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)

1 Angiopoietin-1 (Ang1) is an angiogenic growth factor that binds to the Tie2 receptor on vascular endothelium, promoting blood vessel maturation and integrity. In the present study, we have investigated whether Ang1 also possesses anti-inflammatory properties by determining its effects on endothelial barrier function, neutrophil (PMN) adherence to endothelial cells (EC) and production of the PMN chemotactic factor interleukin-8 (IL-8). 2 Pretreatment of endothelial monolayers with Ang1 attenuated the permeability increase induced by thrombin in both lung microvascular cells and a human endothelial cell line. Similarly, Ang1 prevented the permeability-inducing effects of platelet-activating factor, bradykinin and histamine. 3 Pretreatment of EC with Ang1 also reduced the adherence of PMN to EC stimulated by thrombin. In contrast to its ability to counteract the increase in monolayer permeability brought about by various inflammatory agents, Ang1 did not affect the ability of histamine, PAF, or tumor necrosis factor-alpha to stimulate PMN adherence to EC. 4 In addition to its ability to inhibit PMN adherence, Ang1 diminished IL-8 production from EC challenged with thrombin in a concentration-dependent manner. 5 When EC were preincubated with the specific Rho kinase (ROCK) inhibitor Y-27632, we observed a reduction in PMN adherence in response to thrombin, as well as a decrease in thrombin-stimulated IL-8 production. Coincubation of monolayers with Y-27632 and Ang1 did not further attenuate the above-mentioned responses. However, Ang-1 failed to inhibit the activation of RhoA in response to thrombin, suggesting that inhibition of EC adhesiveness for PMN and IL-8 production by Ang1 does not result from reduced ROCK activation. 6 We conclude that Ang1 can counteract several aspects of the inflammatory response, including endothelial permeability, PMN adherence to EC as well as inhibition of IL-8 production by EC.
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PMID:Angiopoietin-1 inhibits endothelial permeability, neutrophil adherence and IL-8 production. 1277 Sep 38

Rho proteins participate in the regulation of inflammatory gene expression in endothelial cells. We made use of Clostridium difficile toxin B-10643 (TcdB-10463) which inhibites RhoA/Rac1/Cdc42 to analyze their role in expression and regulation of cyclooxygenase-2 (COX-2) in endothelial cells (EC). Pretreatment of EC with TcdB-10643 prevented lipopolysaccharide (LPS)-or tumor necrosis factor-alpha (TNFalpha)-related COX-2 expression but had no effect on COX-1 protein levels. TcdB-10463 preincubation suppressed LPS-dependent nuclear factor-kappaB activation (NF-kappaB). Rho inhibition did not affect COX-1 activity. Inactivation of Rho proteins before LPS stimulation blocked arachidonic acid (AA)-, thrombin-, and Escherichia coli hemolysin (HlyA)-dependent release of COX-2-related 6-ketoprostaglandin F(1alpha), (6k-PGF(1alpha)). In contrast, Rho inhibition did not affect COX-2-dependent 6k-PGF(1alpha) liberation when TcdB-10643 was added 10 h after LPS or TNFalpha stimulation of EC. Therefore, RhoA/Rac1/Cdc42 contribute to NF-kappaB-dependent LPS- and TNFalpha-induced expression of PGHS-2 in EC but had no effect on the activity of expressed COX-1 and COX-2.
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PMID:Rho protein inhibition blocks cyclooxygenase-2 expression by proinflammatory mediators in endothelial cells. 1279 48

The proteinase-activated receptors (PAR) PAR1 and PAR2 mediate responses to thrombin and trypsin-like proteases, respectively. Both receptors are expressed on endothelial cells where they have been reported to transduce a similar set of intracellular responses. In cultured human umbilical vein endothelial cells (HUVEC), we observed a marked difference in shape changes induced by PAR-activating peptides (PAR-APs); unlike PAR1-AP, PAR2-AP failed to stimulate cell rounding. Objectives were to shed light on the mechanisms underlying PAR-mediated cytoskeletal responses. We examined the activation of the Rho family GTPases in HUVEC using highly selective PAR1- and PAR2-APs to do this. Both peptides induced a robust and transient activation of RhoA, with the time course of activation being more sustained for the PAR1-AP. Interestingly, divergent effects on Rac activity were observed. Addition of PAR1-AP inhibited basal Rac activity as well as the phosphorylation of the Rac effector, p21-activated kinase (PAK). In contrast, PAR2-AP induced a modest activation of Rac, phosphorylation of PAK and translocation of cortactin from the cytosol to membrane ruffles, a Rac-dependent event. In vivo, only PAR1-AP rapidly enhanced vascular permeability in a mouse skin assay. We conclude that the differential regulation of the Rac/PAK pathway by PAR1 and PAR2 agonists in endothelial cells points toward distinct roles for these receptors in the control of vascular permeability and blood vessel remodeling.
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PMID:Modulation of Rho GTPase activity in endothelial cells by selective proteinase-activated receptor (PAR) agonists. 1287 83

Thrombin, a serine protease, plays an important role in the progression of atherosclerosis. How atorvastatin could limit the pro-inflammatory response to thrombin was studied in cultured rat aortic smooth muscle cells. The variations in expression of interleukin-6, heme oxygenase-1, p(22phox) and Mox-1 mRNAs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Interleukin-6 release was determined using the B9 cell assay. Nuclear factor-kappa B (NF-kappaB) translocation was analysed by electrophoretic mobility shift assay (EMSA) and RhoA protein translocation by Western blot. Thrombin activated interleukin-6 secretion and mRNA expression in smooth muscle cells in a dose-dependent manner. The greatest effect on mRNA expression was obtained after 1 h of stimulation. Preincubation (72 h) of the cells with various concentrations of atorvastatin prevented this effect. Simultaneous addition of mevalonate overcame this statin effect. Thrombin was without effect on p(22phox) and heme oxygenase-1 mRNA expression but, after 3 h of stimulation, induced a two-fold increase in that of Mox-1. Preincubation with atorvastatin dose-dependently downregulated this Mox-1 mRNA expression. In addition, thrombin induced NF-kappaB translocation and membrane translocation of RhoA in smooth muscle cells which were both prevented by pre-treatment of the cells by atorvastatin. These data demonstrate the ability of atorvastatin to prevent the induction by thrombin of a pro-inflammatory phenotype in smooth muscle cells.
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PMID:Atorvastatin limits the pro-inflammatory response of rat aortic smooth muscle cells to thrombin. 1292 59

Thus far, determining the relative contribution of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca2+-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 +/- 0.02 mol PO4/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 +/- 0.05 and 0.82 +/- 0.05 mol PO4/mol RLC, respectively, within 2.5 min. Ca2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.
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PMID:Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts. 1296 16

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.
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PMID:G13 is an essential mediator of platelet activation in hemostasis and thrombosis. 1452 98

Heterotrimeric Galpha12/13 signals induce cellular responses such as serum response element (SRE)-mediated gene transcription via Rho GTPase. Guanine nucleotide exchange factors (GEFs) are strong candidates for linking Galpha signals to Rho. For example, p115 RhoGEF transduces Galpha13 signals to Rho and inhibits Galpha12/13 signals via the RhoGEF LH domain which links to Galpha subunits. Here, we have evaluated the signaling capacity of Lbc RhoGEF in the context of Galpha12/13 signals. In vitro GEF assays indicate that baculoviral-expressed proto-Lbc has minimal exchange activity, implying that a stimulus is required for Lbc activity in vivo. Expression of a catalytically inactive proto-Lbc mutant in HEK293T cells attenuates Galpha12- and thrombin-induced activation of an SRE transcriptional reporter, and the levels of inhibition observed is similar to that obtained with an analogous p115 RhoGEF mutant. proto-Lbc mutant expression also led to decreased levels of Galpha12-induced RhoA activation in vivo. Complex formation between Galpha12 and Lbc forms was detected. Analysis of the Lbc peptide sequence reveals a previously undetected region which may link to Galpha subunit signals. These findings support a role for Lbc in Galpha12-induced signaling pathways to Rho.
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PMID:Role of Lbc RhoGEF in Galpha12/13-induced signals to Rho GTPase. 1463 90

Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.
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PMID:Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow. 1467 8

Endothelial cells express negligible amounts of tissue factor (TF) that can be induced by thrombin, which is important for acute coronary syndromes. Recent research suggests that endothelial TF expression is positively regulated by RhoA and p38mapk, but negatively by Akt/endothelial nitric oxide synthase (eNOS) pathway. High-density lipoprotein (HDL) is atheroprotective and exerts antiatherothrombotic effect. This study investigated the effect of a reconstituted HDL (rHDL) on endothelial TF expression induced by thrombin and the underlying mechanisms. In cultured human umbilical vein and aortic endothelial cells, thrombin (4 U/mL, 4 hours) increased TF protein level, which was reduced by rHDL (0.1 mg/mL, 43% inhibition, n=3 to 7, P<0.01). Activation of RhoA but not p38mapk by thrombin was prevented by rHDL. rHDL stimulated Akt/eNOS pathway. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 abolished the activation of Akt/eNOS and reversed the inhibitory effect of rHDL on TF expression. Adenoviral expression of the active PI3K mutant (p110) reduced TF expression stimulated by thrombin without inhibiting RhoA activation, whereas expression of the active Akt mutant (m/p) further facilitated TF upregulation by thrombin. Moreover, a dominant-negative Akt mutant (KA) reduced thrombin's effect and did not reverse the rHDL's inhibitory effect on TF expression. Inhibition of eNOS by N(omega)-nitro-L-arginine methyl ester (100 micromol/L) did not affect the rHDL's effect. In conclusion, rHDL inhibits thrombin-induced human endothelial TF expression through inhibition of RhoA and activation of PI3K but not Akt/eNOS. These findings implicate a novel mechanism of antiatherothrombotic effects of HDL.
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PMID:Reconstituted high-density lipoprotein inhibits thrombin-induced endothelial tissue factor expression through inhibition of RhoA and stimulation of phosphatidylinositol 3-kinase but not Akt/endothelial nitric oxide synthase. 1498 29

Thrombin mediates changes in endothelial barrier function and increases endothelial permeability. A feature of thrombin-enhanced endothelial hyperpermeability is contraction of endothelial cells (ECs), accompanied by formation of focal adhesions (FAs). Recently, a G protein-coupled receptor kinase-interacting protein, GIT1, was shown to regulate FA disassembly. We hypothesized that GIT1 modulates thrombin-induced changes in FAs. In human umbilical vein ECs (HUVECs), thrombin recruited GIT1 to FAs, where GIT1 colocalized with FAK and vinculin. Recruitment of GIT1 to FAs was dependent on activation of the small GTPase RhoA, and Rho kinase, as demonstrated by adenoviral transfection of dominant-negative RhoA and treatment with Y-27632. Thrombin stimulated GIT1 tyrosine phosphorylation with a time course similar to FAK phosphorylation in a Rho kinase- and Src-dependent manner. Depletion of GIT1 with antisense GIT1 oligonucleotides had no effect on basal cell morphology, but increased cell rounding and contraction of HUVECs, increased FA formation, and increased FAK tyrosine phosphorylation in response to thrombin, concomitant with increased endothelial hyperpermeability. These data identify GIT1 as a novel mediator in agonist-dependent signaling in ECs, demonstrate that GIT1 is involved in cell shape changes, and suggest a role for GIT1 as a negative feedback regulator that augments recovery of cell contraction.
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PMID:GIT1 mediates thrombin signaling in endothelial cells: role in turnover of RhoA-type focal adhesions. 1501 33


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