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)

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

One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.
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PMID:Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts. 282 18

We developed a HPLC method which separates the following nine inositol-containing compounds of biological interest: inositol, inositol 1-monophosphate, inositol 2- or 4-monophosphate, inositol 1,2-cyclic phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, glycerophosphoinositol, glycerophosphoinositol 4-monophosphate, and glycerophosphoinositol 4,5-bisphosphate. The method shows good resolution and sufficient recovery (70-80%) for each compound. By applying this method to human platelets prelabeled with [3H]inositol and stimulated with thrombin, we found an early increase of inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. Accumulation of glycerophosphoinositol, inositol 1-monophosphate, and an inositol monophosphate which cochromatographs with inositol 2- and inositol 4-monophosphate occurs later. The method is simple, and--after removal of salts from the incubation buffer--can be directly applied to the measurement of aqueous soluble [3H]inositol-labeled compounds in biological samples.
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PMID:Separation of inositol phosphates and glycerophosphoinositol phosphates by high-performance liquid chromatography. 299 20

The analysis of inositol phosphates by anion-exchange HPLC is described. The method employs a citrate buffer gradient to resolve several inositol phosphates including inositol 1-phosphate, inositol 1,4-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3), as well as some of the isomers of these compounds. Since the buffer system does not contain any phosphate, we can use a phosphate assay to examine the chromatographic behavior of phosphate-containing compounds. The method shows good resolution and recovery (greater than 95% for IP2 and IP3). Total analysis time, including reequilibration, is about 90 min. In addition, an isocratic system that can rapidly (less than 10 min) measure IP3 is described. The HPLC system was used to characterize inositol phosphate turnover in thrombin-stimulated platelets and formylmethionyl-leucyl-phenylalanine-stimulated HL-60 cells.
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PMID:Anion-exchange high-performance liquid chromatographic analysis of inositol phosphates. 336 19

Human platelets prelabeled with [3H]inositol were exposed to thrombin. The aqueous soluble inositol phosphates were separated by anion exchange column chromatography, paper chromatography or high-performance liquid chromatography, and identified by cochromatography with authentic standard substances. Thrombin immediately induces the rapid formation of inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. Accumulation of inositol-1-monophosphate and inositol-2-monophosphate occurs later after a time lag of 10 sec. The results indicate that the phospholipase C induced polyphosphoinositide hydrolysis rather than the phosphatidylinositol hydrolysis is the triggering event for platelet activation, and support the concept of inositol 1,4,5-trisphosphate as putative second messenger.
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PMID:Thrombin induces the rapid formation of inositol bisphosphate and inositol trisphosphate in human platelets. 387 4

Human platelets were prelabeled with [3H] inositol and exposed to thrombin or vasopressin. The radioactive inositol monophosphates were separated by high-performance liquid chromatography and identified by cochromatography with unlabeled standard substances. Radioactive inositol 1-monophosphate (Ins 1-P) and inositol 4-monophosphate (Ins 4-P) were detected in unstimulated platelets and accumulated in response to thrombin or vasopressin. Ins 4-P as well as Ins 1-P increased after the formation of inositol 1,4-bisphosphate (Ins 1,4-P2) and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3). Lithium augmented the accumulation of Ins 1-P and Ins 1,4-P2 in stimulated platelets, and also of Ins 4-P in platelets stimulated by vasopressin, but not by thrombin. The results indicate that Ins 1,4-P2 formed in stimulated platelets is partly degraded to Ins 4-P. The significance of Ins 4-P as a marker molecule for the study of inositol phosphate metabolism in stimulated cells is discussed.
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PMID:Evidence for the formation of inositol 4-monophosphate in stimulated human platelets. 399 93

Using a rat pulmonary artery smooth muscle cell line (PAC1), detailed analysis of polyphosphoinositide (PPI) metabolism reveals receptor type-selective patterns in the formation of inositol phosphates and 3-hydroxyphosphorylated PPIs. Responses to several agonists that stimulate hypertrophy or proliferation were examined, and distinct categories of response profile were observed. Thrombin and angiotensin II stimulated the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and the formation of several cytosolic species of inositol phosphates without the activation of PI 3-hydroxykinase. The response to thrombin was distinctive because a very large production of inositol 1,4-bisphosphate was accompanied by hydrolysis of PI 4-phosphate. The response to platelet-derived growth factor (PDGF) was distinguished by the production of the PI 3-hydroxykinase product, PI 3,4,5-trisphosphate, and the appearance of PI 3-hydroxykinase activity in immunoprecipitates. PDGF treatment of PAC1 cultures did not produce accumulation of detectable amounts of inositol 1,4,5-trisphosphate, although a small sustained elevation in the level of inositol monophosphate and a gradual accumulation of inositol 1,3,4-trisphosphate were observed. Characterization of these distinctive responses permitted us to correlate agonist-regulated PPI metabolism with induction of immediate-early genes and stimulation of hypertrophy or proliferation of PAC1 cultures (Rothman, A., Wolner, B., Button, D., and Taylor, P. (1994) J. Biol. Chem. 269, 6399-6404). Thrombin-stimulated PPI turnover and the production of a high level of inositol bisphosphate may be early signals linked to the induction of fosB and PAC1 cell hypertrophy, whereas the activation of PI 3-hydroxykinase and the accumulation of PI 3,4,5-trisphosphate in response to PDGF appear to be associated with mitogenesis.
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PMID:Agonist-selective regulation of polyphosphoinositide metabolism in pulmonary artery smooth muscle cells. 750 2

Previous studies have demonstrated a strict extracellular Ca2+ dependence for the G0 to G1 and G1 to S transition in growth factor-treated T51B rat liver cells that is associated with increased levels of protein kinase C activity. Consequently, we have examined these cells for changes in phospholipid-derived second messengers in response to epidermal growth factor (EGF) and thrombin in order to determine which signals are generated during the initiation of the G0 to G1 transition. Thrombin is coupled to a phosphoinositide hydrolyzing phospholipase C, as we have found a rapid Ca(2+)-independent increase in the levels of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), inositol 1,4-bisphosphate (Ins[1,4]P2), and inositol 4-monophosphate (Ins[4]P), as well as a concomitant, transient elevation in diacylglycerol. No changes in either intracellular or extracellular choline metabolites, or an increase in DNA synthesis, were found in response to thrombin. By contrast, treatment of T51B cells with EGF results in a slower, more prolonged extracellular Ca(2+)-dependent increase in both [3H]-glycerol radiolabeled diacyl-glycerol, and diacylglycerol mass, an increase in choline release into the extracellular medium, and eventually a substantial DNA synthesis. We were, however, unable to detect any changes in phosphatidylinositol (PtdIns) turnover, either by accumulation of inositol phosphates or by changes in phospholipids in response to EGF. These results indicate that DNA synthesis can readily occur in the absence of stimulated PtdIns turnover, and that PtdIns turnover is not sufficient in itself or necessary to induce DNA synthesis and is not necessary for a Ca(2+)-dependent increase in diacylglycerol. Moreover, we have demonstrated that the extracellular Ca(2+)-dependent increase in diacylglycerol levels in response to EGF is associated with an increase in extracellular choline release, which is indicative of an activation of a phosphatidylcholine-linked phospholipase D. These results suggest that diacylglycerol sources other than PtdIns's may be important in the extracellular Ca(2+)-dependent regulation of EGF-mediated cell replication.
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PMID:EGF-induced increase in diacylglycerol, choline release, and DNA synthesis is extracellular calcium dependent. 765 54

Inositol 1,4-bisphosphate (IP2) which rapidly accumulates during cell activation, strongly stimulates an increase in cytoskeletal actin in saponin-permeated platelets, and the effect is insensitive to 5'-Chloro-5'-deoxyadenosine. Within 10 s, the amount of cytoskeletal actin in platelets rapidly increases by 41%, and then slowly increases further. IP2 induces the increase in cytoskeletal actin in a dose-dependent manner. The half-maximal effect requires approximately 2 microM of IP2. Inositol 1,4,5- triphosphate, the messenger for Ca2+ release, causes the increase in cytoskeletal actin, but is less effective than IP2. Inositol 1-monophosphate and inositol 2-monophosphate have no effect on cytoskeletal actin. Phorbol 12-myristate 13-acetate, which has been shown to activate IP3 5'-phosphatase through protein kinase C, stimulates the increase in cytoskeletal actin. Spermine, an inhibitor of IP3 5'-phosphatase, inhibits the thrombin stimulated increase in cytoskeletal actin. These results suggest that IP2 may be a messenger that controls the organization of actin filaments during cell activation. This study presents the first evidence for IP2 as a messenger during cell activation.
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PMID:Increase in cytoskeletal actin induced by inositol 1,4-bisphosphate in saponin-permeated pig platelets. 780 56