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)

The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Thrombin stimulation of human endothelial cell phospholipase D activity. Regulation by phospholipase C, protein kinase C, and cyclic adenosine 3'5'-monophosphate. 131 12

Thrombin, the key regulatory protein of hemostasis, is a potent stimulus for endothelial cell activation, a process implicated in a variety of ischemic, thrombotic, and inflammatory vascular disorders. Activation of the thrombin receptor requires a novel mechanism of receptor proteolysis generating a tethered receptor ligand. Synthetic peptides whose sequences are identical to this newly exposed receptor NH2-terminus reproduce thrombin effects on human and bovine endothelial cell activation. Receptor cleavage by catalytically active alpha-thrombin is tightly coupled to a PI-PLC, with resultant generation of IP3 and DAG, increases in [Ca2+]i, and translocation of PKC (Fig. 3). Both the increase in [Ca2+]i and PKC activation are required for thrombin-stimulated PLA2 and PLD activity, PGI2 synthesis, and barrier dysfunction, the latter occurring as the result of Ca2+ and PKC effects on specific cytoskeletal protein elements and other contractile proteins (Fig. 3). Further investigations are ongoing to identify more clearly not only the precise biochemical intermediates involved in the endothelial cell response to thrombin but also the specific protein kinase systems involved in thrombin-mediated signal transduction in vascular endothelium.
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PMID:Molecular mechanisms of thrombin-induced human and bovine endothelial cell activation. 140 26

Thrombin, the key regulatory protein of hemostasis, has been implicated in a variety of important endothelial cell processes closely linked to endothelial signal transduction mechanisms. An initial event, following receptor binding by catalytically active alpha-thrombin, appears to be the activation of a G-protein-coupled, PI-specific PLC, with resultant generation of IP3 and DAG, with increases in [Ca2+]i, and activation and translocation of PKC (Fig. 9). PKC activation results in down-regulation of PLC, as demonstrated by inhibition of agonist-induced increases in [Ca2+]i, whereas PLA2 activity is up-regulated, with a resultant increase in endothelial PGI2 synthesis. Recently, we have demonstrated that activity of membrane-bound, endothelial PLD, is also up-regulated by PKC activation. In addition to its modulatory role in endothelial cell phospholipase activities, PKC activation appears to play a critical role in thrombin-mediated endothelial barrier dysfunction, likely via specific cytoskeletal protein phosphorylation. A temporal relationship between alpha-thrombin-mediated signal transduction and specific cellular responses, such as PGI2 synthesis and barrier dysfunction, can be established (Fig. 2). Further investigations are ongoing to identify more clearly the precise biochemical intermediates involved in the endothelial cell response to thrombin, as well as the role of differential phosphorylation by various protein kinase systems in thrombin-mediated signal transduction in vascular endothelium.
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PMID:The role of protein kinase C in alpha-thrombin-mediated endothelial cell activation. 157 13

The protein kinase C activators phorbol ester 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (DAG) cause platelet aggregation, secretion, and a rise in aequorin-indicated cytoplasmic Ca2+ ([Ca2+]i), but the importance of this action to platelet activation by these agonists has not been established. We found that the previous addition of PMA or DAG either enhanced or inhibited the platelet response if thrombin was subsequently added, depending on the latter's concentration. The effects of PMA or DAG on the response to thrombin were obtained only if the agonists were added in concentrations sufficient to elevate [Ca2+]i themselves. A [Ca2+]i rise also occurred after the second agonist (thrombin), but its magnitude did not necessarily correlate with subsequent aggregation, secretion, or the activation of protein kinase C as reported by the phosphorylation of a 47-kDa protein (p47). The protein kinase C inhibitor sphingosine inhibited aggregation and p47 phosphorylation caused by PMA or DAG alone or with thrombin, but the [Ca2+]i rise in response to the first agonist was not affected. PMA-induced aggregation and p47 phosphorylation were inhibited by quin2, which also inhibited protein kinase C activity in a cell-free system. We conclude that a rise in aequorin-indicated [Ca2+]i is necessary for PMA or DAG to activate platelets or to alter the subsequent platelet response to thrombin; this [Ca2+]i rise may be a prerequisite for activation of protein kinase C.
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PMID:Response of aequorin-loaded platelets to activators of protein kinase C. 291 35

1. The comparative study of the effect of bradykinin (BK) in young and old IMR-90 human fibroblasts shows that old cells are characterized by a reduced increase in 1,2-diacylglycerol (1,2-DAG) generation upon stimulation after short-term treatment and a significant higher increase after long-term agonist treatment. BK-induced activation of phospholipase D (PLD), the major enzyme involved in sustained 1,2-DAG generation, was 2.5-fold higher in old cells, strongly suggesting that it is involved in the potentiated increase of 1,2-DAG formation. The increased activation of PLD by BK in old cells was specific, since in parallel experiments the effect of thrombin was not significantly different in young and old cells. PLD activity in old cells was only reduced by down-regulation of protein kinase C (PKC) activity, in contrast to what was observed in young cells where it was completely abolished. This indicates that the enzyme activity in old cells was partially PKC-independent. BK was also able to increase the release of [14C]ethanolamine, a water-soluble product of hydrolysis of phosphatidylethanolamine (PtdEtn), through PLD activation in young and old cells. The BK effect was significantly higher in old cells and, very likely, PKC-independent, since phorbol 12-myristate 13-acetate failed to induce PtdEtn hydrolysis. 2. The present results indicate that the PLD/1,2-DAG pathway is specifically potentiated by BK in old fibroblasts, demonstrating that the formation of positive effectors of PKC activation is not necessarily decreased in cellular senescence. It remains to be established whether the increased generation of DAG upon BK stimulation plays any role in the altered PKC signalling pathway which characterizes old fibroblasts.
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PMID:Potentiated bradykinin-induced increase of 1,2-diacylglycerol generation and phospholipase D activity in human senescent fibroblasts. 855 23

Recently, we demonstrated that the N-terminal region of mouse alpha-dystroglycan represents an autonomously folding globular domain, organized into at least two subdomains (Brancaccio et al., Eur. J. Biochem. 246, 166-172, 1997). We have now found a similarity between a part of the alpha-dystroglycan N-terminal sequence (approximately from position 80 to 180) and several protein sequences belonging to the immunoglobulin kappa family. Moreover, we have recombinantly expressed and purified a 31 kDa protein fragment which matches the entire alpha-dystroglycan N-terminal globular domain. To prevent the action of bacterial endogenous proteases and/or thrombin, which cleaves the protein into two fragments at an Arg-Ala trypsin-sensitive site in positions 168-169, we have introduced a single mutation (Arg168-->His), thus making the whole domain more stable and suitable for crystallization. Crystals of this mutant protein were obtained by vapor diffusion using the hanging drop technique, and they diffract to 0.28 nm Bragg spacing.
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PMID:Sequence analysis suggests the presence of an IG-like domain in the N-terminal region of alpha-dystroglycan which was crystallized after mutation of a protease susceptible site (Arg168-->His). 988 1

Incubation of blood platelets with (32)P-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP(2)) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[(32)P]LysoPI was present in resting and activated platelets, whereas [(32)P]lysoPA and [(32)P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [(32)P]PA and [(32)P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [(32)P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [(32)P]CDP-DAG is accompanied by a similar increase in [(32)P]PI and larger increases in [(32)P]PIP and [(32)P]PIP(2) (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.
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PMID:Identification of minor metabolites of phospholipid signal molecules in [(32)P]P(i)-labelled platelets. 1218 13

Utrophin is a component of the platelet membrane cytoskeleton and participates in cytoskeletal reorganization (Earnest, J. P., Santos, G. F., Zuerbig, S., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 27259-27265). Although platelets do not contain dystrophin, the identification of smaller C-terminal isoforms of dystrophin, including Dp71, which are expressed in a wide range of nonmuscle tissues and cell lines, has not been investigated. In this report, we have identified Dp71 protein variants of 55-60 kDa (designated Dp71Delta(110)) in the membrane cytoskeleton of human platelets. Both Dp71Delta(110) and utrophin sediment from lysed platelets along with the high speed detergent-insoluble pellet, which contains components of the membrane cytoskeleton. Like the membrane cytoskeletal proteins vinculin and spectrin, Dp71Delta(110) and utrophin redistributed from the high speed detergent-insoluble pellet to the integrin-rich low speed pellet of thrombin-stimulated platelets. Immunoelectron microscopy provided further evidence that Dp71Delta(110) was localized to the submembranous cytoskeleton. In addition to Dp71Delta(110), platelets contained several components of the dystrophin-associated protein complex, including beta-dystroglycan and syntrophin. To better understand the potential function of Dp71Delta(110), collagen adhesion assays were performed on platelets isolated from wild-type or Dp71-deficient (mdx(3cv)) mice. Adhesion to collagen in response to thrombin was significantly decreased in platelets isolated from mdx(3cv) mice, compared with wild-type platelets. Collectively, our results provide evidence that Dp71Delta(110) is a component of the platelet membrane cytoskeleton, is involved in cytoskeletal reorganization and/or signaling, and plays a role in thrombin-mediated platelet adhesion.
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PMID:Identification of Dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion. 1237 Jan 93

The effects of resveratrol (trans-3,4',5-trihydroxystilbene) on activation responses and the polyphosphoinositide metabolism in human blood platelets have been studied. Resveratrol partially inhibited secretory responses (liberation of dense granule nucleotides and lysosomal acid hydrolases), microparticle formation and protein phosphorylations induced by thrombin. The effects of resveratrol on phosphoinositide metabolites, phosphatidate (PtdOH), phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4(5)-P), phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) were monitored in blood platelets prelabelled with [32P]Pi. Resveratrol not only inhibited the marked increase in levels of PtdOH in platelets activated by thrombin (0.1 U/ml) but it decreased the steady state levels of the other polyphosphoinositide metabolites. The distribution of 32P in phosphoinositides in activated platelets was consistent with inhibition of CDP-DAG inositol transferase and a weak inhibition of PtdIns-4(5)-P kinase. These observations show that resveratrol has a profound effect on phospholipids, particularly on polyphosphoinositide metabolism, and may decrease the amount of PtdIns-4,5-P2 available for signalling in these cells.
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PMID:Resveratrol inhibits polyphosphoinositide metabolism in activated platelets. 1605 Nov 84

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels.
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PMID:Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets. 1771 1


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