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 findings described above illustrate how the src kinase can influence several new pathways of inositol phosphate metabolism, both at the membrane level with the production of novel D-3 phosphoinositides and the activation of PI-3 kinase, and at the cytosolic level by altering the expression of certain inositol polyphosphates, in particular Ins(1,4,5,6)P4. At present, it is difficult to speculate on the role these phenomena play in cellular transformation by src, since the functions of D-3 phosphoinositides and most inositol polyphosphates are unclear. There is evidence, however, that these new pathways of phosphoinositide metabolism occur in response to other types of cellular stimulations besides src transformation. Novel D-3 phosphoinositides are expressed in a variety of nonneoplastic cells, including human platelets treated with thrombin, smooth muscle cells and stimulated neutrophils. In addition, unusual InsP4 isomers such as D/L-Ins(1,4,5,6)P4 are found in chicken erythrocytes, murine macrophages, AR4-2J rat pancreatoma cells and adrenal glomerulosa cells, to name only a few. Recently, associations have been reported between PI-3 kinases and cytoskeletal elements in thrombin- stimulated platelets, and between activated ras proteins in rat liver epithelial cells. The latter discovery is particularly intriguing since GTP-binding proteins such as ras are known to influence cell shape and serve as downstream effector proteins in the signal transduction pathways of numerous growth factor receptors. Thus, one function of novel phosphoinositides and their metabolites may lie at the level of cytoskeletal and cell shape regulation. Clearly, additional roles for phosphoinositides exist in cells besides their traditional use as precursors for the generation of Ins(1,4,5)P3 and diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphoinositides and cell growth. 133 66

The proposed Ca(2+)-signaling actions of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), formed by phosphorylation of the primary Ca(2+)-mobilizing messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), were analyzed in NIH 3T3 and CCL39 fibroblasts transfected with rat brain Ins(1,4,5)P3 3-kinase. In such kinase-transfected cells, the conversion of Ins(1,4,5)P3 to Ins(1,3,4,5)P4 during agonist stimulation was greatly increased, with a concomitant reduction in Ins(1,4,5)P3 levels and attenuation of both the cytoplasmic Ca2+ increase and the Ca2+ influx response. This reduction in Ca2+ signaling was observed during activation of receptors coupled to guanine nucleotide-binding proteins (thrombin and bradykinin), as well as with those possessing tyrosine kinase activity. Single-cell Ca2+ measurements in CCL39 cells revealed that the smaller averaged Ca2+ response of enzyme-transfected cells was due to a marked increase in the number of cells expressing small and slow Ca2+ increases, in contrast to the predominantly large and rapid Ca2+ responses of vector-transfected controls. There was no evidence that high Ins(1,3,4,5)P4 levels promote Ca2+ mobilization, Ca2+ entry, or Ca2+ sequestration. These data indicate that Ins(1,4,5)P3 is the major determinant of the agonist-induced Ca2+ signal in fibroblasts and that Ins(1,3,4,5)P4 does not appear to contribute significantly to this process. Instead, Ins(1,4,5)P3 3-kinase may serve as a negative regulator of the Ca(2+)-phosphoinositide signal transduction mechanism.
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PMID:Agonist-induced calcium signaling is impaired in fibroblasts overproducing inositol 1,3,4,5-tetrakisphosphate. 166 15

The mechanisms involved in platelet aggregation by a monoclonal antibody (mAb) P256 specific for the GPIIb-IIIa complex was investigated following metabolic 32P labelling of platelets. When compared with thrombin, inositol phosphates (InsP) production during P256-induced activation was delayed and no apparent peak, but a small and sustained production of [32P]-Ins(1,4,5)P3 and [32P]-Ins(1,3,4,5)P4, was observed between 20 and 90 s. [32P]-Ins(1,3,4)P3 was also produced with a maximum after 90 s. Addition of the ADP scavenger creatinine phosphate/creatine phosphokinase (CP/CPK) and of the cyclooxygenase inhibitor aspirin together with P256 almost totally abolished InsP formation, whereas platelet aggregation and protein phosphorylation were partially inhibited. F(ab')2 fragments of P256 also aggregated platelets but to a smaller extent than IgG, and without any measurable InsPs. To characterize further P256-induced activation, the phosphorylation of p43, the main substrate of protein kinase C (PKC) and the phosphorylation of tyrosine protein (P-Tyr) was also studied. PKC activation was smaller with P256-IgG than with thrombin but both thrombin and P265-IgG induced a similar profile of P-Tyr involving seven major bands, whereas P256-F(ab')2 only occasionally activated PKC but always significantly phosphorylated a 64,000 molecular weight P-Tyr. The data indicate that the binding of P256 to GPIIb-IIIa, in contrast with thrombin, does not initially lead directly to the activation of the phosphoinositide phospholipase C to produce InsP's but rather involves the activation of protein kinases and also both fragments F(ab')2 and Fc play a specific role in the platelet responses to the mAb. Only the crosstalk between the two pathways evoked by F(ab')2 and Fc respectively allows the activation of all platelet activation systems.
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PMID:Mechanisms involved in platelet activation induced by a monoclonal antibody anti glycoprotein IIb-IIIa: inositol phosphate production is not the primary event. 178 4

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular Ca2+, is metabolized either by dephosphorylation to inositol 1,4-bisphosphate(Ins-(1,4)P2) or by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). It has been shown in vitro that Ins(1,3,4,5)P4 is also dephosphorylated by a 5-phosphomonoesterase to inositol 1,3,4-trisphosphate. However, we have found that exogenous Ins(1,3,4,5)P4 is dephosphorylated to predominantly Ins(1,4,5)P3 in saponin-permeabilized platelets in the presence of KCl (40-160 mM). This inositol polyphosphate 3-phosphomonoesterase activity is independent of Ca2+ (0.1-100 microM), and it was also observed when the ionic strength of the incubation medium was increased with Na+. The action of KCl appears to be due to activation of a 3-phosphomonoesterase as well as an inhibition of the 5-phosphomonoesterase, because the dephosphorylation of Ins(1,4,5)P3 to Ins(1,4)P2 was completely inhibited by KCl. The 3-phosphomonoesterase may be regulated by a protein kinase C, since both thrombin and phorbol dibutyrate increase 3-phosphomonoesterase activity and this is inhibited by staurosporine. The formation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 reported here provides an additional pathway for the formation of the Ca2+-mobilizing second messenger in stimulated cells.
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PMID:Thrombin and phorbol ester stimulate inositol 1,3,4,5-tetrakisphosphate 3-phosphomonoesterase in human platelets. 215 13

Human platelets stimulated with alpha-thrombin or the thromboxane A2 analogue U46619 form the novel phosphatidylinositol (PtdIns) polyphosphate species PtdIns (3,4)P2 and PtdIns(3,4,5)P3 in a time- and dose-dependent manner. In [32P]o-phosphate-labeled platelets, PtdIns(3,4)P2 increases in 2 min to 613% of the basal level in response to 1 unit/ml alpha-thrombin and 295% in response to 5 microM U46619. A dramatic increase is also observed with myo-[3H]inositol-labeled platelets. [32P]PtdIns(3,4,5)P3 is increased with alpha-thrombin and U46619 stimulation by 261 and 183%, respectively. PtdIns(3)P quantities remain nearly equal to those under resting conditions. Neither the basal nor increased levels of PtdIns(3,4)P2 appear to be adequate to account for the rapid elevation of Ins(1,3,4)P3 that we have observed in alpha-thrombin-stimulated platelets. A23187 and/or phorbol 12,13-dibutyrate are not as potent as alpha-thrombin in stimulating changes in PtdIns(3,4)P2 or PtdIns(3,4,5)P3. GTP gamma S (10 microM), however, increases the level of PtdIns(3,4)P2 by 3736% and that of PtdIns(3,4,5)P3 by 456% in saponin-permeabilized platelets incubated with 0.5 mM [gamma-32P]ATP, implying a role for a GTP-binding protein in promoting phosphatidylinositide 3-kinase activity(ies). This is the first report of stimulated generation of 3-phosphorylated phosphoinositides in anucleate cells. A role for these novel phospholipid species in signal transduction, however, awaits elucidation.
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PMID:Human platelets form 3-phosphorylated phosphoinositides in response to alpha-thrombin, U46619, or GTP gamma S. 215 15

Thrombin-stimulated (10 s) human platelets produce Ins(1,4,5)P3 and an additional inositol trisphosphate (InsP3), in approximately a 1:20 ratio. The major InsP3 co-migrates with Ins(1,3,4)P3 on strong-anion-exchange h.p.l.c. To identify this species unequivocally, we treated putative Ins(1,3,4)P3 obtained from thrombin-stimulated myo-[3H]inositol-labelled platelets with NaIO4/NaBH4 or 4-phosphomonoesterase. The products indicate that the major InsP3 is at least 90% D-Ins(1,3,4)P3. D-[3H]Ins(1,3,4)P3 added to saponin-permeabilized platelets is hydrolysed to an InsP2 (7.8%) and phosphorylated by a kinase to yield an inositol polyphosphate (0.9%) in 5 min. The phosphorylation product co-migrates with Ins(1,3,4,6)P4 on Partisphere WAX h.p.l.c. Under similar conditions, L-[3H]Ins(1,3,4)P3 is dephosphorylated but not phosphorylated. Relative phosphatase:kinase ratios are 8.7:1 (Vmax. values) and 0.86:1 (Km values) with respect to D-Ins(1,3,4)P3. The kinase activity is predominantly cytosolic (96.8% of total activity) in freeze-thaw-disrupted platelets, and the accumulation of its product is Ca2(+)-dependent. The activity is identified as a 6-kinase on the basis of its product's insensitivity to 5-phosphomonoesterase, resistance to periodate oxidation and co-migration with standard Ins(1,3,4,6)P4 on h.p.l.c. Incubation of platelets with beta-phorbol dibutyrate (beta-PDBu, 76 nM), causing activation of protein kinase C, results in a 57.5% inhibition (reversible by the protein kinase C inhibitor staurosporine) of Ins(1,3,4,6)P4 accumulation. alpha-PDBu, which does not stimulate protein kinase C, has no effect. Stimulation of intact platelets with thrombin results in the production of Ins(1,3,4,6)P4 (1.4-fold rise in 30 s) and Ins(1,3,4,5)P4, with the latter being the major InsP4 species. Accumulation of Ins(1,3,4,6)P4 is slightly delayed in comparison with Ins(1,3,4)P3 and is relatively small. We propose that the major route of Ins(1,3,4)P3 metabolism in stimulated human platelets is via phosphatase action.
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PMID:Ca2(+)-stimulatable and protein kinase C-inhibitable accumulation of inositol 1,3,4,6-tetrakisphosphate in human platelets. 239 72

We have examined regulation by protein kinase C (Ca2+/phospholipid-dependent enzyme) of thrombin-induced inositol polyphosphate accumulation in human platelets. When platelets are exposed to thrombin for 10 s, the protein kinase C inhibitor staurosporine causes inositol phosphate elevations over control values of 2.7-fold (inositol 1,4,5-trisphosphate (Ins(1,4,5)P3], 1.9-fold (inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4], and 1.2-fold (inositol 1,3,4-trisphosphate). In the same period, phosphatidic acid and diacylglycerol are unaffected. The myosin light chain kinase inhibitor ML-7 has no effect on inositol phosphate accumulations. Staurosporine does not inhibit Ins(1,4,5)P3 3-kinase and 5-phosphomonoesterase activities in saponin-permeabilized platelets incubated with exogenous Ins(1,4,5)P3 unless the platelets have been exposed to thrombin and protein kinase C is consequently activated. The protein kinase C agonist beta-phorbol 12,13-dibutyrate increases the Vmax of the 3-kinase 1.8-fold, with little effect on Km. Our results provide strong evidence for a role for protein kinase C in regulating inositol phosphate levels in thrombin-activated platelets. We propose that endogenously activated protein kinase C removes Ins(1,4,5)P3 by stimulating both 5-phosphomonoesterase and Ins(1,4,5)P3 3-kinase. Initial activation of phospholipase C does not appear to be affected by such protein kinase C. Inhibition of protein kinase C by staurosporine decreases 5-phosphomonoesterase activity. The resulting elevated Ins(1,4,5)P3, as substrate for Ins(1,4,5)P3 3-kinase, promotes production of Ins(1,3,4,5)P4, which also may accumulate through decreased 5-phosphomonoesterase activity and elevated Ca2+ levels. These factors apparently counteract the inhibitory effect on 3-kinase, yielding a net increase in Ins(1,3,4,5)P4.
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PMID:Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphosphates and tetrakisphosphate in human platelets exposed to thrombin. 270 80

Stimulation of human platelets by thrombin leads to rises of both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) within 10 s. The mass of Ins(1,4,5)P3 was measured in platelet extracts after conversion to [3-32P]Ins(1,3,4,5)P4 with Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Basal levels were equivalent to 0.2 microM and rose to 1 microM within 10 s of stimulation by thrombin. The mass of Ins(1,3,4)P3 was more than 10-fold greater than that of Ins(1,4,5)P3 between 10 and 60 s of thrombin stimulation. These results indicate that the majority of InsP3 liberated by phospholipase C in stimulated platelets must be the non-cyclic Ins(1,4,5)P3 in order to allow rapid phosphorylation by Ins(1,4,5)P3 3-kinase to Ins(1,3,4,5)P4 and then dephosphorylation to Ins(1,3,4)P3 by 5-phosphomonoesterase. A significant proportion of the InsP3 extracted from thrombin-stimulated platelets under neutral conditions is resistant to Ins(1,4,5)P3 3-kinase but susceptible after acid treatment, implying the presence of inositol 1,2-cyclic 4,5-trisphosphate (Ins(1,2cyc4,5)P3. The relative proportion of Ins(1,2cyc4,5)P3 increases with time. We suggest that such gradual accumulation is attributable to the relative insensitivity of this compound to hydrolytic and phosphorylating enzymes. Therefore, early Ca2+ mobilization in platelets is more likely to be effected by Ins(1,4,5)P3 than by Ins(1,2cyc4,5)P3.
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PMID:Inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate are minor components of total mass of inositol trisphosphate in thrombin-stimulated platelets. Rapid formation of inositol 1,3,4-trisphosphate. 282 15

We observed that more total inositol trisphosphate (InsP3) was formed when human platelets were stimulated with agonists (15-hydroxy-9,11-azo-prosta-5,13-dienoic acid or thrombin) in the presence of extracellular Ca2+ than in its absence. Analysis of the InsP3 by h.p.l.c. indicated that the increased InsP3 formed in the presence of extracellular Ca2+ was primarily the 1,3,4-trisphosphate [Ins(1,3,4)P3]. In addition, more inositol 1,3,4,5-tetrakisphosphate (InsP4) was formed in the presence of extracellular Ca2+. Experiments conducted with electrically permeabilized platelets demonstrated that conversion of [3H]Ins(1,4,5)P3 to [3H]InsP4 in platelets was Ca2+-dependent, with half-maximal conversion observed at approx. 2.5 microM-Ca2+. By contrast, dephosphorylation of [3H]InsP4 to [3H]Ins(1,3,4)P3 was not activated by Ca2+. A partially purified preparation of Ins(1,4,5)P3 3-kinase from human platelets was found to be insensitive to Ca2+, but addition of calmodulin restored Ca2+-sensitivity to the kinase, increasing its activity about 5-fold. These results show that in human platelets the metabolism of Ins(1,4,5)P3 is regulated by Ca2+-calmodulin, and suggest that the metabolites of Ins(1,4,5)P3 may also have important second-messenger functions in platelets, and are consistent with the hypothesis that the activation of phospholipase C is not dependent on extracellular Ca2+.
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PMID:Calcium modulates the generation of inositol 1,3,4-trisphosphate in human platelets by the activation of inositol 1,4,5-trisphosphate 3-kinase. 284 35

We have investigated factors affecting the activation of phospholipase C in human platelets. Prior exposure of platelets to phorbol esters that stimulated protein kinase C inhibits the activation of phospholipase C in response to a variety of receptor-directed agonists, including alpha- and gamma-thrombin and thromboxane A2 analogues. Such activation has been assayed by measurements of accumulated InsP3 (including Ins(1,4,5)P3 and Ins(1,3,4)P3) and PtdOH. Inhibition is not overcome by Ca2+ ionophores, and substances that block or mimic Na+-H+ exchange neither block nor mimic these inhibitory effects. Cyclic AMP and cyclic GMP, other agents known to inhibit phospholipase C activation, do not accumulate in platelets exposed to phorbol esters. Although a portion of the effects of phorbol ester on InsP3 accumulation may be explained by 5-phosphomonoesterase activity, it is likely that more direct effects on phospholipase C are being exerted as well, and contribute the major inhibitory route. We have examined the susceptibility of adenylyl cyclase-associated Gi and 'Gp'-activated phospholipase C to inhibitory ADP-ribosylation by pertussis toxin-derived enzyme (S1 protomer) administered to saponin-permeabilized platelets. The effects of alpha-thrombin on adenylyl cyclase can be inhibited by up to 50% by S1, at which point inhibition of phospholipase C is barely detectable. Thromboxane A2 analogues, which do not affect adenylyl cyclase (Gi), stimulate phospholipase C; this effect is not impaired by S1. We therefore propose that the inhibitory effects of phorbol esters on the activation of phospholipase C are not mediated primarily by effects on Gi.
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PMID:Regulation of platelet phospholipase C. 290 40


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