, A23187, nor be prevented with inhibitors of arachidonate metabolism. Preincubation of the cells with adrenaline, which inhibited Gs-mediated activation of adenylate cyclase, or pertussis toxin, which inhibited phospholipase C activation, had no effect on thrombin-induced cAMP formation. These results suggest that thrombin can regulate cAMP formation by two different mechanisms. First, thrombin can inhibit adenylate cyclase in a Gi-dependent manner. This effect predominates in HEL-cell membrane preparations, as it does in platelets, but is not detectable when thrombin is added to intact HEL cells. Instead, in intact HEL cells thrombin activates adenylate cyclase. Although clearly receptor-mediated, this response does not appear to involve Gi, Gs, protein kinase C, eicosanoid formation or changes in the cytosolic Ca2+ concentration.
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PMID:Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties. 131 10

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.
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PMID:Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion. 131 May 99

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.
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PMID:Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. 131 81


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