Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin.
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Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin -- PAR1, PAR3, and PAR4 -- are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 account for activation of human platelets by thrombin, or whether PAR3 or still other receptors contribute, is unknown. We have examined the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein were detected in human platelets. Activation of either receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone by antagonist, blocking antibody, or desensitization blocked platelet activation by 1 nM thrombin but only modestly attenuated platelet activation by 30 nM thrombin. Inhibition of PAR4 alone using a blocking antibody had little effect at either thrombin concentration. Strikingly, simultaneous inhibition of both PAR1 and PAR4 virtually ablated platelet secretion and aggregation, even at 30 nM thrombin. These observations suggest that PAR1 and PAR4 account for most, if not all, thrombin signaling in platelets and that antagonists that block these receptors might be useful antithrombotic agents. (+info)
Acceleration of Ca2+ ionophore-induced arachidonic acid liberation by thrombin without the proteolytic action toward the receptor in human platelets.
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We investigated the regulation of arachidonic acid liberation catalyzed by group-IV cytosolic phospholipase A2 (cPLA2) in human platelets upon stimulation with thrombin through interaction with protease-activated receptor-1 (PAR-1) or glycoprotein Ib. Leupeptin, a protease inhibitor, completely inhibited thrombin-induced arachidonic acid liberation and Ca2+ mobilization, with inhibition of its protease activity. However, preincubation with thrombin in the presence of leupeptin potentiated Ca2+ ionophore-induced arachidonic acid liberation. The preincubation did not affect the intracellular Ca2+ level or cPLA2 activity in response to ionomycin. Human leukocyte elastase, which cleaves glycoprotein Ib, did not inhibit the enhancement of arachidonic acid liberation by thrombin in the presence of leupeptin. However, the effect of thrombin with leupeptin was abolished by a peptide corresponding to residues 54-65 of hirudin (hirudin peptide), which impairs the binding of thrombin to PAR-1. Furthermore, Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, which binds to platelets but has no protease activity, also enhanced Ca2+ ionophore-induced arachidonic acid liberation. In contrast, trypsin with leupeptin did not mimic the effect of thrombin with leupeptin, and furthermore trypsin-induced arachidonic acid liberation was insensitive to hirudin peptide. On the basis of the present results, we suggest that thrombin may accelerate cPLA2-catalyzed arachidonic acid liberation through non-proteolytic action toward PAR-1 but not toward glycoprotein Ib in co-operation with the proteolytic action leading to Ca2+ mobilization. (+info)
Pro- and anti-inflammatory actions of thrombin: a distinct role for proteinase-activated receptor-1 (PAR1).
(3/694)
1. Thrombin has well characterized pro-inflammatory actions that have recently been suggested to occur via activation of its receptor, proteinase-activated receptor-1 (PAR1). 2. In the present study, we have compared the effects of thrombin to those of two peptides that selectively activate the PAR1 receptor, in a rat hindpaw oedema model. We have also examined whether or not thrombin can exert anti-inflammatory activity in this model. 3. Both thrombin and the two PAR1 activating peptides induced significant oedema in the rat hindpaw following subplantar injection. 4. The oedema induced by thrombin was abolished by pre-incubation with hirudin, and was markedly reduced in rats in which mast cells were depleted through treatment with compound 48/80 and in rats pretreated with indomethacin. In contrast, administration of the PAR1 activating peptides produced an oedema response that was not reduced by indomethacin and was only slightly reduced in rats pretreated with compound 48/80. 5. Co-administration of thrombin together with a PAR1 activating receptor resulted in a significantly smaller oedema response than that seen with the PAR1 activating peptide alone. This anti-inflammatory effect of thrombin was abolished by pre-incubation with hirudin. 6. These results demonstrate that the pro-inflammatory effects of thrombin occur through a mast-cell dependent mechanism that is, at least in part, independent of activation of the PAR1 receptor. Moreover, thrombin is able to exert anti-inflammatory effects that are also unrelated to the activation of PAR1. (+info)
Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway.
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We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade. (+info)
Thrombin-induced p65 homodimer binding to downstream NF-kappa B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion.
(5/694)
We investigated the mechanisms by which proinflammatory mediator, thrombin, released during intravascular coagulation and tissue injury, induces ICAM-1 (CD54) expression in endothelial cells. Stimulation of HUVEC with thrombin resulted in dose- and time-dependent increases in ICAM-1 mRNA and cell surface expression and in ICAM-1-dependent endothelial adhesivity toward polymorphonuclear leukocytes. Transient transfection of endothelial cells with ICAM-1 promoter luciferase reporter gene (ICAM-1LUC) constructs indicated that deletion of upstream NF-kappa B site (-533 bases from translation start site) had no effect on thrombin responsiveness, whereas mutation/deletion of downstream NF-kappa B site (-223 bases from the translation start site) prevented the activation of ICAM-1 promoter, indicating that the downstream NF-kappa B site is critical for thrombin inducibility. NF-kappa B-directed luciferase activity increased approximately 3-fold when cells transfected with the plasmid pNF-kappa BLUC containing five copies of consensus NF-kappa B site linked to a minimal adenovirus E1B promoter-luciferase gene were exposed to thrombin, indicating that activation of NF-kappa B was essential for thrombin response. Gel supershift assays demonstrated that thrombin induced binding of NF-kappa Bp65 (Rel A) to downstream NF-kappa B site of the ICAM-1 promoter. Thrombin receptor activation peptide, a 14-amino-acid peptide representing the new NH2 terminus of proteolytically activated receptor-1, mimicked thrombin's action in inducing ICAM-1 expression. These data indicate that thrombin activates endothelial ICAM-1 expression and polymorphonuclear leukocyte adhesion by NF-kappa Bp65 binding to the downstream NF-kappa B site of ICAM-1 promoter after proteolytically activated receptor-1 activation. (+info)
Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2.
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1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine. (+info)
Enhancement of vascular permeability by specific activation of protease-activated receptor-1 in rat hindpaw: a protective role of endogenous and exogenous nitric oxide.
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1. To clarify the role of the first thrombin receptor/protease-activated receptor (PAR)-1 in an inflammatory process, we tested and characterized the effect of intraplantar (i.pl.) administration of the highly specific PAR-1 agonist TFLLR-NH2 in rat hindpaw. 2. TFLLR-NH2 administered i.pl. at 0.01-0.03 micromol per paw enhanced vascular permeability in the hindpaw and produced paw oedema in a dose-dependent manner. This effect was almost completely abolished by repeated pretreatment with compound 48/80 to deplete inflammatory mediators in mast cells. 3. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester or N-iminoethyl-L-ornithine, preadministered i.pl., stereospecifically potentiated the i.pl. TFLLR-NH2-induced permeability increase, while the NO donor sodium nitroprusside or NOC-18, given i.pl., suppressed the effect of TFLLR-NH2. 4. These findings demonstrate that specific activation of PAR-1 produces increased vascular permeability accompanied by oedema formation in the rat hindpaw, predominantly via mast cell degranulation, and that endogenous and exogenous NO plays a protective role in the PAR-1-mediated inflammatory event. (+info)
Thrombin-induced platelet activation is inhibited by high- and low-molecular-weight heparin.
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BACKGROUND: Thrombin binds to platelet glycoprotein Ib (Gp Ib), and this interaction contributes to platelet activation. Thrombin ligation to Gp Ib was recently shown to be inhibited by heparin, thus raising the hypothesis, investigated in this article, that heparin might inhibit thrombin-induced platelet activation. METHODS AND RESULTS: Aggregation of gel-filtered platelets by 1 nmol/L thrombin was reduced by both high-molecular-weight (MW) (14 500-Da) and low-MW (4500-Da) heparin, with IC50 values of 1.65+/-0.26 and 5.13+/-0.8 micromol/L, respectively. Homogeneous-MW fractions (16 000- to 13 000-Da range) were used to evaluate the heparin effect on intracytoplasmic calcium release by thrombin. Calcium mobilization by 1 nmol/L thrombin was reduced as a function of heparin concentration, and the inhibitory effect was correlated to the MW of heparin fractions (IC50 values were 1.9+/-0.39, 6.07+/-0.83, and 14. 8+/-0.43 micromol/L for 16 000-, 9000-, and 3000-Da heparin, respectively). Platelet aggregation and calcium mobilization by ADP and by the thrombin receptor-activating peptide were not affected by heparin. The activation of Gp Ib-depleted platelets by alpha-thrombin was not inhibited by heparin. Moreover, platelet stimulation by heparin binding site phosphopyridoxylated thrombin, which has a severe impairment of Gp Ib ligation, was not affected by heparin. Finally, heparin did not interfere with the hydrolysis by thrombin of the protease-activated receptor 1. CONCLUSIONS: These results demonstrated that heparin, by inhibiting the thrombin-Gp Ib interaction, is able to interfere with thrombin-induced platelet activation. The extent of the inhibitory effect is directly related to the MW of heparin fractions. (+info)