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G L Howells - One of the best experts on this subject based on the ideXlab platform.
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immunolocalization of protease activated Receptor 2 in skin Receptor activation stimulates interleukin 8 secretion by keratinocytes in vitro
Immunology, 1998Co-Authors: L Hou, S Kapas, A T Cruchley, M G Macey, Patrick Harriott, C Chinni, S R Stone, G L HowellsAbstract:The Protease-Activated Receptor-2 (PAR-2) is a seven transmembrane domain Receptor related to the thrombin Receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a Receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and Receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions.
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Immunolocalization of protease‐activated Receptor‐2 in skin: Receptor activation stimulates interleukin‐8 secretion by keratinocytes in vitro
Immunology, 1998Co-Authors: L Hou, S Kapas, A T Cruchley, M G Macey, Patrick Harriott, C Chinni, S R Stone, G L HowellsAbstract:The Protease-Activated Receptor-2 (PAR-2) is a seven transmembrane domain Receptor related to the thrombin Receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a Receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and Receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions.
David E. Newby - One of the best experts on this subject based on the ideXlab platform.
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Antithrombotic Effects of Combined PAR (Protease-Activated Receptor)-4 Antagonism and Factor Xa Inhibition.
Arteriosclerosis thrombosis and vascular biology, 2020Co-Authors: Mohammed N. Meah, Jennifer Raftis, Simon Wilson, Vidya Perera, Samira Garonzik, Bindu Murthy, J. Gerry Everlof, Ronald Aronson, Joseph Luettgen, David E. NewbyAbstract:Objective: PAR (Protease-Activated Receptor)-4 antagonism has antiplatelet effects under conditions of high shear stress. We aimed to establish whether PAR4 antagonism had additive antithrombotic a...
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Role of the endothelium in the vascular effects of the thrombin Receptor (Protease-Activated Receptor type 1) in humans.
Journal of the American College of Cardiology, 2008Co-Authors: Ingibjörg J. Guðmundsdóttir, Ninian N. Lang, Nicholas A. Boon, Christopher A. Ludlam, David J. Webb, Keith A.a. Fox, David E. NewbyAbstract:Role of the Endothelium in the Vascular Effects of the Thrombin Receptor (Protease-Activated Receptor Type 1) in HumansIngibjorg J. Guðmundsdottir, Ninian N. Lang, Nicholas A. Boon, Christopher A. ...
Nigel W Bunnett - One of the best experts on this subject based on the ideXlab platform.
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the transient Receptor potential vanilloid 4 trpv4 ion channel mediates protease activated Receptor 1 par1 induced vascular hyperpermeability
Laboratory Investigation, 2020Co-Authors: Scott Peng, Megan S Grace, Arisbel B Gondin, Jeffri S Retamal, Larissa Dill, William Darby, Nigel W BunnettAbstract:Endothelial barrier disruption is a hallmark of tissue injury, edema, and inflammation. Vascular endothelial cells express the G protein-coupled Receptor (GPCR) protease acctivated Receptor 1 (PAR1) and the ion channel transient Receptor potential vanilloid 4 (TRPV4), and these signaling proteins are known to respond to inflammatory conditions and promote edema through remodeling of cell–cell junctions and modulation of endothelial barriers. It has previously been established that signaling initiated by the related protease activated Receptor 2 (PAR2) is enhanced by TRPV4 in sensory neurons and that this functional interaction plays a critical role in the development of neurogenic inflammation and nociception. Here, we investigated the PAR1–TRPV4 axis, to determine if TRPV4 plays a similar role in the control of edema mediated by thrombin-induced signaling. Using Evans Blue permeation and retention as an indication of increased vascular permeability in vivo, we showed that TRPV4 contributes to PAR1-induced vascular hyperpermeability in the airways and upper gastrointestinal tract of mice. TRPV4 contributes to sustained PAR1-induced Ca2+ signaling in recombinant cell systems and to PAR1-dependent endothelial junction remodeling in vitro. This study supports the role of GPCR–TRP channel functional interactions in inflammatory-associated changes to vascular function and indicates that TRPV4 is a signaling effector for multiple PAR family members.
L Hou - One of the best experts on this subject based on the ideXlab platform.
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immunolocalization of protease activated Receptor 2 in skin Receptor activation stimulates interleukin 8 secretion by keratinocytes in vitro
Immunology, 1998Co-Authors: L Hou, S Kapas, A T Cruchley, M G Macey, Patrick Harriott, C Chinni, S R Stone, G L HowellsAbstract:The Protease-Activated Receptor-2 (PAR-2) is a seven transmembrane domain Receptor related to the thrombin Receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a Receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and Receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions.
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Immunolocalization of protease‐activated Receptor‐2 in skin: Receptor activation stimulates interleukin‐8 secretion by keratinocytes in vitro
Immunology, 1998Co-Authors: L Hou, S Kapas, A T Cruchley, M G Macey, Patrick Harriott, C Chinni, S R Stone, G L HowellsAbstract:The Protease-Activated Receptor-2 (PAR-2) is a seven transmembrane domain Receptor related to the thrombin Receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a Receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and Receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions.
Shaun R. Coughlin - One of the best experts on this subject based on the ideXlab platform.
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Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis
Gastroenterology, 2007Co-Authors: Graeme S. Cottrell, Stella Pikios, Brett R. Murphy, Paul J. Wolters, J. Adam Willardsen, Eric Camerer, Shaun R. Coughlin, Silvia Amadesi, George H Caughey, Anders PetersonAbstract:Background & Aims: We studied the role of Protease-Activated Receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. Methods: We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 Receptor antagonist SR140333. Results: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%–28% and tissue and fluid myeloperoxidase by 31%–71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. Conclusions: PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms.
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tissue factor and factor x dependent activation of protease activated Receptor 2 by factor viia
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Eric Camerer, Wei Huang, Shaun R. CoughlinAbstract:Protease-Activated Receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their Receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity.
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Antibodies to Protease-Activated Receptor 3 Inhibit Activation of Mouse Platelets by Thrombin
Blood, 1998Co-Authors: Hiroaki Ishihara, Andrew J Connolly, Dewan Zeng, Carmen Tam, Shaun R. CoughlinAbstract:Recent studies of mice deficient in the thrombin Receptor, Protease-Activated Receptor 1 (PAR1), provided definitive evidence for the existence of a second thrombin Receptor in mouse platelets. We recently identified a new thrombin Receptor designated Protease-Activated Receptor 3 (PAR3). The mRNA encoding a mouse homologue of PAR3 was highly expressed in mouse splenic megakaryocytes, making it a good candidate for the missing mouse platelet thrombin Receptor. We now report that PAR3 protein is expressed on the surface of mouse platelets and that PAR3 antibodies partially inhibit activation of mouse platelets by thrombin but not U46619, a thromboxane Receptor agonist. These observations suggest that PAR3 contributes to mouse platelet activation by thrombin.
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protease activated Receptor 3 is a second thrombin Receptor in humans
Nature, 1997Co-Authors: Hiroaki Ishihara, Yaowu Zheng, Andrew J Connolly, Dewan Zeng, Mark L Kahn, Courtney Timmons, Tracy Tram, Shaun R. CoughlinAbstract:Thrombin is a coagulation protease that activates platelets, leukocytes, endothelial and mesenchymal cells at sites of vascular injury, acting partly through an unusual proteolytically activated G-protein-coupled Receptor1–3. Knockout of the gene encoding this Receptor provided definitive evidence for a second thrombin Receptor in mouse platelets and for tissue-specific roles for different thrombin Receptors4. We now report the cloning and characterization of a new human thrombin Receptor, designated Protease-Activated Receptor 3 (PAR3). PAR3 can mediate throm-bin-triggered phosphoinositide hydrolysis and is expressed in a variety of tissues, including human bone marrow and mouse megakaryocytes, making it a candidate for the sought-after second platelet thrombin Receptor. PAR3 provides a new tool for understanding thrombin signalling and a possible target for therapeutics designed selectively to block thrombotic, inflammatory and proliferative responses to thrombin.
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Conserved Structure and Adjacent Location of the Thrombin Receptor and Protease-Activated Receptor 2 Genes Define a Protease-Activated Receptor Gene Cluster
Molecular medicine (Cambridge Mass.), 1996Co-Authors: Mark L Kahn, Andrew J Connolly, Kenji Ishii, Wen-lin Kuo, Michael Piper, Yu-ping Shi, C. C. Lin, Shaun R. CoughlinAbstract:Background Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin Receptor revealed a G protein-coupled Receptor that is activated by a novel proteolytic mechanism. Recently, a second Protease-Activated Receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin Receptor by sequence and, like the thrombin Receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin Receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of Receptor cleavage. Thus, functionally, the thrombin Receptor and PAR2 constitute a fledgling Receptor family that shares a novel proteolytic activation mechanism. To further explore the relatedness of the two known Protease-Activated Receptors and to examine the possibility that a Protease-Activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin Receptor and PAR2 genes.
