Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of tricyclic antidepressants (imipramine, desipramine, amitriptyline) and several other antidepressants like iprindole, a monoamine oxidase inhibitor phenelzine, trazodone and mianserin as well as cocaine (a potent inhibitor of norepinephrine uptake), and neuroleptics (haloperidol, thioridazine, chlorpromazine) on [3H]inositol phosphate formation was investigated in human platelets. Basal and thrombin-induced [3H]inositol monophosphate ([3H]IP1), [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) production were measured in [3H]myoinositol-labeled platelets in the presence of lithium chloride and in the presence or absence of test drugs. Desipramine, imipramine, amitriptyline and iprindole inhibited thrombin-stimulated formation of [3H]IP2 and [3H]IP3 in human platelets but had no significant effect on [3H]IP1 formation. In contrast, trazodone, mianserin, cocaine and phenelzine had no effect on inositol phosphate formation in thrombin-stimulated human platelets. The neuroleptics thioridazine and chlorpromazine also decreased thrombin-stimulated [3H]IP2 and [3H]IP3 production but not [3H]IP1 in human platelets, whereas haloperidol had no significant effect. The effect of antidepressants and neuroleptics on the level of [3H]phosphatidylinositol ([3H]PI), [3H]phosphatidylinositol 4-phosphate ([3H]PIP) and [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) was also determined. All of the drugs tested except phenelzine and thioridazine increased the accumulation of [3H]PI, [3H]PIP and [3H]PIP2. Thioridazine increased levels of [3H]PI but decreased the level of [3H]PIP and [3H]PIP2, whereas phenelzine had no effect on [3H]PI, [3H]PIP and [3H]PIP2 interconversion in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of antidepressants and neuroleptics on phosphoinositide metabolism in human platelets. 167 74

Prolonged exposure of A-10 cells to Arginine Vasopressin (AVP) resulted in the following responses: (a) loss of vasopressin receptors from the cell surface (30-40%), (b) increased basal levels of inositol and inositol monophosphate, (c) decreased inositol di- and trisphosphate production and decreased intracellular calcium release in response to a second challenge with AVP, (d) attenuation of AVP-mediated inhibition of isoproterenol-stimulated cAMP and ANF-stimulated cGMP accumulation and (e) attenuation of thrombin and ATP-mediated increase in inositol di- and trisphosphate accumulation and intracellular calcium release. All the above responses depended on the time of exposure of the cells to AVP with the responses being attenuated as early as 5-10 min of exposure to AVP. The desensitization also depended on the concentration of AVP used with 50% of maximal desensitization for each response being observed at 5 nM of AVP. This concentration of AVP corresponded well with the Kd of vasopressin for binding to these sites. Desensitization of protein kinase C (PKC) by prolonged exposure of the cells to PDBu or addition of the PKC inhibitor staurosporine during pretreatment with AVP did not prevent AVP-mediated desensitization, suggesting that PKC may not be involved in AVP-mediated desensitization in smooth muscle cells. It is concluded that AVP induced both homologous and heterologous desensitization of phosphatidylinositol turnover and calcium release in smooth muscle cells. The desensitization processes did not appear to be mediated by protein kinase C. The possibility that the locus of the heterologous desensitization may be at the level of substrates such as PI, PIP and PIP2 is discussed.
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PMID:Homologous and heterologous desensitization mediated by vasopressin in smooth muscle cells. 253 42

A new method for high incorporation of [3H]inositol into human platelets is described. The method involves incorporation of [3H]inositol during reversible electropermeabilisation by high voltage discharge, followed by resealing the cells during incubation at 37 degrees C. Between 10- and 20-fold increase of isotope uptake is achieved compared to control intact cells. Permeabilised resealed platelets maintain good responses to thrombin and collagen. Analysis of the incorporation of the label amongst the phosphoinositides shows 70% to be in PI, 20% in PIP, and 10% in PIP2. Stimulation with thrombin and analysis of the formation of IP1, IP2 and IP3 shows the labelling to occur in a hormone-sensitive pool. These studies indicate that reversible electropermeabilisation can be used to achieve good uptake of non-membrane penetrating substances such as inositol.
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PMID:High incorporation of [3H]inositol into phosphoinositides of human platelets during reversible electropermeabilisation. 255 Feb 78

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.
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PMID:Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5'-[gamma-thio]triphosphate and by thrombin in the presence of GTP. 282 Mar 81

Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+-dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-gamma-S. Of the two mitogens, alpha-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only alpha-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, alpha-thrombin but not EGF potentiated GTP-gamma-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples alpha-thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2-phosphodiesterase activity.
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PMID:Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts. 302 38

The specific radioactivity of the phosphodiester and phosphomonoester moieties of the phosphoinositides was determined in resting and thrombin-stimulated platelets that were prelabelled with [32P] Pi. In the unstimulated cells, the specific radioactivity of the monoester phosphates of PIP and PIP2 was similar. Both prolonged incubation at 37 degrees C and upon stimulation with 0.5 U/ml of thrombin, the specific radioactivity of the monoester phosphates decreased 10-15% in both polyphosphoinositides. In the unstimulated cells, the specific radioactivity of the diester phosphates was similar in PI, PIP and PIP2 but amounted only to 3% of the activity of the monoester groups. Prolonged incubation of the unstimulated cells as well as stimulation with thrombin induced a similar 5-6 fold increase in specific radioactivity of the diester phosphate of PI, PIP and PIP2. The results indicate that the phosphoinositides in both resting and thrombin-stimulated platelets exist in a metabolically homogeneous pool.
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PMID:Phosphoinositide metabolism in resting and thrombin-stimulated human platelets. Evidence against metabolic heterogeneity. 303 88

We studied thrombin-induced metabolism of phosphoinositide, protein phosphorylation and platelet aggregation in platelets from 32 NIDDM patients and 12 control subjects. To clarify the effect of diet, sulphonylureas, or insulin treatment, the subjects were divided into three groups based on the type of treatment. Thrombin-induced platelet aggregation was measured with an aggregometer. Low-dose thrombin (0.25 U/ml)-stimulated platelet aggregation in diabetic patients was significantly increased compared with the control subjects. Platelet aggregation in the sulphonylurea and insulin groups was significantly lower than in the diet group. On the other hand, in platelets incubated with [32P]orthophosphate, thrombin-induced incorporation of 32P radioactivity into phosphatidic acid (PA) was significantly lower in the sulphonylurea and insulin groups than in the diet group. Thrombin-induced incorporation of [32P] radioactivity into phosphatidylinositol (PIP) for 10 s was significantly higher in the sulphonylurea group than in the diet group. There were no differences in thrombin-induced 47 kDa protein phosphorylation between platelets from the diet, sulphonylurea, or insulin groups. These results suggest that sulphonylureas and insulin induce suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP2 and production of PA, which leads to inhibition of platelet aggregation.
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PMID:Thrombin-induced platelet aggregation, phosphoinositide metabolism and protein phosphorylation in NIDDM patients treated by diet, sulphonylurea or insulin. 792 50

Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). We now show that treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreased calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulated PIP 5-kinase activity, and this effect was blocked by botulinum C3 exoenzyme, suggesting that Rho was responsible. GTP-bound recombinant Rho stimulated PIP 5-kinase activity, whereas GDP-Rho was much less potent and GTP-bound Rac was ineffective. Microinjected botulinum C3 exoenzyme caused diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reversed the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis could mediate the effects of Rho on the actin cytoskeleton.
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PMID:The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. 795 16

Elevation of cyclic AMP (cAMP) in platelets inhibits agonist-induced, G protein-mediated responses and activation of polyphosphoinositide-specific phospholipase C (PLC) by ill-defined mechanism(s). Signal transduction steps downstream of PLC are inhibited by elevated cAMP, suggesting an inhibitory effect of cAMP, via protein kinase A, on PLC. In [32P]i-prelabeled platelets, forskolin increased intracellular cAMP (104 nmol/1011 cells at 10-5 M forskolin) and [32P]phosphatidylinositol 4-phosphate (Delta[32P]PIP) (30% at 10-7-10-6 M forskolin). The thrombin-induced (0.1 U/ml) increase in production of [32P]PA, 'overshoots' in [32P]PIP and [32P]PIP2 ([32P]phosphatidylinositol 4,5-bisphosphate), and the increase in [32P]PI and secretion of ADP+ATP were abolished by forskolin (10-7 M). Forskolin stimulated total [32P]Pi uptake in resting platelets (48%), increased 32P incorporation into PIP (110%), and inhibited 32P incorporation into PI (50%). The latter inhibition was most likely considerably greater due to the forskolin-induced stimulation of [32P]Pi uptake. The changes in radioactive PA, PIP and PIP2 are regarded as being proportional with their masses in the prelabeled platelets, while the increase in PI (phosphatidylinositol) is regarded as a change in specific radioactivity, and hence in its synthesis. The results suggest that cAMP elevation inhibits the flux in the polyphosphoinositide cycle through both inhibition of PIP 5-kinase and PI synthesis. The inverse relation between forskolin-produced DeltaPIP and [32P]PA production suggests that the PLC reaction is inhibited by elevated cAMP through reduction of substrate (PIP2) resynthesis, and not by inhibition of the PLC enzyme.
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PMID:Elevation of cyclic AMP decreases phosphoinositide turnover and inhibits thrombin-induced secretion in human platelets. 979 33

Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.
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PMID:Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly. 1067 24


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