Protease-activated Receptor-1 Signaling Research Articles

Role of kallikrein 6‐mediated PAR signaling in astrogliosis

A remarkable feature of the expression of kallikrein 6 (KLK6) in the CNS is its abundance in oligodendroglia and neurons, but relative lack of expression in astrocytes. We have shown in cases of CNS injury however, that KLK6 is induced in reactive astrocytes and exhibits prolonged expression in astroglial scar tissue in cases of spinal cord injury, multiple sclerosis and glioblastoma multiforme. In this study, we address the hypothesis that KLK6 plays a role in the promotion of astrogliosis and that this occurs by selective activation of protease activated receptors (PARs). Using primary murine astrocytes we show KLK6 promotes rapid transformation from an epithelioid to a stellate morphology and secretion of interleukin 6, each a hallmark of astrogliosis. The ability of KLK6 to promote stellation was shown to occur as early as 4 hours after a single pulse of KLK6, to be dose‐dependent, and to require activation of PKC and AKT signaling cascades. The stellation promoting activities of KLK6 were further shown to depend at least in part on activation of the thrombin receptor PAR1, since these effects were reduced in astrocytes derived from PAR1‐deficient mice and by a PAR1 specific inhibitor, SCH79797. These results point to novel roles for KLK6‐mediated PAR signaling in the spectrum of events that culminate in astrogliosis. This work was supported by NIH R01 NS052741.

Protease‐activated receptor 2 (PAR2) inactivates the pro‐apoptotic protein, BAD, via ERK1/2 and PI3K activity to decrease apoptosis in colonic epithelial cells

Colonic biopsies from inflamed colon have higher levels of epithelial apoptosis than biopsies from healthy individuals. Therapies that decrease intestinal epithelial apoptosis may decrease the inflammation observed in inflammatory bowel disease. We previously showed that signaling by PAR2, a GPCR activated by serine proteases, delayed IFN‐γ & TNF‐α‐induced apoptosis in colonic epithelial (HT‐29) cells. We aimed to determine the mechanism responsible for PAR2‐induced survival. The PAR2 agonists 2‐furoyl‐LIGRLO (2fLI), SLIGKV and trypsin all significantly reduced cleavage of caspase‐8, 9, 3 as well as PARP in response to IFNγ/TNFα. 2fLI increased phosphorylation of ERK1/2, p90RSK and Akt. 2fLI treatment stimulated the phosphorylation of pro‐apoptotic BAD at Ser112 and Ser136. Selective MAPK pathway inhibitors (U0126 & SL0101) blocked BAD Ser112 phosphorylation, whereas the PI3K‐selective inhibitor (LY294002) blocked BAD Ser136 phosphorylation. Pre‐treatment with 2fLI prior to the addition of IFNγ/TNFα increased BAD phosphorylation at both residues. PAR2 signaling reduces epithelial apoptosis in response to inflammatory cytokines via activation of pathways that inactivate the pro‐apoptotic BAD protein. Our findings may represent a mechanism whereby proteases facilitate epithelial cell survival and colonic healing after inflammation.Funding: Crohn's and Colitis Foundation of Canada

Kallikrein-related peptidase 4 (KLK4) mRNA predicts short-term relapse in colorectal adenocarcinoma patients

The members of the kallikrein-related peptidase (KLK) family are aberrantly expressed in cancer, including colorectal adenocarcinoma. KLK4 is an endogenous activator of protease-activated receptor 1 (PAR1) in HT-29 colorectal adenocarcinoma cells, inducing PAR1 signaling and subsequent ERK1/2 activation. The aim of this study was to analyze KLK4 mRNA expression in colorectal adenocarcinoma and to examine its prognostic value as a novel molecular tissue biomarker in this malignancy. Therefore, total RNA was isolated from primary tumors of 81 colorectal adenocarcinoma patients, cDNA was prepared, and KLK4 mRNA expression analysis was performed using quantitative real-time PCR. KLK4 mRNA was significantly associated with the Dukes stage, tumor invasion, size, and histological grade. Survival analysis demonstrated that KLK4 mRNA expression constitutes an unfavorable prognostic biomarker in colorectal adenocarcinoma, predicting poor disease-free survival (DFS), independently of the nodal status and tumor size. Furthermore, KLK4 mRNA predicts short-term relapse of lymph node-negative patients or those with tumors of early Dukes stage. In conclusion, KLK4 mRNA expression can be regarded as a novel potential tissue biomarker in colorectal adenocarcinoma.

Autophagy Mediates the Delivery of Thrombogenic Tissue Factor to Neutrophil Extracellular Traps in Human Sepsis

BackgroundSepsis is associated with systemic inflammatory responses and induction of coagulation system. Neutrophil extracellular traps (NETs) constitute an antimicrobial mechanism, recently implicated in thrombosis via platelet entrapment and aggregation.Methodology/Principal FindingsIn this study, we demonstrate for the first time the localization of thrombogenic tissue factor (TF) in NETs released by neutrophils derived from patients with gram-negative sepsis and normal neutrophils treated with either serum from septic patients or inflammatory mediators involved in the pathogenesis of sepsis. Localization of TF in acidified autophagosomes was observed during this process, as indicated by positive LC3B and LysoTracker staining. Moreover, phosphatidylinositol 3-kinase inhibition with 3-MA or inhibition of endosomal acidification with bafilomycin A1 hindered the release of TF-bearing NETs. TF present in NETs induced thrombin generation in culture supernatants, which further resulted in protease activated receptor-1 signaling.Conclusions/SignificanceThis study demonstrates the involvement of autophagic machinery in the extracellular delivery of TF in NETs and the subsequent activation of coagulation cascade, providing evidence for the implication of this process in coagulopathy and inflammatory response in sepsis.

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane.

Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency–Associated Developmental Defects by Preventing Matriptase Activation

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1–deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2–deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.

PR3 and elastase alter PAR1 signaling and trigger vWF release via a calcium-independent mechanism from glomerular endothelial cells.

Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH2 and SLIGKV-NH2, respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.

Protease‐activated receptor‐1 cleaved at R46 mediates cytoprotective effects

Activated protein C (aPC) mediates powerful cytoprotective effects through the protease-activated receptor-1 (PAR1) that translate into reduced harm in mouse injury models. However, it remains elusive how aPC-activated PAR1 can mediate cytoprotective effects whereas thrombin activation does the opposite. We hypothesized that aPC and thrombin might induce distinct active conformations in PAR1 causing opposing effects. We analyzed antibody binding to, and cleavage and signalling of PAR1 in either endogenously expressing endothelial or overexpressing 293T cells. In thrombin-cleaved PAR1 neither the tethered ligand nor the hirudin-like domain were available for anti-PAR1 ATAP2 and WEDE15 binding unless the tethered ligand was quenched. In contrast, aPC irreversibly prevented ATAP2 binding while not affecting WEDE15 binding. Reporter constructs with selective glutamine substitutions confirmed R41 as the only thrombin cleavage site in PAR1, whereas aPC preferentially cleaved at R46. Similarly, we report distinct cleavage sites on PAR3, K38 for thrombin and R41 for aPC. A soluble peptide corresponding to R46-cleaved PAR1 enhanced the endothelial barrier function and reduced staurosporine toxicity in endothelial as well as in 293T cells if PAR1 was expressed. Overexpression of PAR1 variants demonstrated that cleavage at R46 but not R41 is required for cytoprotective aPC signaling. We provide a novel concept on how aPC and thrombin mediate distinct effects. We propose that the enzyme-specific cleavage sites induce specific conformations which mediate divergent downstream effects. This unexpected model of PAR1 signaling might lead to novel therapeutic options for the treatment of inflammatory diseases.

Allosteric Modulation of Protease-Activated Receptor Signaling

The protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that are uniquely activated by proteolysis. PARs mediate hemostasis, thrombosis, inflammation, embryonic development and progression of certain malignant cancers. The family of PARs include four members: PAR1, PAR2, PAR3 and PAR4. PARs harbor a cryptic ligand sequence within their N-terminus that is exposed following proteolytic cleavage. The newly formed PAR Nterminus functions as a tethered ligand that binds intramolecularly to the receptor to trigger transmembrane signaling. This unique mechanism of activation would indicate that regardless of the activating protease, cleavage of PARs would unmask a tethered ligand sequence that would induce a similar active receptor conformation and signaling response. However, this is not the case. Recent studies demonstrate that PARs can be differentially activated by synthetic peptide agonists, proteases or through dimerization, that ultimately result in distinct cellular responses. In some cases, allosteric modulation of PARs involves compartmentalization in caveolae, plasma membrane microdomains enriched in cholesterol. Here, we discuss some mechanisms that lead to allosteric modulation of PAR signaling.

A Historical Perspective on Sepsis

In North America, approximately 700,000 cases of sepsis occur each year, with mortality ranging between 30% and 50%. The American Journal of Pathology has featured numerous articles on the topic, revealing mechanistic insights gleaned from both experimental rodent models and human sepsis. Nonetheless, there remains urgent need to determine the basis for sepsis-related complications and how they can be avoided, as well as how they can be most effectively treated once recognized. This historical perspective reviews what we currently understand about the mechanisms of sepsis, as well as the barriers that remain in our treatment strategies.

Activated protein C promotes endothelial barrier protection through biased PAR1 signaling mediated by β‐arrestin and dishevelled‐2 scaffolds

Thrombin, a coagulant protease contributes to inflammatory responses associated with vascular injury and thrombosis. Activated protein C (APC), an anti‐coagulant protease reduces thrombin generation and promotes anti‐inflammatory signaling in endothelial cells. Thrombin and APC signal through the G‐protein coupled protease‐activated receptor1 (PAR1) but elicit opposite effects on endothelial barrier function. The mechanism of APC‐activated PAR1 signaling in endothelial barrier protection is poorly understood. In human endothelial cells, APC activates caveolar PAR1 populations to promote barrier protective responses independent of Gαi signaling. PAR1 is precoupled to β‐arrestins and co‐segregate in caveolin‐1 enriched fractions. APC‐activated PAR1 promotes β‐arrestin‐dependent recruitment and activation of dishevelled‐2 (Dvl‐2), a key mediator of the Wnt/Frizzled signaling pathways. Depletion of β‐arrestins or Dvl‐2 resulted in the loss of APC‐induced Rac1 activation and protection against thrombin‐activated endothelial barrier permeability but not thrombin‐induced RhoA signaling. These findings indicate that PAR1 utilizes β‐arrestins to promote biased signaling and unveils a distinct pathway where β‐arrestins and Dvl‐2 scaffolds promote APC effects on endothelial barrier protection. Funded by R01 HL073328, UC TRDRP 20FT‐0074.

Ubiquitination of protease‐activated receptor‐1 reveals a new mode of signal regulation

Protease‐activated receptor‐1 (PAR1) is irreversibly proteolytically activated by thrombin. The regulatory mechanisms that control PAR1 signaling are critical for proper cellular responses. Here, we show that PAR1 is ubiquitinated after agonist stimulation. However, ubiquitin is not essential for PAR1 internalization and lysosomal degradation. This study seeks to identify the PAR1 ubiquitin modifying enzymes and the function of receptor ubiquitination. A candidate siRNA screen revealed a NEDD4 family E3 ligase to be a critical mediator of agonist‐stimulated PAR1 ubiquitination. NEDD4 formed a stable interaction with PAR1 after agonist stimulation. To determine if PAR1 signaling regulated NEDD4 recruitment we examined the role Ca2+. Treatment with the Ca2+ chelator BAPTA blocks PAR1‐ stimulated Ca2+ mobilization, NEDD4 recruitment and receptor ubiquitination. Furthermore, agonist induced PAR1 ubiquitination mediates the recruitment and activation of MAPK signaling. This study is the first example of GPCR stimulation, in this in case PAR1, regulating the activation and recruitment of NEDD4 E3 ligase to mediate it’s own modification, which is critical for activation of MAPK signaling. Supported by NIH R01 HL073328

PROTEASE ACTIVATED RECEPTOR 2 (PAR2)‐MEDIATED ACTIVATION OF PROTEIN KINASE A

Mast cell degranulation and evoked protease‐activated receptor 2 (PAR2) activity has been implicated in inflammation and chronic pain. Both protein kinase C and cAMP‐dependent protein kinase (PKA) transduce activated PAR2 signaling. However, it is unclear how PKA is activated by PAR2. In this study, we tested the hypothesis that activated PAR2 modulates the activation of PKA in a temporal manner. Acute treatment of HEK293 cells with PAR2 agonists SLIGRL−NH2 or the small molecule AC264613 resulted in a time and dose dependent inhibition of cAMP accumulation that could be reversed by administering a PAR2 antagonist FSLLRY−NH2. Furthermore, prolonged exposure of PAR2 to its agonists led to a paradoxical time‐dependent upregulation of intracellular cAMP levels. These effects are abrogated by pre‐treatment with pertussis toxin and suggest that PAR2 is coupled to the Gi protein. These results suggest a mechanism for the activation of PKA and affirm PAR2 as a significant regulator of inflammation and pain signaling.

An antagonist of human protease activated receptor‐2 attenuates PAR2 signaling, macrophage activation, mast cell degranulation, and collagen‐induced arthritis in rats

Multiple serine proteases exert proinflammatory actions by signaling through protease-activated receptor-2 (PAR2) on the cell surface. Although inhibitors of individual proteases are anti-inflammatory, we sought to discover whether the first potent antagonist of their common target PAR2 might be beneficial in treating chronic arthritis-like inflammatory disease. Using a fluorescence assay, a novel compound, GB88, was shown to antagonize PAR2-induced intracellular Ca(2+) release in human monocyte-derived macrophages, being 1000 times more potent than a control compound, ENMD-1068 (IC(50) 1.6 ± 0.5 μM vs. 1.2 ± 0.4 mM, respectively). In Wistar rats, GB88 was orally bioavailable (F=55%, T(max) 4 h, C(max) 1.7 μM, 10 mg/kg). GB88 inhibited the acute paw edema induced in Wistar rats by intraplantar λ-carrageenan or PAR2 agonists 2-furoyl-LIGRLO-NH(2) or mast cell β-tryptase, without inhibiting proteolytic activity of tryptase in vitro. In the chronic collagen-induced model of arthritis in rats, GB88 (10 mg/kg) was disease modifying and ameliorated pathological and histopathological changes (edema, pannus formation, synovial hyperplasia, collagen degradation, macrophage invasion, mast cell degranulation) compared to untreated arthritic controls. The results suggest that an orally active PAR2 antagonist is effective in treating chronic arthritis in rats through inhibiting macrophage infiltration, mast cell degranulation, and β-tryptase-PAR2 signaling in joint inflammation.

Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway, and it promotes developmental and tumour angiogenesis. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r(-/-)) but not PAR2-deficient (F2rl1(-/-)) mice, and inhibition of thrombin showed that thrombin-PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF-PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.

Prohibitins are involved in protease‐activated receptor 1‐mediated platelet aggregation

Prohibitins (PHBs), comprising the two homologous members PHB1 and PHB2, are ubiquitously expressed and highly conserved. The membrane PHBs have been reported to be involved in typhoid fever, obesity, and cancer metastasis. Proteomic studies have revealed the presence of PHBs in human platelets, but the roles of PHBs during platelet aggregation are unknown. To investigate the role of PHBs in platelet aggregation. PHB1 and PHB2 were detected on the surfaces of human platelets by flow cytometry and confocal microscopy. The PHBs were distributed in lipid rafts, as determined by sucrose density centrifugation. In addition, the PHBs were associated with protease-activated receptor 1 (PAR1), as determined by Bm-TFF2 (a PAR1 agonist)-affinity chromatography, coimmunoprecipitation, and confocal microscopy. The platelet aggregation, α(IIb) β(3) activation, granular secretion and calcium mobilization stimulated by low concentrations of thrombin (0.05 U mL(-1)) or PAR1-activating peptide (PAR1-AP) (20 μm) were reduced or abolished as a result of the blockade of PHBs by anti-PHB antibodies or their Fab fragments; however, the same results were not obtained with induction by high concentrations of thrombin (0.6 U mL(-1)) or protease-activated receptor 4-activating peptide (300 μm). The calcium mobilization in MEG-01 megakaryocytes stimulated by PAR1-AP was significantly suppressed by PHB depletion with RNA interference against PHB1 and PHB2. PHBs are localized on the human platelet membrane and are involved in PAR1-mediated platelet aggregation. Until recently, PHBs were unknown as regulators of PAR1 signaling, and they may be effective targets for antiplatelet therapy.

Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways

IntroductionThrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs).MethodsThrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity.ResultsOsteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation.ConclusionOur results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways.

The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.

Non-Canonical Cleavage of Protease Activated Receptor 1 (PAR1) by Activated Protein C Provides Novel Insights Into the Repertoire of Cytoprotective and Proinflammatory PAR1 Signaling

Abstract 534 Activated protein C (APC) exerts direct cytoprotective activities on cells that are required for APC's ability to reduce mortality in murine sepsis models and that likely have potential contributions to mortality reduction in patients with severe sepsis. Although multiple receptors have been implicated to promote APC direct cytoprotective activities on various cell types, the endothelial protein C receptor (EPCR) and PAR1 play pivotal roles in our current understanding of the molecular mechanisms for APC's direct effects on cells. According to the current generally accepted model, binding of APC to EPCR on the endothelial cell membrane facilitates activation of PAR1 and induction of signaling that ultimately results in cytoprotective effects. However, this model raises the dilemma of how thrombin and APC can often have seemingly opposing effects when activating PAR1. To gain insights into mechanisms for the contrasting effects of APC vs. thrombin signaling, PAR1 activation by APC was studied in greater detail. We hypothesized that APC might cleave PAR1 not only at Arg41 but also at Arg46 with distinctly different consequences than caused by Arg41 cleavage and that this alternative cleavage could distinguish APC's from thrombin's effects. Previously we found that, in the presence of EDTA, APC cleaved a synthetic PAR1 N-terminal peptide (TR33-66) at Arg41. Now we have found that, in buffers containing CaCl2, APC cleaved the PAR1 TR33-66 peptide at Arg41 and also at an additional site distal from Arg41. Proteolysis of the TR33-66 peptide with APC resulted in fragments corresponding to TR33-41 and TR42-66 similar to thrombin. But in contrast to thrombin, a third fragment was generated by APC and the TR42-66 fragment disappeared over time with the concomitant accumulation of a novel peptide. Furthermore, incubation of thrombin-cleaved TR33-66 with APC resulted in additional proteolysis of TR42-66 and generation of the same novel fragment, indicating the possibility of a second APC cleavage site in PAR1 that was distinct and distal from Arg41. Isolation of the novel proteolytic fragments and their MALDI-TOF analysis identified Arg46 as the second APC cleavage site in the TR33-66 peptide. When cells containing wild-type EPCR were transfected with SEAP-PAR1 wild type and mutant constructs, both thrombin and APC cleaved wt-PAR1. As anticipated, efficient cleavage by thrombin was observed for R46Q-PAR1 but not R41Q-PAR1 or R41Q/R46Q-PAR1. In contrast, APC readily cleaved both R41Q-PAR1 and R46Q-PAR1 whereas cleavage of R41Q/R46Q-PAR1 by APC was negligible. APC mediated cleavage of R41Q-PAR1 and R46Q-PAR1 required the presence of functional EPCR and was not supported by the APC-binding-defective E86A-EPCR mutant. These results indicate that on cells Arg46 in PAR1 can serve as a second cleavage site for APC. Since the new PAR1 N-terminus after proteolysis acts as a tethered ligand for receptor activation, cleavage at Arg41 vs. Arg46 could potentially create structurally distinct agonists, which might help explain the divergent patterns for PAR1-mediated cytoprotective APC signaling vs. proinflammatory IIa signaling. In studies to see if the APC-induced new PAR1 N-terminus starting at Asn47 could promote signaling, we found that a synthetic peptide with the PAR1 47–66 N-terminal sequence (NPND-peptide) increased Akt phosphorylation at Ser473 in endothelial cells over 30 min whereas neither a control scrambled sequence (47-66)-peptide nor a TRAP peptide had a similar remarkable effect on Akt phosphorylation. Moreover, the NPND-peptide, but neither the scrambled sequence-related peptide nor a TRAP peptide, inhibited staurosporine-induced endothelial cell apoptosis. Thus, it appears that the new N-terminus generated by APC's cleavage at Arg46 in PAR1 generates a novel tethered ligand that can induce cytoprotective APC-like but not thrombin-like signaling characteristics. In summary, APC is capable of a unique, functionally significant cleavage of PAR1 at Arg46 that can initiate distinctive signaling compared to canonical cleavage of PAR1 at Arg41. This implies activated PAR1 is a GPCR with multiple active conformations capable of multiple, agonist selective activity profiles which may help explain the divergent patterns for cytoprotective APC signaling vs. proinflammatory thrombin signaling. Disclosures: Mosnier: Scripps Research institute: Employment, Patents & Royalties. Griffin:ZZBiotech LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Scripps Research Institute: Employment, Patents & Royalties.

Inhibition of Thrombin Receptor Signaling on α-Smooth Muscle Actin + CD34 + Progenitors Leads to Repair After Murine Immune Vascular Injury

The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.