Deficiency of protease-activated receptor (PAR) 1 and PAR2 exacerbates collagen-induced arthritis in mice via differing mechanisms.

OBJECTIVES: Protease-activated receptor (PAR) 1 and PAR2 have been implicated in RA, however their exact role is unclear. Here, we detailed the mechanistic impact of these receptors on the onset and development of inflammatory arthritis in murine CIA and antigen-induced arthritis (AIA) models. METHODS: CIA or AIA was induced in PAR1 or PAR2 gene knockout (KO) and matched wild type mice. The onset and development of arthritis was monitored clinically and histologically. Immune cells, cytokines and MMPs were detected by ELISA, zymography, flow cytometry, western blot or immunohistochemistry. RESULTS: In CIA, PAR1KO and PAR2KO exacerbated arthritis, in opposition to their effects in AIA. These deficient mice had high plasma levels of IL-17, IFN-γ, TGF-ß1 and MMP-13, and lower levels of TNF-α; T cells and B cells were higher in both KO spleen and thymus, and myeloid-derived suppressor cells were lower only in PAR1KO spleen, when compared with wild type cells. Th1, Th2 and Th17 cells were lower in PAR1KO spleens cells, whereas Th1 and Th2 cells were lower and Th17 cells higher in both KO thymus cells, when compared with wild type cells. PAR1KO synovial fibroblasts proliferated faster and produced the most abundant MMP-9 amongst three type cells in the control, lipopolysaccharides or TNF stimulated conditions. CONCLUSION: This is the first study demonstrated that deficiency of PAR1 or PAR2 aggravates inflammatory arthritis in CIA. Furthermore, the protective functions of PAR1 and PAR2 in CIA likely occur via differing mechanisms involving immune cell differentiation and cytokines/MMPs.

Ischemic stroke in PAR1 KO mice: Decreased brain plasmin and thrombin activity along with decreased infarct volume.

BACKGROUND: Ischemic stroke is a common and debilitating disease with limited treatment options. Protease activated receptor 1 (PAR1) is a fundamental cell signaling mediator in the central nervous system (CNS). It can be activated by many proteases including thrombin and plasmin, with various down-stream effects, following brain ischemia. METHODS: A permanent middle cerebral artery occlusion (PMCAo) model was used in PAR1 KO and WT C57BL/6J male mice. Mice were evaluated for neurological deficits (neurological severity score, NSS), infarct volume (Tetrazolium Chloride, TTC), and for plasmin and thrombin activity in brain slices. RESULTS: Significantly low levels of plasmin and thrombin activities were found in PAR1 KO compared to WT (1.6±0.4 vs. 3.2±0.6 ng/µl, p<0.05 and 17.2±1.0 vs. 21.2±1.0 mu/ml, p<0.01, respectively) along with a decreased infarct volume (178.9±14.3, 134.4±13.3 mm3, p<0.05). CONCLUSIONS: PAR1 KO mice have smaller infarcts, with lower thrombin and plasmin activity levels. These findings may suggest that modulation of PAR1 is a potential target for future pharmacological treatment of ischemic stroke.

Blocking the Thrombin Receptor Promotes Repair of Demyelinated Lesions in the Adult Brain.

Myelin loss limits neurological recovery and myelin regeneration and is critical for restoration of function. We recently discovered that global knock-out of the thrombin receptor, also known as Protease Activated Receptor 1 (PAR1), accelerates myelin development. Here we demonstrate that knocking out PAR1 also promotes myelin regeneration. Outcomes in two unique models of myelin injury and repair, that is lysolecithin or cuprizone-mediated demyelination, showed that PAR1 knock-out in male mice improves replenishment of myelinating cells and remyelinated nerve fibers and slows early axon damage. Improvements in myelin regeneration in PAR1 knock-out mice occurred in tandem with a skewing of reactive astrocyte signatures toward a prorepair phenotype. In cell culture, the promyelinating effects of PAR1 loss of function are consistent with possible direct effects on the myelinating potential of oligodendrocyte progenitor cells (OPCs), in addition to OPC-indirect effects involving enhanced astrocyte expression of promyelinating factors, such as BDNF. These findings highlight previously unrecognized roles of PAR1 in myelin regeneration, including integrated actions across the oligodendrocyte and astroglial compartments that are at least partially mechanistically linked to the powerful BDNF-TrkB neurotrophic signaling system. Altogether, findings suggest PAR1 may be a therapeutically tractable target for demyelinating disorders of the CNS.SIGNIFICANCE STATEMENT Replacement of oligodendroglia and myelin regeneration holds tremendous potential to improve function across neurological conditions. Here we demonstrate Protease Activated Receptor 1 (PAR1) is an important regulator of the capacity for myelin regeneration across two experimental murine models of myelin injury. PAR1 is a G-protein-coupled receptor densely expressed in the CNS, however there is limited information regarding its physiological roles in health and disease. Using a combination of PAR1 knock-out mice, oligodendrocyte monocultures and oligodendrocyte-astrocyte cocultures, we demonstrate blocking PAR1 improves myelin production by a mechanism related to effects across glial compartments and linked in part to regulatory actions toward growth factors such as BDNF. These findings set the stage for development of new clinically relevant myelin regeneration strategies.

Proteinase-activated receptor 1 antagonism ameliorates experimental pulmonary hypertension.

AIMS: Pulmonary hypertension (PH) is characterized by progressive increases in pulmonary vascular resistance (PVR). Thrombotic lesions are common pathological findings. The pulmonary artery has a unique property regarding the vasoconstrictive response to thrombin, which is mediated by proteinase-activated receptor 1 (PAR1). We aim to elucidate the role of PAR1 in the development and progression of PH. METHODS AND RESULTS: A rat model of monocrotaline-induced PH and a mouse model of hypoxia (Hx)-induced PH were used to investigate the effects of atopaxar (a PAR1 antagonist) and PAR1 knockout on haemodynamic parameters, right ventricular hypertrophy (RVH), vascular remodelling and survival. In perfused lung preparations, the pressor response to PAR1 agonist was significantly augmented in monocrotaline-induced PH. Both the preventive and therapeutic administration of atopaxar significantly inhibited the increase in PVR and the development of RVH and prolonged survival. A real-time PCR revealed that the level of PAR1 mRNA in the pulmonary artery was significantly higher than that in any of the systemic arteries examined in control rats, and the level was significantly up-regulated in monocrotaline-induced PH. PAR1 gene knockout significantly attenuated the haemodynamic and histological findings in the mouse model of Hx-induced PH. CONCLUSION: The specific expression of PAR1 in the pulmonary artery and its up-regulation were suggested to play a critical role in the development and progression of experimental PH in murine models. PAR1 is a potential therapeutic target for the treatment of PH.

PAR2 (Protease-Activated Receptor 2) Deficiency Attenuates Atherosclerosis in Mice.

OBJECTIVE: PAR2 (protease-activated receptor 2)-dependent signaling results in augmented inflammation and has been implicated in the pathogenesis of several autoimmune conditions. The objective of this study was to determine the effect of PAR2 deficiency on the development of atherosclerosis. APPROACH AND RESULTS: PAR2 mRNA and protein expression is increased in human carotid artery and mouse aortic arch atheroma versus control carotid and aortic arch arteries, respectively. To determine the effect of PAR2 deficiency on atherosclerosis, male and female low-density lipoprotein receptor-deficient (Ldlr-/-) mice (8-12 weeks old) that were Par2+/+ or Par2-/- were fed a fat- and cholesterol-enriched diet for 12 or 24 weeks. PAR2 deficiency attenuated atherosclerosis in the aortic sinus and aortic root after 12 and 24 weeks. PAR2 deficiency did not alter total plasma cholesterol concentrations or lipoprotein distributions. Bone marrow transplantation showed that PAR2 on nonhematopoietic cells contributed to atherosclerosis. PAR2 deficiency significantly attenuated levels of the chemokines Ccl2 and Cxcl1 in the circulation and macrophage content in atherosclerotic lesions. Mechanistic studies using isolated primary vascular smooth muscle cells showed that PAR2 deficiency is associated with reduced Ccl2 and Cxcl1 mRNA expression and protein release into the supernatant resulting in less monocyte migration. CONCLUSIONS: Our results indicate that PAR2 deficiency is associated with attenuation of atherosclerosis and may reduce lesion progression by blunting Ccl2- and Cxcl1-induced monocyte infiltration.

Protease-activated receptor-1 deficiency protects against streptozotocin-induced diabetic nephropathy in mice.

Endogenously administered activated protein C ameliorates diabetic nephropathy (DN) in a protease-activated receptor-1 (PAR-1)-dependent manner, suggesting that PAR-1 activation limits the progression of DN. Activation of PAR-1 in fibroblast-like cells, however, induces proliferation and extracellular matrix production, thereby driving fibrotic disease. Considering the key role of mesangial proliferation and extracellular matrix production during DN, PAR-1 may in fact potentiate diabetes-induced kidney injury. To determine the net effect of PAR-1 in DN, streptozotocin-induced DN was studied in wild type and PAR-1 deficient mice. Subsequent mechanistic insight was obtained by assessing profibrotic responses of mesangial and tubular epithelial cells in vitro, following PAR-1 stimulation and inhibition. Despite having similar glucose levels, PAR-1 deficient mice developed less kidney damage after induction of diabetes, as evidenced by diminished proteinuria, plasma cystatin C levels, expansion of the mesangial area, and tubular atrophy. In vitro, PAR-1 signaling in mesangial cells led to increased proliferation and expression of matrix proteins fibronectin and collagen IV. Conversely, a reduction in both proliferation and fibronectin deposition was observed in diabetic PAR-1 deficient mice. Overall, we show that PAR-1 plays an important role in the development of DN and PAR-1 might therefore be an attractive therapeutic target to pursue in DN.

From the Cover: Coagulation-Driven Hepatic Fibrosis Requires Protease Activated Receptor-1 (PAR-1) in a Mouse Model of TCDD-Elicited Steatohepatitis.

Emerging evidence supports a role for environmental chemical exposure in the pathology of non-alcoholic fatty liver disease (NAFLD), a disease process tightly linked to increased activity of the blood coagulation cascade. Exposure of C57BL/6 mice to the persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) recapitulates features of the NAFLD spectrum, including steatosis, hepatic injury, inflammation, and fibrosis. We assessed coagulation cascade activation, and determined the role of the thrombin receptor protease activated receptor-1 (PAR-1) in experimental TCDD-elicited NAFLD. Chronic exposure to TCDD (30 µg/kg every 4 days for 28 days) was associated with intrahepatic coagulation, indicated by increased plasma thrombin-antithrombin levels and hepatic fibrin(ogen) deposition. PAR-1 deficiency diminished TCDD-elicited body weight loss and relative liver weight was reduced in TCDD-exposed PAR-1-/- mice compared with TCDD-exposed wild-type mice. PAR-1 deficiency did not affect TCDD-induced hepatic steatosis or hepatocellular injury, as indicated by serum alanine aminotransferase activity. Despite a lack of effect on these 2 features of NAFLD pathology, PAR-1 deficiency was associated with a reduction in hepatic inflammation evident in liver histopathology, and reflected by a reduction in serum levels of the proinflammatory cytokine interleukin-6. Moreover, TCDD-driven hepatic collagen deposition was markedly reduced in PAR-1-deficient mice. These results indicate that experimental TCDD-elicited steatohepatitis is associated with coagulation cascade activation and PAR-1-driven hepatic inflammation and fibrosis.

Microglia Activation and Polarization After Intracerebral Hemorrhage in Mice: the Role of Protease-Activated Receptor-1.

Polarized microglia play a dual (beneficial/detrimental) role in neurological diseases. However, the status and the factors that modulate microglia polarization in intracerebral hemorrhage (ICH) remain unclear. In the present study, we investigated the role of protease-activated receptor-1 (PAR-1, a thrombin receptor) in ICH-induced microglia polarization in mice. Male wild-type (WT) and PAR-1 knockout (PAR-1 KO) mice received an infusion of 30-µL autologous blood or saline into the right basal ganglia. Mice were euthanized at different time points and the brains were used for Western blotting and immunohistochemistry. Some mice had magnetic resonance imaging. We found that ICH induced microglia activation and polarization. M1 phenotypic markers were markedly increased and reached a peak as early as 4 h, remained high at 3 days and decreased 7 days after ICH. M2 phenotypic markers were upregulated later than M1 markers reaching a peak at day 1 and declining by day 7 after ICH. PAR-1 was upregulated after ICH and expressed in the neurons and microglia. ICH induced less brain swelling and neuronal death in PAR-1 KO mice, and this was associated with less M1 polarization and reduced proinflammatory cytokine levels in the brain. In conclusion, these results suggest that polarized microglia occur dynamically after ICH and that PAR-1 plays a role in the microglia activation and polarization.

Colon Cancer Growth and Dissemination Relies upon Thrombin, Stromal PAR-1, and Fibrinogen.

Thrombin-mediated proteolysis is a major determinant of metastasis, but is not universally important for primary tumor growth. Here, we report that colorectal adenocarcinoma represents one important exception whereby thrombin-mediated functions support both primary tumor growth and metastasis. In contrast with studies of multiple nongastrointestinal cancers, we found that the growth of primary tumors formed by murine and human colon cancer cells was reduced in mice by genetic or pharmacologic reduction of circulating prothrombin. Reduced prothrombin expression was associated with lower mitotic indices and invasion of surrounding tissue. Mechanistic investigations revealed that thrombin-driven colonic adenocarcinoma growth relied upon at least two targets of thrombin-mediated proteolysis, protease-activated receptor-1 (PAR-1) expressed by stromal cells and the extracellular matrix protein, fibrinogen. Colonic adenocarcinoma growth was reduced in PAR-1-deficient mice, implicating stromal cell-associated PAR-1 as one thrombin target important for tumor outgrowth. Furthermore, tumor growth was dramatically impeded in fibrinogen-deficient mice, offering the first direct evidence of a critical functional role for fibrinogen in malignant tumor growth. Tumors harvested from fibrinogen-deficient mice displayed a relative reduction in cell proliferative indices, as well as increased tumor necrosis and decreased tumor vascular density. Collectively, our findings established a functional role for thrombin and its targets PAR-1 and fibrinogen in the pathogenesis of colonic adenocarcinoma, supporting tumor growth as well as local invasion and metastasis.

Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma.

BACKGROUND: Prior work showed that whole blood, plasma, and serum injections are damaging to the neonatal rodent brain in a model of intracerebral/periventricular hemorrhage. Thrombin alone is also damaging. In adult animal models of hemorrhagic stroke, the protease-activated (thrombin) receptor PAR1 mediates some of the brain damage. We hypothesized that PAR1 interference will reduce the adverse effects of blood products on immature rodent brain and cells. RESULTS: Cultured oligodendrocyte precursor cells from rats and mice were exposed to blood plasma with and without the PAR1 antagonists SCH-79797 or BMS-200261. In concentrations previously shown to have activity on brain cells, neither drug showed evidence of protection against the toxicity of blood plasma. Newborn mice (wild type, heterozygous, and PAR1 knockout) were subjected to intracerebral injection of autologous whole blood into the periventricular region of the frontal lobe. Cell proliferation, measured by Ki67 immunoreactivity in the subventricular zone, was suppressed at 1 and 2 days, and was not normalized in the knockout mice. Cell apoptosis, measured by activated caspase 3 immunoreactivity, was not apparent in the subventricular zone. Increased apoptosis in periventricular striatal cells was not normalized in the knockout mice. CONCLUSION: Interference with the thrombin-PAR1 system does not reduce the adverse effects of blood on germinal cells of the immature rodent brain. PAR1 interference is unlikely to be a useful treatment for reducing the brain damage that accompanies periventricular (germinal matrix) hemorrhage, a common complication of premature birth.

Contractile properties of slow and fast skeletal muscles from protease activated receptor-1 null mice.

INTRODUCTION: Protease-activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow-twitch soleus muscles and fast-twitch extensor digitorum longus (EDL) muscles from PAR-1 null and littermate control mice. METHODS: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. RESULTS: Soleus muscles from PAR-1 null mice exhibited longer contraction times, a leftward shift in the force-stimulation frequency relationship, and decreased fatiguability compared with controls. PAR-1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR-1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. CONCLUSIONS: The absence of PAR-1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991-998, 2014.

Proteinase activated receptor 1 mediated fibrosis in a mouse model of liver injury: a role for bone marrow derived macrophages.

Liver fibrosis results from the co-ordinated actions of myofibroblasts and macrophages, a proportion of which are of bone marrow origin. The functional effect of such bone marrow-derived cells on liver fibrosis is unclear. We examine whether changing bone marrow genotype can down-regulate the liver's fibrotic response to injury and investigate mechanisms involved. Proteinase activated receptor 1 (PAR1) is up-regulated in fibrotic liver disease in humans, and deficiency of PAR1 is associated with reduced liver fibrosis in rodent models. In this study, recipient mice received bone marrow transplantation from PAR1-deficient or wild-type donors prior to carbon tetrachloride-induced liver fibrosis. Bone marrow transplantation alone from PAR1-deficient mice was able to confer significant reductions in hepatic collagen content and activated myofibroblast expansion on wild-type recipients. This effect was associated with a decrease in hepatic scar-associated macrophages and a reduction in macrophage recruitment from the bone marrow. In vitro, PAR1 signalling on bone marrow-derived macrophages directly induced their chemotaxis but did not stimulate proliferation. These data suggest that the bone marrow can modulate the fibrotic response of the liver to recurrent injury. PAR1 signalling can contribute to this response by mechanisms that include the regulation of macrophage recruitment.

Deficiency of protease-activated receptor-1 limits bacterial dissemination during severe Gram-negative sepsis (melioidosis).

Protease-activated receptor-1 (PAR-1) is a G-coupled transmembrane receptor expressed by multiple cell types present in the lungs that can be activated by various proteases generated during acute inflammation. In this study we aimed to investigate the role of PAR-1 during melioidosis, a common cause of (pneumo)sepsis in Southeast Asia in a murine model of intranasal inoculation of the causative pathogen Burkholderia (B.) pseudomallei. Our results show that endogenous PAR-1 facilitates bacterial growth and dissemination during murine melioidosis, which is associated with increased cell influxes. However, these observations have no impact on survival.

Critical role for PAR1 in kallikrein 6-mediated oligodendrogliopathy.

Kallikrein 6 (KLK6) is a secreted serine protease preferentially expressed by oligodendroglia in CNS white matter. Elevated levels of KLK6 occur in actively demyelinating multiple sclerosis (MS) lesions and in cases of spinal cord injury (SCI), stroke, and glioblastoma. Taken with recent evidence establishing KLK6 as a CNS-endogenous activator of protease-activated receptors (PARs), we hypothesized that KLK6 activates a subset of PARs to regulate oligodendrocyte physiology and potentially pathophysiology. Here, primary oligodendrocyte cultures derived from wild type or PAR1-deficient mice and the murine oligodendrocyte cell line, Oli-neu, were used to demonstrate that Klk6 (rodent form) mediates loss of oligodendrocyte processes and impedes morphological differentiation of oligodendrocyte progenitor cells (OPCs) in a PAR1-dependent fashion. Comparable gliopathy was also elicited by the canonical PAR1 agonist, thrombin, as well as PAR1-activating peptides (PAR1-APs). Klk6 also exacerbated ATP-mediated oligodendrogliopathy in vitro, pointing to a potential role in augmenting excitotoxicity. In addition, Klk6 suppressed the expression of proteolipid protein (PLP) RNA in cultured oligodendrocytes by a mechanism involving PAR1-mediated Erk1/2 signaling. Microinjection of PAR1 agonists, including Klk6 or PAR1-APs, into the dorsal column white matter of PAR1(+/+) but not PAR1(-/-) mice promoted vacuolating myelopathy and a loss of immunoreactivity for myelin basic protein (MBP) and CC-1(+) oligodendrocytes. These results demonstrate a functional role for Klk6-PAR1 signaling in oligodendroglial pathophysiology and suggest that antagonists of PAR1 or its protease agonists may represent new modalities to moderate demyelination and to promote myelin regeneration in cases of CNS white matter injury or disease.

Tumor MMP-1 activates endothelial PAR1 to facilitate vascular intravasation and metastatic dissemination.

Intravasation, the active entry of primary tumor cells into the vasculature, remains the least studied step in the metastatic cascade. Protease-mediated escape and stromal invasion of tumor cells represent widely accepted processes leading up to the intravasation step. However, molecular factors that contribute directly to tumor cell vascular penetration have not been identified. In this study, the in vivo role of the collagenolytic protease, MMP-1, in cancer cell intravasation and metastasis was analyzed by using a highly disseminating variant of human HEp3 epidermoid carcinoma, HEp3-hi/diss. Although naturally acquired or experimentally induced MMP-1 deficiency substantially suppressed HEp3-hi/diss intravasation, supplementation of recombinant MMP-1 to MMP-1-silenced primary tumors restored their impaired vascular dissemination. Surprisingly, abrogation of MMP-1 production and activity did not significantly affect HEp3-hi/diss migration or matrix invasion, suggesting noncollagenolytic mechanisms underlying MMP-1-dependent cell intravasation. In support of such noncollagenolytic mechanisms, MMP-1 silencing in HEp3-hi/diss cells modulated the microarchitecture and integrity of the angiogenic vasculature in a novel microtumor model. Concomitantly, MMP-1 deficiency led to decreased levels of intratumoral vascular permeability, tumor cell intravasation, and metastatic dissemination. Taking advantage of PAR1 deficiency of HEp3-hi/diss cells, we further show that endothelial PAR1 is a putative nontumor-cell/nonmatrix target, activation of which by carcinoma-produced MMP-1 regulates endothelial permeability and transendothelial migration. The inhibitory effects of specific PAR1 antagonists in live animals have also indicated that the mechanisms of MMP-1-dependent vascular permeability in tumors involve endothelial PAR1 activation. Together, our findings mechanistically underscore the contribution of a tumor MMP-1/endothelial PAR1 axis to actual intravasation events manifested by aggressive carcinoma cells.

Protease-activated receptor (PAR)2, but not PAR1, is involved in collateral formation and anti-inflammatory monocyte polarization in a mouse hind limb ischemia model.

AIMS: In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model. METHODS AND RESULTS: PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered. CONCLUSION: PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patients.

Regulation of neuronal plasticity and fear by a dynamic change in PAR1-G protein coupling in the amygdala.

Fear memories are acquired through neuronal plasticity, an orchestrated sequence of events regulated at circuit and cellular levels. The conventional model of fear acquisition assumes unimodal (for example, excitatory or inhibitory) roles of modulatory receptors in controlling neuronal activity and learning. Contrary to this view, we show that protease-activated receptor-1 (PAR1) promotes contrasting neuronal responses depending on the emotional status of an animal by a dynamic shift between distinct G protein-coupling partners. In the basolateral amygdala of fear-naive mice PAR1 couples to Gαq/11 and Gαo proteins, while after fear conditioning coupling to Gαo increases. Concurrently, stimulation of PAR1 before conditioning enhanced, but afterwards it inhibited firing of basal amygdala neurons. An initial impairment of the long-term potentiation (LTP) in PAR1-deficient mice was transformed into an increase in LTP and enhancement of fear after conditioning. These effects correlated with more frequent 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor-mediated miniature post synaptic events and increased neuronal excitability. Our findings point to experience-specific shifts in PAR1-G protein coupling in the amygdala as a novel mechanism regulating neuronal excitability and fear.

Protease-activated receptor-1 modulates hippocampal memory formation and synaptic plasticity.

Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity.

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.