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)

Thrombin orchestrates cellular events after injury to the vascular system and extravasation of blood into surrounding tissues. The pathophysiological response to thrombin is mediated by protease-activated receptor-1 (PAR-1), a seven-transmembrane G protein-coupled receptor expressed in the nervous system that is identical to the thrombin receptor in platelets, fibroblasts, and endothelial cells. Once activated by thrombin, PAR-1 induces rapid and dramatic changes in cell morphology, notably the retraction of growth cones, axons, and dendrites in neurons and processes in astrocytes. The signal is conveyed by a series of localized ATP-dependent reactions directed to the actin cytoskeleton. How cells meet the dynamic and localized energy demands during signal transmission is unknown. Using the yeast two-hybrid system, we identified an interaction between PAR-1 cytoplasmic tail and the brain isoform of creatine kinase, a key ATP-generating enzyme that regulates ATP within subcellular compartments. The interaction was confirmed in vitro and in vivo. Reducing creatine kinase levels or its ATP-generating potential inhibited PAR-1-mediated cellular shape changes as well as a PAR-1 signaling pathway involving the activation of RhoA, a small G protein that relays signals to the cytoskeleton. Thrombin-stimulated intracellular calcium release was not affected. Our results suggest that creatine kinase is bound to PAR-1 where it may be poised to provide bursts of site-specific high-energy phosphate necessary for efficient receptor signal transduction during cytoskeletal reorganization.
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PMID:Creatine kinase, an ATP-generating enzyme, is required for thrombin receptor signaling to the cytoskeleton. 1105 Feb 37

Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase-dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2). Here, we present evidence that both proteins undergo PI 3-kinase-dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCgamma2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)-labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.
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PMID:Phosphatidylinositol 3-kinase-dependent translocation of phospholipase Cgamma2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation. 1115 84

Loss of membrane potential (membrane depolarization) is one of the earliest and most striking responses of quiescent cells to stimulation with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and thrombin. Membrane depolarization is due to the activation of a chloride conductance. While this response has received relatively little attention in the past, it is clear that the acute loss of membrane potential may have important physiological consequences. However, the dissection of the underlying G protein pathway and the establishment of cause-effect relationships have remained elusive to date. Here we report that, in neuronal cells, the depolarizing chloride current invariably accompanies GPCR-induced activation of RhoA and subsequent neurite retraction, and neither of these events requires phosphoinositide hydrolysis or Ca2+ mobilization. Through antibody microinjections and a genetic approach, we demonstrate that activation of the chloride conductance is mediated by Galpha(13) in a RhoA-independent manner in both neuronal cells and fibroblasts. We further show that, in neuronal cells, this newly described Galpha(13) pathway may profoundly modulate membrane excitability during RhoA-regulated neurite remodeling.
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PMID:Galpha(13) mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells. 1123 Nov 29

The C termini of G protein alpha subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a G alpha(i) peptide to bind the A(1) adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998 ) J. Biol. Chem. 273, 14912--14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of G alpha(i) could specifically inhibit downstream responses to agonist stimulation of the muscarinic M(2) receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610--6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of G alpha subunits. Minigene vectors expressing G alpha C-terminal peptides (G alpha(i), G alpha(q), G alpha(12), and G alpha(13)) or the control minigene vector, which expresses the G alpha(i) peptide in random order (G(iR)), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal G alpha minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.
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PMID:G alpha minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation. 1127 83

The growth-stimulating effects of thrombin are mediated primarily via activation of a G protein-coupled receptor, PAR-1. Because PAR-1 has no intrinsic tyrosine kinase activity, yet requires tyrosine phosphorylation events to induce mitogenesis, we investigated the role of the Janus tyrosine kinases (JAKs) in thrombin-mediated signaling. JAK2 was activated rapidly in rat vascular smooth muscle cells (VSMC) treated with thrombin, and signal transducers and activators of transcription (STAT1 and STAT3) were phosphorylated and translocated to the nucleus in a JAK2-dependent manner. AG-490, a JAK2-specific inhibitor, and a dominant negative JAK2 mutant inhibited thrombin-induced ERK2 activity and VSMC proliferation suggesting that JAK2 is upstream of the Ras/Raf/MEK/ERK pathway. To elucidate the functional significance of JAK-STAT activation, we studied the effect of thrombin on heat shock protein (Hsp) expression, based upon the following: 1) reports that thrombin stimulates reactive oxygen species production in VSMC; 2) the putative role of Hsps in modulating cellular responses to reactive oxygen species; and 3) the presence of functional STAT1/3-binding sites in Hsp70 and Hsp90beta promoters. Indeed, thrombin up-regulated Hsp70 and Hsp90 protein expression via enhanced binding of STATs to cognate binding sites in the Hsp70 and Hsp90 promoters. Together, these results suggest that JAK-STAT pathway activation is necessary for thrombin-induced VSMC growth and Hsp gene expression.
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PMID:Thrombin regulates vascular smooth muscle cell growth and heat shock proteins via the JAK-STAT pathway. 1127 37

Cellular effects of thrombin are mediated by members of a new subfamily of G protein-coupled receptors designated proteinase-activated receptors (PARs) with the prototype PAR-1. Investigation of PAR-1-induced signaling has been shown to be very important in clarifying thrombin's role in cell metabolism, differentiation, and growth. We evaluated connection of PAR-1 with the cAMP/PKA pathway in SNB-19 glioblastoma cells. Alpha-thrombin and the synthetic PAR-1 agonist SFLLRN stimulated PKA as shown by increased PKA activity and translocation of the catalytic PKA alpha subunits (PKA(cat)alpha) into the nucleus. However, no effect on cAMP could be observed. PKA(cat)alpha was found to be associated with nuclear factor-kappa B (NF-kappaB) p65 and its inhibitor protein IkappaB in SNB-19 cells. After PAR-1 stimulation, this association was markedly diminished. We conclude that PAR-1 mediates PKA activation without altering cAMP levels but includes NF-kappaB-associated PKA(cat)alpha in SNB-19 glioblastoma cells. This is the first evidence for a cAMP-independent PKA signaling by a G protein-coupled receptor.
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PMID:A novel PAR-1-type thrombin receptor signaling pathway: cyclic AMP-independent activation of PKA in SNB-19 glioblastoma cells. 1135 44

We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Galpha(i) and Galpha(12)/Galpha(13) subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.
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PMID:The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways. 1136 Jan 79

Thrombin, the terminal serine protease in the coagulation cascade, is a proinflammatory molecule in vivo and induces endothelial activation in vitro. The cellular signaling mechanisms involved in this function are unknown. The role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in thrombin-induced chemokine production was studied. Phosphorylation of both p38 MAPK and its substrate, ATF-2, was observed in human umbilical vein endothelial cells (HUVECs) stimulated with thrombin, with a maximum after 5 minutes of stimulation. Using the selective p38 MAPK inhibitor SB203580, there was a significant decrease in thrombin-induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) protein production and messenger RNA steady-state levels. In addition, SB203580 decreased IL-8 and MCP-1 production induced by the thrombin receptor-1 agonist peptide (TRAP), suggesting functional links between the thrombin G protein-coupled receptor and the p38 MAPK pathway. Furthermore, endothelial activation in the presence of SB203580 decreased the chemotactic activity of thrombin-stimulated HUVEC supernatant on neutrophils and monocytic cells. In contrast, the p42/p44 MAPK pathway did not appear to be involved in thrombin- or TRAP-induced endothelial chemokine production, because there was no reduction in the presence of the p42/p44-specific inhibitor PD98059. These results demonstrate that the p38 rather than p42/44 MAPK signaling pathway plays an important role in thrombin-induced endothelial proinflammatory activation and suggest that inhibition of p38 MAPK may be an interesting target for anti-inflammatory strategies in vascular diseases combining thrombosis and inflammation. (Blood. 2001;98:667-673)
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PMID:The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment. 1146 65

The coagulation protease thrombin triggers fibrin formation, platelet activation, and other cellular responses at sites of tissue injury. We report a role for PAR1, a protease-activated G protein-coupled receptor for thrombin, in embryonic development. Approximately half of Par1-/- mouse embryos died at midgestation with bleeding from multiple sites. PAR1 is expressed in endothelial cells, and a PAR1 transgene driven by an endothelial-specific promoter prevented death of Par1-/- embryos. Our results suggest that the coagulation cascade and PAR1 modulate endothelial cell function in developing blood vessels and that thrombin's actions on endothelial cells-rather than on platelets, mesenchymal cells, or fibrinogen-contribute to vascular development and hemostasis in the mouse embryo.
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PMID:A role for thrombin receptor signaling in endothelial cells during embryonic development. 1153 66

The serine protease thrombin has been proposed to be involved in neuromuscular plasticity. Its specific receptor "protease activated receptor-1" (PAR-1), a G protein-coupled receptor, has been shown to be expressed in myoblasts but not after fusion (Suidan et al., 1996 J Biol Chem 271:29162-29169). In the present work we have investigated the expression of PAR-1 during rat skeletal muscle differentiation both in vitro and in vivo. Primary cultures of rat foetal skeletal muscle, characterized by their spontaneous contractile activity, were used for exploration of PAR-1 by RT-PCR, immunocytochemistry and Western blotting. Our results show that PAR-1 mRNA and protein are both present in myoblasts and myotubes. Incubation of myotubes loaded with fluo-3-AM in presence of thrombin (200 nM) or PAR-1 agonist peptide (SFLLRN, 500 microM), induced the intracellular release of calcium indicating the activation of PAR-1. Blockade of contractile activity by tetrodotoxin (TTX, 6 nM) did not modify either PAR-1 synthesis or its cellular localization. Investigation of PAR-1 on rat muscle cryostat sections at Day 18 of embryogenesis and postnatal Days 1, 5, and 10 indicated that this protein is first expressed in the cytoplasm and that it later localizes to the membrane. Moreover, its expression correlates with myosin heavy chain transitions occurring during post-natal period and is restricted to primary fibers. Taken together, these results suggest that PAR-1 expression is not related to contractile activity but to myogenic differentiation.
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PMID:Expression of the thrombin receptor (PAR-1) during rat skeletal muscle differentiation. 1159


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