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)

p38 mitogen-activated protein kinase (MAPK) was identified in platelets on the basis of (a) its reactivity with antibodies to C-terminal and N-terminal peptides, and (b) its ability to activate MAPK-activated protein kinase-2, which phosphorylates the small heat shock protein, hsp27. p38 MAPK was activated in platelets by collagen fibers, a collagen-related cross-linked peptide, thrombin, or the thromboxane analogue U46619. A highly specific inhibitor of p38 MAPK, a pyridinyl imidazole known as SB203580, inhibited the platelet enzyme in vitro (IC50 approximately 0.5 microM). At similar concentrations it also inhibited agonist-stimulated phosphorylation of hsp27 in platelets, and platelet aggregation and secretion induced by minimal aggregatory concentrations of collagen or U46619, but not thrombin. Inhibition of aggregation was overcome by increasing agonist dose. SB203580 might act by inhibiting thromboxane generation, but this was only inhibited by 10-20% at low agonist concentrations. p38 MAPK provides a crucial signal, which is necessary for aggregation caused by minimal concentrations of collagen fibers or U46619. Thrombin or high doses of these agonists generate signals that bypass the enzyme, or render the enzyme no longer rate-limiting.
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PMID:Role for p38 mitogen-activated protein kinase in platelet aggregation caused by collagen or a thromboxane analogue. 863 72

Recently identified c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase are activated by stimuli of various cellular stresses, cytokines, and growth factors. Strong activation of JNK was reported in the regenerating liver, implicating JNK in growth stimulation of hepatocytes. However, it is not known which factors regulate JNK activity in liver cells. In this study, we examined activation of JNK and p38 in HepG2 cells stimulated with heterotrimeric G protein-coupled receptor agonists known as mitogens. Thrombin, lysophosphatidic acid (LPA), and bradykinin (BK) stimulated extracellular signal-regulated protein kinase to similar extents, indicating that HepG2 cells have cell surface receptors for these agonists, which are coupled to intracellular signaling pathways. In contrast, only thrombin strongly activated JNK and p38. Thrombin-induced activation of JNK and p38 peaked at 30 minutes and 15 minutes with maximal stimulation of 13- and 4-fold increases, respectively. LPA and BK failed to activate JNK at all and activated p38 only slightly. Interestingly, thrombin-induced JNK activation was inhibited by protein kinase C down-regulation and the addition of a specific protein kinase C inhibitor. Short-term stimulation of cells with an active phorbol ester also induced JNK activation in HepG2 cells. These results indicate that thrombin is a relatively strong activator for JNK and p38 and might play a role in the regulation of activities of JNK and p38 in liver cells.
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PMID:Thrombin activates two stress-activated protein kinases, c-Jun N-terminal kinase and p38, in HepG2 cells. 958 92

Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.
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PMID:Thrombopoietin potentiates agonist-stimulated activation of p38 mitogen-activated protein kinase in human platelets. 1040 23

In platelets and other secretory cells, protein kinase C (PKC) plays a role in exocytosis stimulated by physiological extracellular signals, although its linkage to the secretory machinery is poorly understood. We investigated whether Rab6, a GTP-binding protein that fractionates with platelet alpha-granules, may be involved in linking these processes. We found that Rab6 contains two PKC consensus phosphorylation sites that are evolutionarily conserved. In platelets metabolically labelled with [(32)P]P(i), Rab6 phosphorylation was induced by phorbol esters or by thrombin. This phosphorylation was blocked by a specific PKC inhibitor (Ro-31-8220), but not by a p38 mitogen-activated protein kinase inhibitor (PD-169316). Physiological stimulation of platelets caused a PKC-dependent translocation of Rab6 from platelet particulate fractions, nearly doubling the fraction of Rab6 in the cytosol. A human Rab6 isoform (Rab6C) that is preferentially expressed in human platelet RNA was cloned and its phosphorylation by PKC was characterized. Rab6C incorporated up to 2 mol of [(32)P]P(i) per mol of active protein. Rab6C bound GDP and GTP with K(d) values of 113+/-12 and 119+/-27 nM respectively, and hydrolysed GTP at a rate of 100+/-15 micromol of GTP/mol of Rab6C per min. PKC phosphorylation of Rab6C increased the affinity for GTP by 3-fold, although it had lesser effects on GDP (1.6-fold). Phosphorylation did not alter the GTPase activity. In summary, thrombin activation of platelets leads to PKC-dependent phosphorylation of Rab6 and a translocation of Rab6 to the cytosol. We suggest that PKC phosphorylation may be an important mechanism through which Rab functional interactions in vesicle trafficking and secretion can be altered in response to an external stimulus.
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PMID:Rab6 is phosphorylated in thrombin-activated platelets by a protein kinase C-dependent mechanism: effects on GTP/GDP binding and cellular distribution. 1045 22

Physiological concentrations of low-density lipoprotein (LDL) sensitize blood platelets to alpha-thrombin- and collagen-induced secretion, and after prolonged contact trigger secretion independent of other agonists. Here we report that LDL activates the small GTPases Rap1 and Ral but not Ras, as assessed by specific precipitation of the GTP-bound enzymes. In unstirred suspensions, the inhibitor SB203580 blocks Rap1 activation by 60-70%, suggesting activation via p38 mitogen-activated protein kinase and a second, unidentified route. Inhibitors of cyclooxygenase (indomethacin) and the thromboxane A(2) (TxA(2)) receptor (SQ30741) induce complete inhibition, indicating that Rap1 activation is the result of TxA(2) formation. Stirring reveals a second, TxA(2)-independent Rap1 activation, which correlates quantitatively with a slow induction of dense granule secretion. Both pathways are unaffected by inhibitors of ligand binding to integrin alpha(IIb)beta(3). The results suggest that Rap1 and Ral, but not Ras, may take part in signalling routes initiated by LDL that initially enhance the sensitivity of platelets to other agonists and later trigger LDL-dependent secretion.
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PMID:Low-density lipoprotein activates the small GTPases Rap1 and Ral in human platelets. 1086 Dec 33

Heat-shock proteins are found in organisms as diverse as slime moulds, bacteria, plants and higher eukarycotes. They play fundamental roles in cell function, ranging from protein folding to transmembrane protein movement, to serving as scaffolds or frameworks for the assembly of enzyme signalling complexes such as the steroid receptors. Intracellular concentrations may be high, in the range of structural proteins such as actin, with which they often interact. Therefore, it is not surprising that heat-shock proteins are present in blood platelets, and recent studies point to important roles in platelet function. The small heat-shock protein, hsp27, becomes phosphorylated following cell stimulation with thrombin and associates with the actin-rich cytoskeleton. Phosphorylation results from activation of a protein kinase cascade involving the p38 mitogen-activated protein kinase (MAPK), the MAPKAP-K2 kinase, as well as PRAK, or p38-regulated protein kinase. Intriguingly, platelet hsp27 can associate with platelet factor XIII, suggesting a role for regulation of transglutaminase activity in stabilizing fibrin-platelet clots. The higher molecular-weight heat-shock proteins hsc70 and hsp90 are also present in platelets, being found in a large phosphorylated complex that contains the catalytic and myosin-targeting subunits of protein phosphatase 1 (PP1). Platelet adhesion to collagen via the alpha 2 beta 1 integrin causes the rapid dissociation of this complex and dephosphorylation of components. These results suggest that hsc70 and hsp90 can serve as signalling scaffolds, helping regulate function, including platelet adhesion and spreading via modulation of protein phosphatase activity. Hsp27, on the other hand, may be more involved in controlling actin polymerization during the platelet shape change and subsequent aggregation.
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PMID:Heat-shock proteins and platelet function. 1093 76

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood. We have previously reported that thrombin activates p38 mitogen-activated protein kinase (p38 MAPK) by a tyrosine kinase-dependent mechanism, and that p38 MAPK has a role in thrombin-induced mitogenic response in rat VSMC. In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin-induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose-dependent manner, whereas it had no effect on the response to platelet-derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA-AM and GF109203X, which suggest a requirement for intracellular Ca(2+) increase and protein kinase C. We next examined the effect of AG1478 on thrombin-induced DNA synthesis. AG1478 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. In contrast, PDGF-induced DNA synthesis was not affected by AG1478. In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin-induced proliferation of VSMC.
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PMID:Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway. 1130 36

Endothelin-1 (ET-1) has been implicated in the pathogenesis of renal inflammation. This study investigated the mechanisms underlying the synergistic upregulation of preproET-1 gene expression in human mesangial cells after co-stimulation with thrombin and tumor necrosis factor alpha (TNFalpha). Whereas thrombin induced a moderate upregulation of preproET-1 mRNA, co-stimulation with TNFalpha resulted in a strong and protracted upregulation of this mRNA species. Thrombin+TNFalpha-induced upregulation of preproET-1 expression was found to require p38 mitogen-activated protein kinase and protein kinases C, whereas activation of extracellular signal-regulated kinase, c-Jun-N-terminal kinase, or intracellular Ca(2+) release were not required. Actinomycin D chase experiments suggested that enhanced stability of preproET-1 mRNA did not account for the increase in transcript levels. PreproET-1 promoter analysis demonstrated that the 5'-flanking region of preproET-1 encompassed positive regulatory elements engaged by thrombin. Negative modulation of thrombin-induced activation exerted by the distal 5' portion of preproET-1 promoter (-4.4 kbp to 204 bp) was overcome by co-stimulation with TNFalpha, providing a possible mechanism underlying the synergistic upregulation of preproET-1 expression by these two agonists. In conclusion, human mesangial cell expression of preproET-1 may be increased potently in the presence of two common proinflammatory mediators, thereby providing a potential mechanism for ET-1 production in inflammatory renal disease.
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PMID:PreproEndothelin-1 expression in human mesangial cells: evidence for a p38 mitogen-activated protein kinase/protein kinases-C-dependent mechanism. 1137 37

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [alpha-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.
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PMID:G-protein-coupled-receptor activation of the smooth muscle calponin gene. 1143 13

The heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) is activated under hypoxic conditions, resulting in the upregulation of its target genes plasminogen activator inhibitor-1 (PAI-1) and vascular endothelial growth factor (VEGF). PAI-1 and VEGF are also induced in response to vascular injury, which is characterized by the activation of platelets and the coagulation cascade as well as the generation of reactive oxygen species (ROS). However, it is not known whether HIF-1 is also stimulated by thrombotic factors. We investigated the role of thrombin, platelet-associated growth factors, and ROS derived from the p22(phox)-containing NADPH oxidase in the activation of HIF-1 and the induction of its target genes PAI-1 and VEGF in human vascular smooth muscle cells (VSMCs). Thrombin, platelet-derived growth factor-AB (PDGF-AB), and transforming growth factor-beta(1) (TGF-beta(1)) upregulated HIF-1alpha protein in cultured and native VSMCs. This response was accompanied by nuclear accumulation of HIF-1alpha as well as by increased HIF-1 DNA-binding and reporter gene activity. The thrombin-induced expression of HIF-1alpha, PAI-1, and VEGF was attenuated by antioxidant treatment as well as by transfection of p22(phox) antisense oligonucleotides. Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol-3-kinase significantly decreased thrombin-induced HIF-1alpha, PAI-1, and VEGF expression. These findings demonstrate that the HIF-1 signaling pathway can be stimulated by thrombin and platelet-associated growth factors and that a redox-sensitive cascade activated by ROS derived from the p22(phox)-containing NADPH oxidase is crucially involved in this response.
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PMID:Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase. 1144 Sep 77


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