Duodenase activates rat peritoneal mast cells via protease-activated receptors of type 1

It was found that duodenase, a serine protease from the bovine duodenum, activates rat peritoneal mast cells (PMC) in vitro presumably via protease-activated receptors (PARs). Like thrombin (a serine protease from the blood coagulation system) and the PAR1 agonist peptide (PAR1-AP), duodenase was shown to accelerate the secretion of β-hexosaminidase (a marker of cell degranulation) by PMC in a dose-dependent manner. The blockage of the proteolytic activity of duodenase toward the substrate Tos-Gly-Pro-Lys-pNA by the soybean Bauman-Birk protease inhibitor substantially reduced (by 40%) the ability of duodenase to stimulate the secretory activity of PMC. Pretreatment of PMC with duodenase decreased the β-hexosaminidase secretion induced by thrombin and PAR1-AP by 35 and 41.7 %, respectively, and abolished the antiinflammatory effect of activated protein C. At the same time, pretreatment of PMC with duodenase did not affect the secretion of β-hexosaminidase induced by compound 48/80, a nonspecific degranulator of mast cells. Duodenase, unlike PAR1-AP (30–100 μM), in a broad concentration range (10–100 nM) did not induce aggregation of human platelets, but suppressed the platelet aggregation elicited by PAR1-AP.     

The role of PAR1 in protective action of activated protein C during nonimmune activation of mast cells

Activated protein C (APC) regulates the functional activity of mast cells by reducing release of β-hexosaminidase, the marker of mast cell degranulation. APC modulated not only spontaneous secretion from mast cells, but also secretion induced by the degranulators, proteinase-activated receptor agonist peptide (PAR1-AP) and compound 48/80. PAR1 desensitization by thrombin abolished the decrease of β-hexosaminidase secretion induced by low APC concentrations (≤1.5 nM). APC inactivated by phenylmethylsulfonyl fluoride (PMSF), did non mimic the enzyme action on mast cells. Duodenase (the duodenal proteinase) activated peritoneal mast cell via PAR1. APC abolished the proinflammatory effect of duodenase and PAR1-AP by reducing release of mast cell mediators. The effect of APC could be attributed to nitric oxide generation by mast cells because in the presence of L-NAME the secretory function restored. These data suggest involvement of mast cell PAR1 into regulatory mechanism responsible for the anti-inflammatory effect of APC.     

Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts.

Duodenase is a 29-kDa serine endopeptidase that displays selective trypsin- and chymotrypsin-like substrate specificity. This enzyme has been localized to epitheliocytes of Brunner's glands, and as described here, to mast cells within the intestinal mucosa and lungworm-infected lung, implying an important additional role in inflammation and tissue remodelling. In primary cultures of pulmonary artery fibroblasts, duodenase induced a concentration-dependent increase in [3H]thymidine incorporation with a maximal effect observed at 30 nm. Pretreating duodenase with soybean trypsin inhibitor abolished DNA synthesis, confirming that proteolytic activity was an essential requirement for this response. PAR1, PAR2 and PAR4 activating peptides were unable to induce [3H]thymidine incorporation in pulmonary artery fibroblasts. Likewise, pretreatment of fibroblasts with TNFalpha, known to up-regulate PAR2 expression in other systems, and IL-1beta, did not enhance the potential of duodenase to induce DNA synthesis. Furthermore, duodenase increased GTPgammaS binding to fibroblast membranes indicating that a G-protein-coupled receptor may mediate the effects of duodenase. Duodenase-induced DNA synthesis and GTPgammaS binding were both found to be inhibited by pertussis toxin, implying a role for Gi/o. Selective inhibitors of MEK1 (PD98059) and protein kinase C (GF109203X) only partially inhibited duodenase-induced DNA synthesis, but both wortmannin (100 nm) and LY294002 (10 microm) inhibited this response completely, indicating a key role for PtdIns 3-kinase. Furthermore, duodenase induced a 2.3 plus minus 0.1-fold increase in PtdIns 3-kinase activity in p85 immunoprecipitates, which was sensitive to inhibition by wortmannin. These results suggest that duodenase can induce pulmonary artery fibroblast DNA synthesis in a PtdIns 3-kinase-dependent manner via a G-protein-coupled receptor which is activated by a proteolytic mechanism.     

Interaction of viper venom serine peptidases with thrombin receptors on human platelets

The serine peptidases, thrombocytin and PA-BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA-BJ, and thrombocytin on the degradation of the soluble N-terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41-Ser42. In addition, a rapid cleavage of the peptide bond at Arg46-Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA-BJ and thrombocytin both cleaved at 41-42 and 46-47 peptide bonds, and fragment 42-103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.     

Protease-activated receptor-2 regulates vascular endothelial growth factor expression in MDA-MB-231 cells via MAPK pathways

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor that is cleaved and activated by serine proteases including the coagulation protease factor VIIa (FVIIa). There is evidence that PAR2 function contributes to angiogenesis, but the mechanisms involved are poorly defined. Here we show that PAR2 activation in human breast cancer cells leads to the upregulation of vascular endothelial growth factor (VEGF). Activation of PAR2 with agonist peptide (AP), trypsin or FVIIa results in a robust increase of VEGF message and protein. Incubation of cells with PAR1-AP, PAR3-AP, PAR4-AP, or thrombin has only a modest effect on VEGF production. Cleavage blocking antibodies show that FVIIa-mediated VEGF production is PAR2 mediated. Mitogen-activated protein kinase (MAPK) pathway inhibitors U0126 and SB203580 inhibit PAR2-mediated VEGF production. Incubation of cells with PAR2-AP leads to significant extracellular regulated kinase1/2 (ERK1/2) and p38 MAPK phosphorylation and activation. Collectively, these data suggest that PAR2 signaling through MAPK pathways leads to the production of proangiogenic VEGF in breast cancer cells.     

A new concept of action of hemostatic proteases on inflammation,neurotoxicity, and tissue regeneration

Key hemostatic serine proteases such as thrombin and activated protein C (APC) are signaling molecules controlling blood coagulation and inflammation, tissue regeneration, neurodegeneration, and some other processes. By interacting with protease-activated receptors (PARs), these enzymes cleave a receptor exodomain and liberate new amino acid sequence known as a tethered ligand, which then activates the initial receptor and induces multiple signaling pathways and cell responses. Among four PAR family members, APC and thrombin mainly act via PAR1, and they trigger divergent effects. APC is an anticoagulant with antiinflammatory and cytoprotective activity, whereas thrombin is a protease with procoagulant and proinflammatory effects. Hallmark features of APC-induced effects result from acting via different pathways: limited proteolysis of PAR1 localized in membrane caveolae with coreceptor (endothelial protein C receptor) as well as its targeted proteolytic action at a receptor exodomain site differing from the canonical thrombin cleavage site. Hence, a new noncanonical tethered PAR1 agonist peptide (PAR1-AP) is formed, whose effects are poorly investigated in inflammation, tissue regeneration, and neurotoxicity. In this review, a concept about a role of biased agonism in effects exerted by APC and PAR1-AP via PAR1 on cells involved in inflammation and related processes is developed. New evidence showing a role for a biased agonism in activating PAR1 both by APC and PAR1-AP as well as induction of antiinflammatory and cytoprotective cellular responses in experimental inflammation, wound healing, and excitotoxicity is presented. It seems that synthetic PAR1 peptide-agonists may compete with APC in controlling some inflammatory and neurodegenerative diseases.     

Protease-activating receptor-4 induces full platelet spreading on a fibrinogen matrix: involvement of ERK2 and p38 and Ca2+ mobilization

Although the involvement of protease-activating receptor PAR1 and PAR4 is well established in platelet aggregation, their role in platelet adhesion and spreading has yet to be characterized. We investigated platelet adhesion and spreading on a fibrinogen matrix after PAR1 and PAR4 stimulation in correlation with the activation of two MAPKs, ERK2 and p38. Of the two PAR-activating peptides (PAR-APs), PAR1-AP and PAR4-AP, which both induce adhesion, only PAR4-AP induced full platelet spreading. Although both PAR1-AP and PAR4-AP induced ADP secretion, which is required for platelet spreading, only PAR4-AP induced sustained Ca(2+) mobilization. In these conditions of PAR4 induction, ERK2 and p38 activation were involved in platelet spreading but not in platelet adhesion. p38 phosphorylation was dependent on ADP signaling through P2Y12, its receptor. ERK2 phosphorylation was triggered through integrin alphaIIbbeta3 outside-in signaling and was dependent on the Rho pathway. ERK2 and p38 activation induced phosphorylation of the myosin light chain and actin polymerization, respectively, necessary for cytoskeleton reorganization. These findings provide the first evidence that thrombin requires PAR4 for the full spreading response. ERK2 and p38 and sustained Ca(2+) mobilization, involved in PAR4-induced platelet spreading, contribute to the stabilization of platelet thrombi at sites of high thrombin production.     

Modulation of hippocampal neuron survival by thrombin and factor Xa

Effects of thrombin, factor Xa (FXa), and protease-activated receptor 1 and 2 agonist peptides (PAR1-AP and PAR2-AP) on survival and intracellular Ca2+ homeostasis in hippocampal neuron cultures treated with cytotoxic doses of glutamate were investigated. It is shown that at low concentrations (相似文献   

Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells

Thrombin and related protease-activated receptors 1, 2, 3, and 4 (PAR1–4) play a multifunctional role in many types of cells including endothelial cells. Here, using RT-PCR and immunofluorescence staining, we showed for the first time that PAR1–4 are expressed on primary human brain microvascular endothelial cells (HBMEC). Digital fluorescence microscopy and fura 2 were used to monitor intracellular Ca²⁺ concentration ([Ca²⁺]_i) changes in response to thrombin and PAR1-activating peptide (PAR1-AP) SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca²⁺]_i rise that was inhibited by pretreatment of HBMEC with the phospholipase C inhibitor U-73122 and the sarco(endo)plasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin. Thrombin induced transient [Ca²⁺]_i increase, whereas PAR1-AP exhibited sustained [Ca²⁺]_i rise. The PAR1-AP-induced sustained [Ca²⁺]_i rise was significantly reduced in the absence of extracellular calcium or in the presence of an inhibitor of store-operated calcium channels, SKF-96365. Restoration of extracellular Ca²⁺ to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca²⁺ resulted in significant [Ca²⁺]_i rise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented [Ca²⁺]_i rise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca²⁺]_i rise to high PAR1-AP concentration. Additionally, treatment with thrombin decreased transendothelial electrical resistance in HBMEC, whereas PAR1-AP was without significant effect. These findings suggest that, in contrast to thrombin, stimulation of PAR1 by untethered peptide SFFLRN results in stimulation of store-operated Ca²⁺ influx without significantly affecting brain endothelial barrier functions. store-operated calcium influx; desensitization; transendothelial electrical resistance; digital imaging     

Thrombin induces IL-6 but not TNFalpha secretion by mouse mast cells: threshold-level thrombin receptor and very low level FcepsilonRI signaling synergistically enhance IL-6 secretion

Mast cells become activated in multiple diseases wherein thrombin generation is often clinically apparent, but the effect of thrombin on cytokine release by mast cells remains unexplored. Thus, we examined IL-6 and TNFalpha release by thrombin-challenged mast cells. Thrombin and the protease-activated receptor (PAR)-1 peptide TRAP(14) induced these cells to secrete IL-6 in a dose-dependent fashion. Mast cells secreted > or =2800 pg IL-6/10(6) cells over 24 h, but only low levels of serotonin and no significant TNFalpha. Furthermore, at near-background levels of allergen, threshold doses of alpha-thrombin synergistically enhanced the IL-6 response (by up to 100-fold), but high-dose costimulation led to a simple additive response. Both the PI(3)- and sphingosine-kinase signaling pathways contributed importantly to the thrombin response. Our data thus clearly demonstrate that low-level thrombin and FcepsilonRI signaling can synergize to augment mast cell IL-6 responses, and that thrombin also differentially induces cytokine secretion by mast cells.     

Trypsin induces activation and inflammatory mediator release from human eosinophils through protease-activated receptor-2.

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.     

The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.     

PAR1-mediated RhoA activation facilitates CCL2-induced chemotaxis in PC-3 cells

Patients with advanced prostate cancer often exhibit increased activation of the coagulation system. The key activator of the coagulation cascade is the serine protease thrombin which is capable of eliciting numerous cellular responses. We previously reported that the thrombin receptor PAR1 is overexpressed in prostate cancer. To investigate further the role of PAR1 in prostate cancer metastasis, we examined the effects of thrombin activation on cell adhesion and motility in PC-3 prostate cancer cells. Activation of PAR1-induced dynamic cytoskeletal reorganization and reduced PC-3 binding to collagen I, collagen IV, and laminin (P < 0.01) but not fibronectin. Expression of the cell surface integrin receptors did not change as assessed by flow cytometry. Immunofluorescence microscopy revealed that PAR1 stimulation caused reorganization of the focal adhesions, suggesting that PAR1 activation in PC-3 cells may be modulating cell adhesion through integrin function but not expression. Furthermore, RhoA was activated upon stimulation with thrombin with subsequent cell contraction, decreased cell adhesion, and induced migration towards monocyte chemoattractant protein 1 (MCP-1; CCL2). Thus, it appears that thrombin stimulation plays a role in prostate cancer metastasis by decreasing cell adhesion to the extracellular matrix and positioning the cell in a "ready state" for migration in response to a chemotactic signal. Further exploration is needed to determine whether PAR1 activation affects other signaling pathways involved in prostate cancer.     

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibα of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca²⁺ mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbα by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbα binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbα proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbα cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbα.     

Thrombin and PAR-1 acitvating peptide increase iNOS expression in cytokine-stimulated C6 glioma cells

Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1-10 U/mL) and PAR1-AP (1-100 microM) induced a significant concentration-dependent increase both of IFN-gamma- (250 U/mL) or TNF-alpha- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS-PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulog(TM) (60 microg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response.     

Induction of IL-6 release from human T cells by PAR-1 and PAR-2 agonists

Proteinase-activated receptors (PAR) have been recognized as playing an important role in inflammation and immune response. However, little is known of the expression and function of PAR on human T cells. In this study, the expression of PAR on highly purified human T cells was determined and the secretion of IL-6 from cultured T cells in response to serine proteinases and agonist peptides of PAR was examined. The results showed that T cells express PAR-1, PAR-2 and PAR-3 proteins and genes. Thrombin, trypsin and tryptase, but not elastase, were able to stimulate concentration-dependent secretion of IL-6 from T cells following a 16 h incubation period. The specific inhibitors of thrombin, trypsin and tryptase inhibited the actions of these proteinases on T cells, indicating that the enzymatic activity is essential for their actions. Agonist peptides of PAR SFLLR-NH2, TFLLRN-NH2 and SLIGKV-NH2, but not TFRGAP-NH2, GYPGQV-NH2 and AYPGKF-NH2, are also capable of inducing IL-6 release from T cells. In conclusion, induction of IL-6 secretion from T cells by thrombin, trypsin and tryptase is probably through the activation of PAR, suggesting that serine proteinases are involved in the regulation of immune response of the body.     

Osteosarcoma cell-calcium signaling through tissue factor-factor VIIa complex and factor Xa

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.     

Trypsin induces tumour necrosis factor-alpha secretion from a human leukemic mast cell line

Trypsin activating both proteinase-activated receptor (PAR) 2 and PAR4 plays an important role in inflammation. We have investigated the potential of trypsin to induce TNF-alpha secretion from the human leukemic mast cell line (HMC-1). HMC-1 cells co-express both PAR2 and PAR4, and their agonist trypsin signals to HMC-1 cells. Trypsin (100 nm), SLIGKV-NH(2) (100 microm, corresponding to the PAR2 tethered ligand), or GYPGQV-NH(2) (100 microm, corresponding to the PAR4 tethered ligand) induced tumour necrosis factor (TNF)-alpha secretion from HMC-1 cells. TNF-alpha secretion by trypsin was significantly blocked by pretreatment with 50 microm PD098059, MEK-1 inhibitor. Furthermore, trypsin stimulated the activation of extracellular signal-regulated kinase (ERK) in HMC-1 cells without any detectable activation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase homologue. These results show that trypsin may induce TNF-alpha secretion following activation of ERK via both PAR2 and PAR4 on HMC-1 cells.     

The role of PAR family receptors in activation of mast cells in the norm and in acute inflammation in rats

Dose-dependent release of beta-hexoaminidase induced with thrombin was shown to be mediated by the PAR-1. This was further confirmed by means of agonist, antagonist and PAR desensitization. Acceleration of the mast cell mediator secretion by the Xa factor and PAR-2 agonist, was revealed. An increase in the mast cell release induced by thrombin and TRAP-6 was shown in the acute peritonitis model.     

Inflammatory mediators modulate thrombin and cathepsin-G signaling in human bronchial fibroblasts by inducing expression of proteinase-activated receptor-4

Human lung fibroblasts express proteinase-activated receptor-1 (PAR1), PAR2 and PAR3, but not PAR4. Because PAR2 has inflammatory effects on human primary bronchial fibroblasts (HPBF), we asked 1) whether the inflammatory mediators TNF-alpha and LPS could modify HPBF PAR expression and 2) whether modified PAR expression altered HPBF responsiveness to PAR agonists in terms of calcium signaling and cell growth. TNF-alpha and LPS induced PAR4 mRNA expression (RT-PCR) at 6 h and 24 h, respectively. TNF-alpha and LPS also upregulated PAR2 mRNA expression with similar kinetics but had negligible effect on PAR1 and PAR3. Flow cytometry for PAR1, PAR2, and PAR3 also demonstrated selective PAR2 upregulation in response to TNF-alpha and LPS. Intracellular calcium signaling to SLIGKV-NH2 (a selective PAR2-activating peptide; PAR2-AP) and AYPGQV-NH2 (PAR4-AP) revealed that TNF-alpha and LPS induced maximal responses to these PAR agonists at 24 h and 48 h, respectively. Upregulation of PAR2 by TNF-alpha heightened HPBF responses to trypsin, while PAR4 induction enabled cathepsin-G-mediated calcium signaling. Cathepsin-G also disarmed PAR1 and PAR2 in HPBF, while tryptase disarmed PAR2. Induction of PAR4 also enabled thrombin to elicit a calcium signal through both PAR1 and PAR4, as determined by a desensitization assay. In cell growth assays the PAR4 agonists cathepsin-G and AYPGQV-NH2 reduced HPBF cell number only in TNF-alpha-treated HPBF. Moreover, the mitogenic effect of thrombin (a PAR1/PAR4 agonist) but not the PAR1-AP TFLLR-NH2, was ablated in TNF-alpha-treated HPBF. These findings point to an important mechanism, whereby cellular responses to thrombin and cathepsin-G can be modified during an inflammatory response.