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.     

Anatomical localization of protease-activated receptor-1 and protease-mediated neuroglial crosstalk on peri-synaptic astrocytic endfeet

We studied the localization, activation and function of protease-activated receptor 1 (PAR-1) at the CNS synapse utilizing rat brain synaptosomes and slices. Confocal immunofluoresence and transmission electron microscopy in brain slices with pre-embedding diaminobenzidine (DAB) immunostaining found PAR-1 predominantly localized to the peri-synaptic astrocytic endfeet. Structural confocal immunofluorescence microscopy studies of isolated synaptosomes revealed spherical structures stained with anti-PAR-1 antibody which co-stained mainly for glial-filament acidic protein compared with the neuronal markers synaptophysin and PSD-95. Immunoblot studies of synaptosomes demonstrated an appropriate major band corresponding to PAR-1 and activation of the receptor by a specific agonist peptide (SFLLRN) significantly modulated phosphorylated extracellular signal-regulated kinase. A significant membrane potential depolarization was produced by thrombin (1 U/mL) and the PAR-1 agonist (100 μM) and depolarization by high K(+) elevated extracellular thrombin-like activity in the synaptosomes preparation. The results indicate PAR-1 localized to the peri-synaptic astrocytic endfeet is most likely activated by synaptic proteases and induces cellular signaling and modulation of synaptic electrophysiology. A protease mediated neuron-glia pathway may be important in both physiological and pathological regulation of the synapse.     

Injury-Related Factors and Conditions Down-Regulate the Thrombin Receptor (PAR-1) in a Human Neuronal Cell Line

Abstract: Previous studies have demonstrated that thrombin can induce potent effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR-1). Mechanisms of PAR-1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR-1 regulation in neural cells. In the present study, PAR-1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti-PAR-1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high-affinity, specific binding to a single affinity class of receptors: K_D = 3.13 and 5.25 nM, B_max = 190.1 and 67.8 fmol/mg of protein for ¹²⁵I-ATAP2 and ¹²⁵I-SPAN12, respectively. Specificity for PAR-1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR-1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin-induced PAR-1 cleavage and internalization, with protein synthesis-dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR-1 expression. Significant decreases in PAR-1 expression were induced by the protein kinase C activator phorbol 12-myristate 13-acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (32% at 3 h), and the injury-related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally with PAR-1 cell surface expression.     

Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors

5-Hydroxytryptamine 2A (5-HT(2A)) serotonin receptors are important for a variety of functions including vascular smooth muscle contraction, platelet aggregation, and the modulation of perception, cognition, and emotion. In a search for 5-HT(2A) receptor-interacting proteins, we discovered that caveolin-1 (Cav-1), a scaffolding protein enriched in caveolae, complexes with 5-HT(2A) receptors in a number of cell types including C6 glioma cells, transfected HEK-293 cells, and rat brain synaptic membrane preparations. To address the functional significance of this interaction, we performed RNA interference-mediated knockdown of Cav-1 in C6 glioma cells, a cell type that endogenously expresses both 5-HT(2A) receptors and Cav-1. We discovered that the in vitro knockdown of Cav-1 in C6 glioma cells nearly abolished 5-HT(2A) receptor-mediated signal transduction as measured by calcium flux assays. RNA interference-mediated knockdown of Cav-1 also greatly attenuated endogenous Galpha(q)-coupled P2Y purinergic receptor-mediated signaling without altering the signaling of PAR-1 thrombin receptors. Cav-1 appeared to modulate 5-HT(2A) signaling by facilitating the interaction of 5-HT(2A) receptors with Galpha(q). These studies provide compelling evidence for a prominent role of Cav-1 in regulating the functional activity of not only 5-HT(2A) serotonin receptors but also selected Galpha(q)-coupled receptors.     

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by alpha-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibalpha also has a high affinity binding site for alpha-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbalpha may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by alpha-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k(cat)/K(m) value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 +/- 0.1 x 10(7) m(-1) sec(-1). The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbalpha on platelet membranes by mocarhagin treatment reduced the k(cat)/K(m) value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k(cat)/K(m) value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbalpha, reduced the apparent k(cat)/K(m) value by about 5-fold. 4) Displacement of alpha-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparent k(cat)/K(m) value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparent k(cat)/K(m) value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a "cofactor" for PAR-1 activation by thrombin.     

Mesotrypsin, a brain trypsin, activates selectively proteinase-activated receptor-1, but not proteinase-activated receptor-2, in rat astrocytes

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.     

Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.     

Thrombin stimulates the expression of PDGF in lung epithelial cells

Several growth factors, including platelet-derived growth factor (PDGF), have been implicated in the mechanism of lung and airway remodeling. In the present study, we evaluated whether thrombin may promote lung and airway remodeling by increasing PDGF production from lung and airway epithelial cells. Conditioned medium (CM) was prepared by treating epithelial cells with increasing concentrations of thrombin; before use in the assays, CM was treated with hirudin until complete inhibition of thrombin activity. CM from epithelial cells stimulated the proliferation of lung fibroblasts and bronchial smooth muscle cells. Anti-PDGF antibody significantly inhibited this CM proliferative activity, implicating PDGF in this effect. Enzyme immunoassay and RT-PCR demonstrated that thrombin induced the secretion and expression of PDGF from bronchial and alveolar epithelial cells. RT-PCR showed that epithelial cells express the thrombin receptors protease-activated receptor (PAR)-1, PAR-3, and PAR-4. The PAR-1 agonist peptide was also found to induce PDGF secretion from epithelial cells, suggesting that the cellular effect of thrombin occurs via a PAR-1-mediated mechanism. Overall, this study showed for the first time that thrombin may play an important role in the process of lung and airway remodeling by stimulating the expression of PDGF via its cellular receptor, PAR-1.     

Studies on the receptors mediating responses of osteoblasts to thrombin

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2'-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.     

Up-regulation of proteinase-activated receptor 1 expression in astrocytes during HIV encephalitis

Proteinase-activated receptor 1 (PAR-1) is a G protein-coupled receptor that is activated by thrombin and is implicated in the pathogenesis of inflammation. Although PAR-1 is expressed on immunocompetent cells within the brain such as astrocytes, little is known about its role in the pathogenesis of inflammatory brain diseases. Herein, we investigated PAR-1 regulation of brain inflammation by stimulating human astrocytic cells with thrombin or the selective PAR-1-activating peptide. Activated cells expressed significantly increased levels of IL-1 beta, inducible NO synthase, and PAR-1 mRNA. Moreover, supernatants of these same cells were neurotoxic, which was inhibited by an N-methyl-D-aspartate receptor antagonist. Striatal implantation of the PAR-1-activating peptide significantly induced brain inflammation and neurobehavioral deficits in mice compared with mice implanted with the control peptide or saline. Since HIV-related neurological disease is predicated on brain inflammation and neuronal injury, the expression of PAR-1 in HIV encephalitis (HIVE) was investigated. Immunohistochemical analysis revealed that PAR-1 and (pro)-thrombin protein expression was low in control brains, but intense immunoreactivity was observed on astrocytes in HIVE brains. Similarly, PAR-1 and thrombin mRNA levels were significantly increased in HIVE brains compared with control and multiple sclerosis brains. These data indicated that activation and up-regulation of PAR-1 probably contribute to brain inflammation and neuronal damage during HIV-1 infection, thus providing new therapeutic targets for the treatment of HIV-related neurodegeneration.     

Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.     

Thrombin regulates the function of human blood dendritic cells

Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions.     

Thrombin regulates matrix metalloproteinase-9 expression in human monocytes

We investigated whether thrombin, the final activator of coagulation cascade, regulates expression of matrix metalloproteinases (MMP)-9 in human monocytes.We show that thrombin stimulation induced MMP-9 secretion of monocytes dose- and time-dependently as revealed by gelatin zymography. Real-time RT-PCR and Western blot analysis demonstrated that thrombin up-regulated mRNA and protein levels of MMP-9. Pre-incubation with anti-protease-activated receptor (PAR)-1 or anti-PAR-3 antibody partially inhibited the thrombin-induced MMP-9 secretion. Simultaneous incubation with both showed synergistic effect, indicating the involvement of both receptors in this thrombin effect. BAPTA, a Ca²⁺ chelator, abolished the thrombin-induced MMP-9 secretion, indicating the requirement of Ca²⁺ mobilization in this process. Inhibition of thrombin-induced MMP-9 secretion by either MEK inhibitor or p38 kinase inhibitor revealed that the thrombin effect was mediated by both ERK1/2 and p38 pathways. The activation of NFκB by thrombin as demonstrated by electromobility shift assay was also shown to be critical to the thrombin-induced MMP-9 up-regulation.     

Protein kinase Cbeta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells

We studied the effects of protein kinase C (PKC) activation onendothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca²⁺ concentration inhuman dermal microvascular endothelial cells (HMEC) exposed to-thrombin or phorbol ester,12-O-tetradecanoylphorbol-13-acetate (TPA).Immunofluorescence showed that thrombin and TPA reduced the cellsurface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1desensitization. In contrast, prior activation of PKC with TPA produceddesensitization to thrombin and histamine, indicatingheterologous PAR-1 desensitization. Treatment of cells withstaurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC isozymes(PKC_I andPKC_II) by transducing cellswith antisense cDNA of PKC_Iprevented the TPA-induced decrease in cell surface PAR-1 expression andrestored ~60% of the cytosolic Ca²⁺ signal in response tothrombin. In contrast, depletion of PKC isozymes did not affect theloss of cell surface PAR-1 and induction of homologous PAR-1desensitization by thrombin. Therefore, homologous PAR-1desensitization by thrombin occurs independently of PKC isozymes,whereas the PKC-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.

     

Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1

Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin-induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up-regulated secretion of the chemokine growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) in primary rat astrocytes in a concentration-dependent manner. However, we found no increase of interleukin (IL)-6, IL-1beta and tumour necrosis factor-alpha secretion. Protease-activated receptor 1 (PAR-1)-induced GRO/CINC-1 release was mainly mediated by c-Jun N-terminal kinase (JNK) activation. Extracellular signal-regulated kinase 1/2 might be partially involved, but not p38 mitogen-activated protein kinase. Further studies demonstrated that PAR-1 activation, as well as application of recombinant GRO/CINC-1, protected astrocytes from C(2)-ceramide-induced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB-332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR-1. These results demonstrate for the first time that PAR-1 plays an important role in anti-apoptosis in the brain by regulating the release of chemokine GRO/CINC-1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.     

Coordinate activation of human platelet protease-activated receptor-1 and -4 in response to subnanomolar alpha-thrombin

We previously demonstrated that human platelets activated with SFLLRN release PAR-1 activation peptide, PAR-1-(1-41), even in the presence of hirudin. This observation suggests that during their activation, platelets generate a protease that activates PAR-1. In this study, PAR-1 and -4 activation peptides were detected 10 s after 相似文献   

Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.     

Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4

It is commonly accepted that thrombin exerts its proinflammatory properties through the activation of proteinase-activated receptor (PAR)-1, although two other thrombin receptors have been discovered: PAR-3 and PAR-4. In this study, we have investigated the mechanisms and the receptors involved in thrombin-induced leukocyte/endothelial cell interactions by using selective agonists and antagonists of thrombin receptors in an in vivo intravital microscopy system. Topical addition of selective PAR-1 agonists to rat mesenteric venules failed to reproduce the increased leukocyte rolling and adhesion observed after thrombin topical addition. When added together with the selective PAR-1 antagonist RWJ-56110, thrombin was still able to provoke increased leukocyte rolling and adherence. The thrombin-induced leukocyte rolling and adherence was not affected by pretreatment of rats with an anti-platelet serum. Selective PAR-4-activating peptide was able to reproduce the effects of thrombin on leukocyte rolling and adhesion. Intraperitoneal injection of PAR-4-activating peptide also caused a significant increase in leukocyte migration into the peritoneal cavity. In rat tissues, PAR-4 expression was detected both on endothelium and isolated leukocytes. Taken together, these results showed that in rat mesenteric venules, thrombin exerts proinflammatory properties inducing leukocyte rolling and adherence, by a mechanism independent of PAR-1 activation or platelet activation. However, PAR-4 activation either on endothelial cells or on leukocytes might be responsible for the thrombin-induced effects. These findings suggest that PAR-4 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence and recruitment, and that in addition to PAR-1, PAR-4 could be involved in proinflammatory properties of thrombin.     

The occupancy of endothelial protein C receptor by its ligand modulates the par-1 dependent signaling specificity of coagulation proteases

Several recent studies have demonstrated that the activation of protease-activated receptor 1 (PAR-1) by thrombin and activated protein C (APC) on cultured vascular endothelial cells elicits paradoxical proinflammatory and antiinflammatory responses, respectively. Noting that the protective intracellular signaling activity of APC requires the interaction of the protease with its receptor, endothelial protein C receptor (EPCR), we recently hypothesized that the occupancy of EPCR by protein C may also change the PAR-1-dependent signaling specificity of thrombin. In support of this hypothesis, we demonstrated that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that the occupancy of EPCR by the Gla-domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through the coupling of PAR-1 to the pertussis toxin sensitive G(i) -protein. Thus, when EPCR is bound by protein C, a PAR-1-dependent protective signaling response in cultured endothelial cells can be mediated by either thrombin or APC. This article will briefly review the mechanism by which the occupancy of EPCR by its natural ligand modulates the PAR-1-dependent signaling specificity of coagulation proteases.     

Thrombin enhancement of interleukin-1 expression in mononuclear cells: involvement of proteinase-activated receptor-1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is considered a pro-inflammatory molecule. We have previously demonstrated that differentiated monocytes express the proteolytically activated receptor for thrombin (PAR-1) and that thrombin enhances the release of interleukin (IL)-6 in human monocytes. In the present study we show that thrombin upregulates the production of both IL-1alpha and IL-1beta in phytohemagglutin (PHA)-activated human peripheral blood mononuclear cells (PBMC). Treating PHA-activated PBMC with the PAR-1 activation peptide, SFLLRN, mimics the effects of thrombin on IL-1alpha and IL-1beta production. Thus, it appears that these pro-inflammatory effects induced by thrombin may be mediated through activation of PAR-1. ELISA and RNase protection assays indicate that thrombin and SFLLRN peptide upregulates IL-1 expression at both protein and mRNA levels. Thrombin directly affects monocyte IL-1 expression, since treatment of differentiated U937 cells with thrombin and SFLLRN enhances IL-1 production. These results may help explain how thrombin can enhance IL-1 expression in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to inflammatory responses observed in several pathophysiological conditions.