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Liu S, Shen Y, Chen J, Ruan Z, Hua L, Wang K, Xi X, Mao J. The critical role of platelets in venous thromboembolism: Pathogenesis, clinical status, and emerging therapeutic strategies. Blood Rev 2025:101302. [PMID: 40382294 DOI: 10.1016/j.blre.2025.101302] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2025] [Revised: 05/08/2025] [Accepted: 05/11/2025] [Indexed: 05/20/2025]
Abstract
Venous thromboembolism (VTE), encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE), is a complex vascular disorder with high morbidity and mortality, driven by Virchow's Triad: blood stasis, hypercoagulability, and endothelial injury. VTE is now recognized as an inflammatory process involving multiple components. Platelets are involved in the process of VTE, contributing to thrombosis initiation, progression, resolution and recurrence through coagulation activation, and interactions with immune and endothelial cells. Anticoagulation remains the cornerstone of VTE treatment; however, antiplatelet agents like aspirin have demonstrated therapeutic potential, particularly following major orthopedic surgeries. Furthermore, emerging platelet-targeted therapies and biomarkers offer new opportunities for improving VTE diagnosis and treatment. This review explores the evolving role of platelets in VTE pathophysiology, assesses current antiplatelet strategies, and highlights novel therapeutic approaches. Advancing platelet research in VTE may lead to safer, more effective interventions, optimizing outcomes for patients with this life-threatening condition.
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Affiliation(s)
- Shuang Liu
- Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Yan Shen
- Research Center for Experimental Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Jiayi Chen
- Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Zheng Ruan
- Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Li Hua
- School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Kankan Wang
- Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
| | - Xiaodong Xi
- Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
| | - Jianhua Mao
- Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Collaborative Innovation Center of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
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Knauss EA, Guci J, Luc N, Disharoon D, Huang GH, Gupta AS, Nieman MT. Mice with reduced protease-activated receptor 4 reactivity show decreased venous thrombosis and platelet procoagulant activity. J Thromb Haemost 2025; 23:1278-1288. [PMID: 39798922 PMCID: PMC11992619 DOI: 10.1016/j.jtha.2024.12.031] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2024] [Revised: 12/26/2024] [Accepted: 12/27/2024] [Indexed: 01/15/2025]
Abstract
BACKGROUND Hypercoagulation and thrombin generation are major risk factors for venous thrombosis. Sustained thrombin signaling through protease-activated receptor (PAR) 4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3, which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a genome wide association studies meta-analysis. OBJECTIVES The goal of this study was to determine the mechanism for the association of rs2227376 with a reduced risk of VTE using mice with a homologous mutation (PAR4-P322L). METHODS Venous thrombosis was examined using our recently generated PAR4-P322L mice in the inferior vena cava stasis and stenosis models. Coagulation and clot stability were measured using rotational thromboelastometry. Thrombin-generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry. RESULTS Mice heterozygous (PAR4P/L) or homozygous (PAR4L/L) at position 310 had reduced sizes of venous clots at 48 hours. PAR4P/L and PAR4L/L platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, in addition to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation. CONCLUSION The leucine allele in extracellular loop 3, PAR4-322L, leads to fewer procoagulant platelets, decreased endogenous thrombin potential, and reduced platelet-neutrophil aggregation. This decreased ability to generate thrombin and bind to neutrophils offers a mechanism for PAR4's role in VTE, highlighting a key role for PAR4 signaling.
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Affiliation(s)
- Elizabeth A Knauss
- Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA
| | - Johana Guci
- Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA
| | - Norman Luc
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA
| | - Dante Disharoon
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA
| | - Grace H Huang
- Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA
| | - Anirban Sen Gupta
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA
| | - Marvin T Nieman
- Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
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Rajala R, Griffin CT. Endothelial protease-activated receptor 4: impotent or important? Front Cardiovasc Med 2025; 12:1541879. [PMID: 39935714 PMCID: PMC11810968 DOI: 10.3389/fcvm.2025.1541879] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2024] [Accepted: 01/09/2025] [Indexed: 02/13/2025] Open
Abstract
The protease thrombin, which increases its levels with various pathologies, can signal through the G protein-coupled receptors protease-activated receptors 1 and 4 (PAR1/PAR4). PAR1 is a high-affinity receptor for thrombin, whereas PAR4 is a low-affinity receptor. Finding functions for PAR4 in endothelial cells (ECs) has been an elusive goal over the last two decades. Several studies have demonstrated a lack of functionality for PAR4 in ECs, with many claiming that PAR4 function is confined mostly to platelets. A recent study from our lab identified low expressing but functional PAR4 in hepatic ECs in vivo. We also found that PAR4 likely has a higher signaling potency than PAR1. Given this potency, ECs seem to limit PAR4 signaling except for extreme cases. As a result, we claim PAR4 is not an impotent receptor because it is low expressing, but rather PAR4 is low expressing because it is a very potent receptor. Since we have finally shown PAR4 to be present and functional on ECs in vivo, it is important to outline why such controversy arose over the last two decades and, more importantly, why the receptor was undervalued on ECs. This timely review aims to inspire investigators in the field of vascular biology to study the regulatory aspect of endothelial PAR4 and its relationship with the more highly expressed PAR1.
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Affiliation(s)
- Rahul Rajala
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, United States
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
- Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
| | - Courtney T. Griffin
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, United States
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
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4
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Li PF, Lu X, Zhou YQ, Wang K, Yang P, Chen XH, Xu F. Predictive Value of Systemic Immune Inflammation Index, Aggregate Index of Systemic Inflammation, and Systemic Inflammation Response Index in Lower Extremity Deep Venous Thrombosis Following Severe Trauma. CHINESE MEDICAL SCIENCES JOURNAL = CHUNG-KUO I HSUEH K'O HSUEH TSA CHIH 2024; 39:241-248. [PMID: 39789927 DOI: 10.24920/004411] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/12/2025]
Abstract
OBJECTIVES Venous thromboembolism is a highly prevalent condition after polytrauma, and recognized as an important factor contributing to poor prognosis. The aim of this study was to investigate the risk factors for lower extremity deep venous thrombosis (LEDVT) in a severely traumatized population and to evaluate their predictive value for LEDVT. METHODS This was a retrospective, single-center observational study. All subjects were severely traumatized patients who were admitted to the Traumatic Intensive Care Unit from January 2021 to May 2024. Based on Doppler ultrasound findings of both lower extremities from the time of injury to 30 days post-injury, patients who developed LEDVT were enrolled in the LEDVT group, and those who did not develop LEDVT were enrolled in the NLEDVT group. Demographic, clinical, and laboratory data were collected upon admission. Multivariable logistic regression analysis was performed to identify risk factors for LEDVT. Receiver operating characteristic (ROC) curve was used to evaluate the overall fit of the final model. RESULTS There were 56 patients enrolled in the LEDVT group and 81 patients in the NLEDVT group.Age, Aggregate Index of Systemic Inflammation (AISI), Systemic Inflammation Response Index (SIRI), ICU length of stay, and albumin were identified as independent risk factors for LEDVT (all P < 0.05). The area under their ROC curves were 0.604, 0.657, 0.694, 0.668, and 0.405, respectively. Combined model for early clinical prediction of LEDVT in severely traumatized patients by age, SIRI, AISI, and albumin resulted in an area under the ROC curve of 0.805 (95%CI: 0.73-0.88, SE = 0.037). CONCLUSIONS The combination of age, SIRI, AISI, and albumin has a predictive value for LEDVT in severely traumatized patients.
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Affiliation(s)
- Peng-Fei Li
- Department of Emergency Medicine, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu, China
| | - Xin Lu
- Department of Emergency Medicine, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu, China
| | - Yu-Qian Zhou
- Department of Emergency Medicine, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu, China
| | - Ke Wang
- Department of Emergency Medicine, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu, China
| | - Peng Yang
- Department of Emergency Medicine, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu, China
| | - Xiong-Hui Chen
- Department of Emergency Medicine, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu, China
| | - Feng Xu
- Department of Emergency Medicine, The First Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu, China.
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Knauss EA, Guci J, Luc N, Disharoon D, Huang GH, Gupta AS, Nieman MT. Mice with Reduced PAR4 Reactivity show Decreased Venous Thrombosis and Platelet Procoagulant Activity. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.10.14.617127. [PMID: 39463946 PMCID: PMC11507748 DOI: 10.1101/2024.10.14.617127] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 10/29/2024]
Abstract
Background Hypercoagulation and thrombin generation are major risk factors for venous thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single-nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3 (P310L), which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis. Objective The goal of this study is to determine the mechanism for the association of rs2227376 with reduced risk for VTE in using mice with a homologous mutation (PAR4-P322L). Methods Venous thrombosis was examined using our recently generated PAR4-P322L mice in the inferior vena cava stasis and stenosis models. Coagulation and clot stability was measured using rotational thromboelastometry (ROTEM). Thrombin generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry. Results PAR4P/L and PAR4L/L had reduced incidence and size of venous clots at 48 hours. PAR4P/L and PAR4L/L platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, which led to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation. Conclusions The leucine allele in extracellular loop 3, PAR4-322L leads to fewer procoagulant platelets and decreased endogenous thrombin potential. This decreased ability to generate thrombin offers a mechanism for PAR4's role in VTE highlighting a key role for PAR4 signaling.
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Affiliation(s)
- Elizabeth A. Knauss
- Case Western Reserve University, School of Medicine, Department of Pharmacology, Cleveland, OH United States
| | - Johana Guci
- Case Western Reserve University, School of Medicine, Department of Pharmacology, Cleveland, OH United States
| | - Norman Luc
- Case Western Reserve University, Department of Biomedical Engineering, Cleveland, OH United States
| | - Dante Disharoon
- Case Western Reserve University, Department of Biomedical Engineering, Cleveland, OH United States
| | - Grace H. Huang
- Case Western Reserve University, School of Medicine, Department of Pharmacology, Cleveland, OH United States
| | - Anirban Sen Gupta
- Case Western Reserve University, Department of Biomedical Engineering, Cleveland, OH United States
| | - Marvin T. Nieman
- Case Western Reserve University, School of Medicine, Department of Pharmacology, Cleveland, OH United States
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Andrianova I, Kowalczyk M, Denorme F. Protease activated receptor-4: ready to be part of the antithrombosis spectrum. Curr Opin Hematol 2024; 31:238-244. [PMID: 38814792 DOI: 10.1097/moh.0000000000000828] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/01/2024]
Abstract
PURPOSE OF REVIEW Cardiovascular disease is a major cause of death worldwide. Platelets play a key role in this pathological process. The serine protease thrombin is a critical regulator of platelet reactivity through protease activated receptors-1 (PAR1) and PAR4. Since targeting PAR4 comes with a low chance for bleeding, strategies blocking PAR4 function have great antithrombotic potential. Here, we reviewed the literature on platelet PAR4 with a particular focus on its role in thromboinflammation. RECENT FINDINGS Functional PAR4 variants are associated with reduced venous thrombosis risk (rs2227376) and increased risk for ischemic stroke (rs773902). Recent advances have allowed for the creation of humanized mouse lines in which human PAR4 is express instead of murine PAR4. This has led to a better understanding of the discrepancies between human and murine PAR4. It also made it possible to introduce single nucleotide polymorphisms (SNPs) in mice allowing to directly test the in vivo functional effects of a specific SNP and to develop in vivo models to study mechanistic and pharmacologic alterations induced by a SNP. SUMMARY PAR4 plays an important role in cardiovascular diseases including stroke, myocardial infarction and atherosclerosis. Targeting PAR4 hold great potential as a safe antithrombotic strategy.
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Affiliation(s)
- Izabella Andrianova
- Department of Emergency Medicine, Washington University School of Medicine, St. Louis, Missouri
| | - Mia Kowalczyk
- Molecular Medicine Program, University of Utah, Salt Lake City, Utah, USA
| | - Frederik Denorme
- Department of Emergency Medicine, Washington University School of Medicine, St. Louis, Missouri
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7
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Han X, Knauss EA, Fuente MDL, Li W, Conlon RA, LePage DF, Jiang W, Renna SA, McKenzie SE, Nieman MT. A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity. J Thromb Haemost 2024; 22:1715-1726. [PMID: 38508397 PMCID: PMC12036797 DOI: 10.1016/j.jtha.2024.03.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Revised: 02/26/2024] [Accepted: 03/11/2024] [Indexed: 03/22/2024]
Abstract
BACKGROUND Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. OBJECTIVES The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). METHODS A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbβ3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. RESULTS PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbβ3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. CONCLUSION PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.
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Affiliation(s)
- Xu Han
- Case Western Reserve University School of Medicine, Department of Pharmacology, Cleveland, Ohio, USA
| | - Elizabeth A Knauss
- Case Western Reserve University School of Medicine, Department of Pharmacology, Cleveland, Ohio, USA
| | - Maria de la Fuente
- Case Western Reserve University School of Medicine, Department of Pharmacology, Cleveland, Ohio, USA
| | - Wei Li
- Department of Biomedical Sciences, Joan C. Edwards School of Medicine at Marshall University, Huntington, West Virginia, USA
| | - Ronald A Conlon
- Case Transgenic and Targeting Facility, Case Western Reserve University, Cleveland, Ohio, USA
| | - David F LePage
- Case Transgenic and Targeting Facility, Case Western Reserve University, Cleveland, Ohio, USA
| | - Weihong Jiang
- Case Transgenic and Targeting Facility, Case Western Reserve University, Cleveland, Ohio, USA
| | - Stephanie A Renna
- Department of Medicine, The Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Steven E McKenzie
- Department of Medicine, The Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Marvin T Nieman
- Case Western Reserve University School of Medicine, Department of Pharmacology, Cleveland, Ohio, USA.
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Smith S, Cassada JB, Von Bredow L, Erreger K, Webb EM, Trombley TA, Kalbfleisch JJ, Bender BJ, Zagol-Ikapitte I, Kramlinger VM, Bouchard JL, Mitchell SG, Tretbar M, Shoichet BK, Lindsley CW, Meiler J, Hamm HE. Discovery of Protease-Activated Receptor 4 (PAR4)-Tethered Ligand Antagonists Using Ultralarge Virtual Screening. ACS Pharmacol Transl Sci 2024; 7:1086-1100. [PMID: 38633591 PMCID: PMC11020070 DOI: 10.1021/acsptsci.3c00378] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 02/29/2024] [Accepted: 03/04/2024] [Indexed: 04/19/2024]
Abstract
Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound. From the single-hit compound, we developed a novel series of PAR4 antagonists. Subsequent lead optimization via simultaneous virtual library searches and structure-based rational design efforts led to potent antagonists of thrombin-induced activation. Interestingly, this series of antagonists was active against PAR4 activation by the native protease thrombin cleavage but not the synthetic PAR4 agonist peptide AYPGKF.
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Affiliation(s)
- Shannon
T. Smith
- Department
of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
| | - Jackson B. Cassada
- Department
of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
| | - Lukas Von Bredow
- Warren
Center for Neuroscience Drug Discovery, Nashville, Tennessee 37067, United States
- Institute
for Drug Discovery, Leipzig University Medical
School, Leipzig 04109, Germany
| | - Kevin Erreger
- Department
of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
| | - Emma M. Webb
- Department
of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
| | - Trevor A. Trombley
- Warren
Center for Neuroscience Drug Discovery, Nashville, Tennessee 37067, United States
| | - Jacob J. Kalbfleisch
- Department
of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
- Warren
Center for Neuroscience Drug Discovery, Nashville, Tennessee 37067, United States
| | - Brian J. Bender
- Department
of Pharmaceutical Chemistry, University
of California San Francisco, San Francisco, California 94158, United States
| | - Irene Zagol-Ikapitte
- Warren
Center for Neuroscience Drug Discovery, Nashville, Tennessee 37067, United States
| | - Valerie M. Kramlinger
- Department
of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
- Warren
Center for Neuroscience Drug Discovery, Nashville, Tennessee 37067, United States
| | - Jacob L. Bouchard
- Warren
Center for Neuroscience Drug Discovery, Nashville, Tennessee 37067, United States
| | - Sidnee G. Mitchell
- Department
of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
| | - Maik Tretbar
- Institute
for Drug Discovery, Leipzig University Medical
School, Leipzig 04109, Germany
| | - Brian K. Shoichet
- Department
of Pharmaceutical Chemistry, University
of California San Francisco, San Francisco, California 94158, United States
| | - Craig W. Lindsley
- Department
of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
- Department
of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
- Warren
Center for Neuroscience Drug Discovery, Nashville, Tennessee 37067, United States
| | - Jens Meiler
- Department
of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
- Department
of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
- Institute
for Drug Discovery, Leipzig University Medical
School, Leipzig 04109, Germany
| | - Heidi E. Hamm
- Department
of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States
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Qu G, Li X, Jin R, Guan D, Ji J, Li S, Shi H, Tong P, Gan W, Zhang A. MicroRNA-26a alleviates tubulointerstitial fibrosis in diabetic kidney disease by targeting PAR4. J Cell Mol Med 2024; 28:e18099. [PMID: 38164021 PMCID: PMC10844712 DOI: 10.1111/jcmm.18099] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2023] [Revised: 11/01/2023] [Accepted: 11/25/2023] [Indexed: 01/03/2024] Open
Abstract
Our previous study found that miR-26a alleviates aldosterone-induced tubulointerstitial fibrosis (TIF). However, the effect of miR-26a on TIF in diabetic kidney disease (DKD) remains unclear. This study clarifies the role and possible mechanism of exogenous miR-26a in controlling the progression of TIF in DKD models. Firstly, we showed that miR-26a was markedly decreased in type 2 diabetic db/db mice and mouse tubular epithelial cells (mTECs) treated with high glucose (HG, 30 mM) using RT-qPCR. We then used adeno-associated virus carrying miR-26a and adenovirus miR-26a to enhance the expression of miR-26a in vivo and in vitro. Overexpressing miR-26a alleviated the TIF in db/db mice and the extracellular matrix (ECM) deposition in HG-stimulated mTECs. These protective effects were caused by reducing expression of protease-activated receptor 4 (PAR4), which involved in multiple pro-fibrotic pathways. The rescue of PAR4 expression reversed the anti-fibrosis activity of miR-26a. We conclude that miR-26a alleviates TIF in DKD models by directly targeting PAR4, which may provide a novel molecular strategy for DKD therapy.
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Affiliation(s)
- Gaoting Qu
- Department of Pediatric NephrologyThe Second Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Xingyue Li
- Department of Pediatric NephrologyThe Second Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Ran Jin
- Department of Pediatric NephrologyThe Second Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Dian Guan
- Department of Pediatric SurgeryThe First Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Jialing Ji
- Department of PediatricsThe Fourth Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Shanwen Li
- Department of Pediatric NephrologyThe Second Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Huimin Shi
- Department of Pediatric NephrologyThe Second Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Pingfan Tong
- Department of PediatricsThe Fourth Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Weihua Gan
- Department of Pediatric NephrologyThe Second Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
| | - Aiqing Zhang
- Department of PediatricsThe Fourth Affiliated Hospital of Nanjing Medical UniversityNanjingP.R. China
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10
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Wang Q, Qin Y, Ma J, Zhou K, Xia G, Li Y, Xie L, Afful RG, Lan Q, Huo X, Zou J, Yang H. An early warning indicator of mortality risk in patients with COVID-19: the neutrophil extracellular traps/neutrophilic segmented granulocyte ratio. Front Immunol 2024; 15:1287132. [PMID: 38348024 PMCID: PMC10859410 DOI: 10.3389/fimmu.2024.1287132] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2023] [Accepted: 01/15/2024] [Indexed: 02/15/2024] Open
Abstract
Background Neutrophil extracellular traps (NETs) play a key role in thrombus formation in patients with coronavirus disease 2019 (COVID-19). However, the existing detection and observation methods for NETs are limited in their ability to provide quantitative, convenient, and accurate descriptions of in situ NETs. Therefore, establishing a quantitative description of the relationship between NETs and thrombosis remains a challenge. Objective We employed morphological observations of blood cells and statistical analyses to investigate the correlation between the NETs/neutrophilic segmented granulocyte ratio and mortality risk in patients with COVID-19. Methods Peripheral blood samples were collected from 117 hospitalized patients with COVID-19 between November 2022 and February 2023, and various blood cell parameters were measured. Two types of smudge cells were observed in the blood and counted: lymphatic and neutral smudge cells. Statistical data analysis was used to establish COVID-19 mortality risk assessment indicators. Results Morphological observations of neutrophilic smudge cells revealed swelling, eruption, and NETs formation in the neutrophil nuclei. Subsequently, the NETs/neutrophilic segmented granulocyte ratio (NNSR) was calculated. A high concentration of NETs poses a fatal risk for thrombus formation in patients. Statistical analysis indicated that a high NNSR was more suitable for evaluating the risk of death in patients with COVID-19 compared to elevated fibrinogen (FIB) and D-dimer (DD) levels. Conclusion Observing blood cell morphology is an effective method for the detection of NETs, NNSR are important markers for revealing the mortality risk of patients with COVID-19.
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Affiliation(s)
- Qiong Wang
- The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, Nanjing Medical University, Wuxi, China
| | - Yu Qin
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Jingyun Ma
- The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, Nanjing Medical University, Wuxi, China
| | - Kehao Zhou
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Guiping Xia
- The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, Nanjing Medical University, Wuxi, China
| | - Ya Li
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Li Xie
- School of Internet of Things Engineering, Jiangnan University, Wuxi, China
| | - Richmond Godwin Afful
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Qian Lan
- School of Internet of Things Engineering, Jiangnan University, Wuxi, China
| | - Xingyu Huo
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
| | - Jian Zou
- The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, Nanjing Medical University, Wuxi, China
| | - Hailin Yang
- Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
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11
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Han X, Knauss EA, de la Fuente M, Li W, Conlon RA, LePage DF, Jiang W, Renna SA, McKenzie SE, Nieman MT. A Mouse Model of the Protease Activated Receptor 4 (PAR4) Pro310Leu Variant has Reduced Platelet Reactivity. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.01.569075. [PMID: 38077081 PMCID: PMC10705540 DOI: 10.1101/2023.12.01.569075] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/17/2023]
Abstract
Background Protease activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated a single nucleotide polymorphism in extracellular loop 3 (ECL3), PAR4-P310L (rs2227376) leads to a hypo-reactive receptor. Objectives The goal of this study was to determine how the hypo-reactive PAR4 variant in ECL3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). Methods A point mutation was introduced into the PAR4 gene, F2rl3, via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbβ3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model. Results PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbβ3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. Conclusions PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.
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Affiliation(s)
- Xu Han
- Department of Pharmacology, Case Western Reserve University, Cleveland, OH United States
| | - Elizabeth A. Knauss
- Department of Pharmacology, Case Western Reserve University, Cleveland, OH United States
| | - Maria de la Fuente
- Department of Pharmacology, Case Western Reserve University, Cleveland, OH United States
| | - Wei Li
- Department of Biomedical Sciences, Joan C. Edwards School of Medicine at Marshall University, Huntington, WV United States
| | - Ronald A Conlon
- Case Transgenic and Targeting Facility, Case Western Reserve University, Cleveland, OH United States
| | - David F. LePage
- Case Transgenic and Targeting Facility, Case Western Reserve University, Cleveland, OH United States
| | - Weihong Jiang
- Case Transgenic and Targeting Facility, Case Western Reserve University, Cleveland, OH United States
| | - Stephanie A. Renna
- Department of Medicine, The Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA United States
| | - Steven E. McKenzie
- Department of Medicine, The Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA United States
| | - Marvin T. Nieman
- Department of Pharmacology, Case Western Reserve University, Cleveland, OH United States
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12
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Renna SA, McKenzie SE, Michael JV. Species Differences in Platelet Protease-Activated Receptors. Int J Mol Sci 2023; 24:ijms24098298. [PMID: 37176005 PMCID: PMC10179473 DOI: 10.3390/ijms24098298] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2023] [Revised: 04/21/2023] [Accepted: 05/02/2023] [Indexed: 05/15/2023] Open
Abstract
Protease-activated receptors (PARs) are a class of integral membrane proteins that are cleaved by a variety of proteases, most notably thrombin, to reveal a tethered ligand and promote activation. PARs are critical mediators of platelet function in hemostasis and thrombosis, and therefore are attractive targets for anti-platelet therapies. Animal models studying platelet PAR physiology have relied heavily on genetically modified mouse strains, which have provided ample insight but have some inherent limitations. The current review aims to summarize the notable PAR expression and functional differences between the mouse and human, in addition to highlighting some recently developed tools to further study human physiology in mouse models.
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Affiliation(s)
- Stephanie A Renna
- Department of Medicine, The Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA 19107, USA
| | - Steven E McKenzie
- Department of Medicine, The Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA 19107, USA
| | - James V Michael
- Department of Medicine, The Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, PA 19107, USA
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13
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Abstract
Mutations of polycystin-1 (PC1) are the major cause (85% of cases) of autosomal dominant polycystic kidney disease (ADPKD), which is the fourth leading cause of kidney failure. PC1 is thought to function as an atypical G protein-coupled receptor, yet the mechanism by which PC1 regulates G-protein signaling remains poorly understood. A significant portion of ADPKD mutations of PC1 encode a protein with defects in maturation or reduced function that may be amenable to functional rescue. In this work, we have combined complementary biochemical and cellular assay experiments and accelerated molecular simulations, which revealed an allosteric transduction pathway in activation of the PC1 C-terminal fragment. Our findings will facilitate future rational drug design efforts targeting the PC1 signaling function. Polycystin-1 (PC1) is an important unusual G protein-coupled receptor (GPCR) with 11 transmembrane domains, and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal, and membrane-embedded, C-terminal fragments (CTF), and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless, and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain, and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074–S3585 and R3848–E4078 predicted from the GaMD simulations were validated by mutagenesis experiments. Together, these complementary analyses have provided insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1.
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14
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Stoller ML, Basak I, Denorme F, Rowley JW, Alsobrooks J, Parsawar K, Nieman MT, Yost CC, Hamilton JR, Bray PF, Campbell RA. Neutrophil cathepsin G proteolysis of protease-activated receptor 4 generates a novel, functional tethered ligand. Blood Adv 2022; 6:2303-2308. [PMID: 34883511 PMCID: PMC9006282 DOI: 10.1182/bloodadvances.2021006133] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2021] [Accepted: 11/16/2021] [Indexed: 12/04/2022] Open
Abstract
Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease-activated receptor (PAR) amino termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.
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Affiliation(s)
- Michelle L. Stoller
- University of Utah Molecular Medicine Program, University of Utah, Salt Lake City, UT
| | - Indranil Basak
- Department of Biochemistry, University of Otago, Dunedin, New Zealand
| | - Frederik Denorme
- University of Utah Molecular Medicine Program, University of Utah, Salt Lake City, UT
| | - Jesse W. Rowley
- University of Utah Molecular Medicine Program, University of Utah, Salt Lake City, UT
- Division of Pulmonary, Department of Internal Medicine, University of Utah, Salt Lake City, UT
| | - James Alsobrooks
- Department of Medicine, University of Virginia, Charlottesville, VA
| | - Krishna Parsawar
- Analytical and Biological Mass Spectrometry Core Facility, University of Arizona, Tucson, AZ
| | - Marvin T. Nieman
- Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, OH
| | - Christian Con Yost
- University of Utah Molecular Medicine Program, University of Utah, Salt Lake City, UT
- Division of Neonatology, Department of Pediatric Medicine, University of Utah, Salt Lake City, UT
| | - Justin R. Hamilton
- Australian Centre for Blood Diseases, Monash University, Melbourne, Australia; and
| | - Paul F. Bray
- University of Utah Molecular Medicine Program, University of Utah, Salt Lake City, UT
- Division of Hematology and Hematologic Malignancies, and
| | - Robert A. Campbell
- University of Utah Molecular Medicine Program, University of Utah, Salt Lake City, UT
- Division of General Medicine, Department of Internal Medicine, University of Utah, Salt Lake City, UT
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15
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Yu X, Li S, Zhu X, Kong Y. Inhibitors of protease activated receptor 4 (PAR4): a review of recent patents (2013-2021). Expert Opin Ther Pat 2022; 32:153-170. [PMID: 35081321 DOI: 10.1080/13543776.2022.2034786] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
INTRODUCTION Protease-activated receptor 4 (PAR4), belonging to a subfamily of G-protein-coupled receptors (GPCR), is expressed on the surface of Human platelets, and the activation of it can lead to platelets aggregation. Studies demonstrated that PAR4 inhibition protect mice from arterial/arteriolar thrombosis, pulmonary embolism and cerebral infarct, while do not affect the haemostatic responses integrity. Therefore, PAR4 has been a promising target for the development of anti-thrombotic agents. AREAS COVERED This review covers recent patents and literature on PAR4 and their application published between 2013 and 2021. EXPERT OPINION PAR4 is a promising anti-thrombotic target and PAR4 inhibitors are important biologically active compounds for the treatment of thrombosis. Most the recent patents and literature focus on PAR4 selective inhibitors, and BMS-986120 and BMS-986141, which were developed by BMS, have entered clinical trials. With the deep understanding of the crystal structures and biological functions of PAR4, we believe that many other novel types of molecules targeting PAR4 would enter the clinical studies or the market.
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Affiliation(s)
- Xiangying Yu
- School of Life & Technology, China Pharmaceutical University, Nanjing, 210009, PR China
| | - Shanshan Li
- Institute of Medicinal & Chemistry, China Pharmaceutical University, Nanjing, 210009, PR China
| | - Xiong Zhu
- Institute of Medicinal & Chemistry, China Pharmaceutical University, Nanjing, 210009, PR China
| | - Yi Kong
- School of Life & Technology, China Pharmaceutical University, Nanjing, 210009, PR China
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16
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Selvadurai MV, Riaz M, Xie S, Tonkin A, McNeil JJ, Lacaze P, Hamilton J. The PAR4 platelet thrombin receptor variant rs773902 does not impact the incidence of thrombotic or bleeding events in a healthy older population. Thromb Haemost 2021; 122:1130-1138. [PMID: 34852379 DOI: 10.1055/a-1711-1395] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
BACKGROUND Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor important for thrombosis and a target of anti-platelet drug development. A frequently occurring single nucleotide polymorphism (SNP; rs773902) causes a PAR4 sequence variant (NC_000019.10:p.Ala120Thr) whereby platelets from Thr120-expressing individuals are hyper-responsive to PAR4 agonists versus platelets from Ala120-expressing individuals. However, whether this enhanced platelet responsiveness translates to increased thrombotic risk or decreased bleeding risk remains unknown. OBJECTIVES To examine the association of rs773902 with adjudicated cardiovascular events and aspirin use in a randomized trial population of healthy older individuals. PATIENTS/METHODS We analyzed 13,547 participants in the ASPirin in Reducing Events in the Elderly (ASPREE) trial. Participants had no previous cardiovascular events at enrollment and were randomized to either 100 mg daily aspirin or placebo for a median follow-up of 4.7 years. Total genotypes were 8,761 (65%) GG (Ala120 variant), 4,303 (32%) heterozygotes, and 483 (4%) AA (Thr120 variant). Cox proportional hazard regression tested the relationship between rs773902 and thrombotic events (major adverse cardiovascular events [MACE] and ischemic stroke [IS]) and bleeding (major hemorrhage [MHEM] and intracranial bleeding [ICB]). RESULTS No statistically significant association was observed overall or by treatment group between rs773902 and any thrombotic or bleeding event examined. Further, there was no significant interaction between rs773902 and treatment for any of MACE, IS, MHEM, or ICB. CONCLUSIONS This post-hoc analysis of a prospective cohort study suggests that, despite sensitizing platelet activation, the rs773902 PAR4 variant is not associated with thrombotic cardiovascular or bleeding events in a healthy older population.
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Affiliation(s)
- Maria V Selvadurai
- Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
| | - Moeen Riaz
- Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
| | - Sophia Xie
- Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
| | | | | | | | - Justin Hamilton
- Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
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17
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Mwirigi J, Kume M, Hassler SN, Ahmad A, Ray PR, Jiang C, Chamessian A, Mseeh N, Ludwig BP, Rivera BD, Nieman MT, Van de Ven T, Ji RR, Dussor G, Boitano S, Vagner J, Price TJ. A Role for Protease Activated Receptor Type 3 (PAR3) in Nociception Demonstrated Through Development of a Novel Peptide Agonist. THE JOURNAL OF PAIN 2021; 22:692-706. [PMID: 33429107 PMCID: PMC8197731 DOI: 10.1016/j.jpain.2020.12.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/16/2020] [Revised: 12/02/2020] [Accepted: 12/20/2020] [Indexed: 02/07/2023]
Abstract
The protease activated receptor (PAR) family is a group of G-protein coupled receptors (GPCRs) activated by proteolytic cleavage of the extracellular domain. PARs are expressed in a variety of cell types with crucial roles in homeostasis, immune responses, inflammation, and pain. PAR3 is the least researched of the four PARs, with little known about its expression and function. We sought to better understand its potential function in the peripheral sensory nervous system. Mouse single-cell RNA sequencing data demonstrates that PAR3 is widely expressed in dorsal root ganglion (DRG) neurons. Co-expression of PAR3 mRNA with other PARs was identified in various DRG neuron subpopulations, consistent with its proposed role as a coreceptor of other PARs. We developed a lipid tethered PAR3 agonist, C660, that selectively activates PAR3 by eliciting a Ca2+ response in DRG and trigeminal neurons. In vivo, C660 induces mechanical hypersensitivity and facial grimacing in WT but not PAR3-/- mice. We characterized other nociceptive phenotypes in PAR3-/- mice and found a loss of hyperalgesic priming in response to IL-6, carrageenan, and a PAR2 agonist, suggesting that PAR3 contributes to long-lasting nociceptor plasticity in some contexts. To examine the potential role of PAR3 in regulating the activity of other PARs in sensory neurons, we administered PAR1, PAR2, and PAR4 agonists and assessed mechanical and affective pain behaviors in WT and PAR3-/- mice. We observed that the nociceptive effects of PAR1 agonists were potentiated in the absence of PAR3. Our findings suggest a complex role of PAR3 in the physiology and plasticity of nociceptors. PERSPECTIVE: We evaluated the role of PAR3, a G-protein coupled receptor, in nociception by developing a selective peptide agonist. Our findings suggest that PAR3 contributes to nociception in various contexts and plays a role in modulating the activity of other PARs.
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Affiliation(s)
- Juliet Mwirigi
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas
| | - Moeno Kume
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas
| | - Shayne N Hassler
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas
| | - Ayesha Ahmad
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas
| | - Pradipta R Ray
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas
| | - Changyu Jiang
- Duke University School of Medicine, Department of Anesthesiology, Pharmacology, and Cancer Biology, Durham, North Carolina
| | - Alexander Chamessian
- Duke University School of Medicine, Department of Anesthesiology, Pharmacology, and Cancer Biology, Durham, North Carolina
| | - Nakleh Mseeh
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas
| | - Breya P Ludwig
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas
| | - Benjamin D Rivera
- Department of Physiology, University of Arizona, Asthma and Airway Disease Research Center, Tucson, Arizona
| | - Marvin T Nieman
- Case Western Reserve University School of Medicine, Department of Pharmacology, Cleveland, Ohio
| | - Thomas Van de Ven
- Duke University School of Medicine, Department of Anesthesiology, Pharmacology, and Cancer Biology, Durham, North Carolina
| | - Ru-Rong Ji
- Duke University School of Medicine, Department of Anesthesiology, Pharmacology, and Cancer Biology, Durham, North Carolina
| | - Gregory Dussor
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas
| | - Scott Boitano
- Department of Physiology, University of Arizona, Asthma and Airway Disease Research Center, Tucson, Arizona
| | - Josef Vagner
- University of Arizona, Bio5 Research Institute, Tucson, Arizona
| | - Theodore J Price
- University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, Richardson, Texas.
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18
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Gui Y, Yuan S. Epigenetic regulations in mammalian spermatogenesis: RNA-m 6A modification and beyond. Cell Mol Life Sci 2021; 78:4893-4905. [PMID: 33835194 PMCID: PMC11073063 DOI: 10.1007/s00018-021-03823-9] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Revised: 03/04/2021] [Accepted: 03/25/2021] [Indexed: 02/06/2023]
Abstract
Emerging evidence shows that m6A, one of the most abundant RNA modifications in mammals, is involved in the entire process of spermatogenesis, including mitosis, meiosis, and spermiogenesis. "Writers" catalyze m6A formation on stage-specific transcripts during male germline development, while "erasers" remove m6A modification to maintain a balance between methylation and demethylation. The different functions of RNA-m6A transcripts depend on their recognition by "readers". m6A modification mediates RNA metabolism, including mRNA splicing, translation, and degradation, as well as the maturity and biosynthesis of non-coding RNAs. Sperm RNA profiles are easily affected by environmental exposure and can even be inherited for several generations, similar to epigenetic inheritance. Here, we review and summarize the critical role of m6A in different developmental stages of male germ cells, to understand of the mechanisms and epigenetic regulation of m6A modifications. In addition, we also outline and discuss the important role of non-coding RNAs in spermatogenesis and RNA modifications in epigenetic inheritance.
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Affiliation(s)
- Yiqian Gui
- Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Shuiqiao Yuan
- Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
- Shenzhen Huazhong University of Science and Technology Research Institute, Shenzhen, 518057, China.
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19
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Bouck EG, de la Fuente M, Zunica ER, Li W, Mumaw MM, Nieman MT. Murine cadherin-6 mediates thrombosis in vivo in a platelet-independent manner. Res Pract Thromb Haemost 2021; 5:125-131. [PMID: 33537536 PMCID: PMC7845066 DOI: 10.1002/rth2.12458] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2020] [Accepted: 10/26/2020] [Indexed: 11/07/2022] Open
Abstract
BACKGROUND Platelet adhesion is the critical process mediating stable thrombus formation. Previous reports of cadherin-6 on human platelets have demonstrated its role in platelet aggregation and thrombus formation. OBJECTIVES We aimed to further characterize the importance of cadherin-6 in thrombosis in vivo. METHODS Cadherin-6 platelet expression was evaluated by western blotting, flow cytometry, and immunoprecipitation. Thrombosis was evaluated using the FeCl3 and Rose Bengal carotid artery models in C57Bl6 mice treated with anti-cadherin-6 or IgG and wild-type or Cdh6-/- mice. Platelet function was compared in wild-type and Cdh6-/- mice using tail-clip assays, aggregometry, and flow cytometry. RESULTS Human platelet expression of cadherin-6 was confirmed at ~3000 copies per platelet. Cdh6-/- mice or those treated with anti-cadherin-6 antibody showed an increased time to occlusion in both thrombosis models. Cadherin-6 was not expressed on mouse platelets, and there were no differences in tail bleeding times, platelet aggregation, or platelet activation in wild-type versus Cdh6-/- mice. CONCLUSIONS Cadherin-6 plays an essential role in thrombosis in vivo. However, cadherin-6 is not expressed on murine platelets. These data are in contrast to human platelets, which express a functional cadherin-6/catenin complex. The essential, platelet-independent role for cadherin-6 in hemostasis may allow it to be an effective and safe therapeutic target.
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Affiliation(s)
- Emma G. Bouck
- Department of PharmacologyCase Western Reserve UniversityClevelandOHUSA
| | | | | | - Wei Li
- Deparmtent of Biomedical SciencesMarshall University Joan C. Edwards School of MedicineHuntingtonWVUSA
| | - Michele M. Mumaw
- Department of PharmacologyCase Western Reserve UniversityClevelandOHUSA
| | - Marvin T. Nieman
- Department of PharmacologyCase Western Reserve UniversityClevelandOHUSA
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20
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Han X, de la Fuente M, Nieman MT. Complement factor C4a does not activate protease-activated receptor 1 (PAR1) or PAR4 on human platelets. Res Pract Thromb Haemost 2021; 5:104-110. [PMID: 33537534 PMCID: PMC7845074 DOI: 10.1002/rth2.12459] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2020] [Revised: 10/22/2020] [Accepted: 11/04/2020] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND Protease-activated receptor (PAR) 1 and PAR4 are key thrombin signal mediators for human platelet activation and aggregation in response to vascular injury. They are primarily activated by thrombin cleavage of the N-terminus to expose a tethered ligand. In addition to the canonical activation by thrombin, a growing panel of proteases can also elicit PAR1- or PAR4-mediated signal transduction. Recently, complement factor C4a was reported as the first endogenous agonist for both PAR1 and PAR4. Further, it is the first endogenous nontethered ligand that activates PAR1 and PAR4. These studies were conducted with human microvascular cells; the impact of C4a on platelet PARs is unknown. OBJECTIVES The goal of this study was to interrogate PAR1 and PAR4 activation by C4a on human platelets. METHODS Platelet-rich plasma (PRP) was isolated from healthy donors. PRP was stimulated with C4a, and the platelet aggregation was measured. Human embryonic kidney (HEK) 293 Flp-In T-rex cells were used to further test if C4a stimulation can initiate PAR1- or PAR4-mediated Gαq signaling, which was measured by intracellular calcium mobilization. RESULTS C4a failed to elicit platelet aggregation via PAR1- or PAR4-mediated manner. In addition, no PAR1- or PAR4-mediated calcium mobilization was observed upon C4a stimulation on HEK293 cells. CONCLUSIONS Complement factor C4a does not activate PAR1 or PAR4 on human platelets. These data show that PAR1 and PAR4 activation by C4a on microvascular cells likely requires a cofactor, which reinforces the concept of cell type-specific regulation of protease signaling.
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Affiliation(s)
- Xu Han
- Department of PharmacologyCase Western Reserve UniversityClevelandOHUSA
| | | | - Marvin T. Nieman
- Department of PharmacologyCase Western Reserve UniversityClevelandOHUSA
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21
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Popping the lid on PAR4 activation. Blood 2020; 136:2101-2102. [DOI: 10.1182/blood.2020007334] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
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22
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Han X, Nieman MT. The domino effect triggered by the tethered ligand of the protease activated receptors. Thromb Res 2020; 196:87-98. [PMID: 32853981 DOI: 10.1016/j.thromres.2020.08.004] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Revised: 07/23/2020] [Accepted: 08/03/2020] [Indexed: 12/20/2022]
Abstract
Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that have a unique activation mechanism. Unlike other GPCRs that can be activated by free ligands, under physiological conditions, PARs are activated by the tethered ligand, which is a part of their N-terminus that is unmasked by proteolysis. It has been 30 years since the first member of the family, PAR1, was identified. In this review, we will discuss this unique tethered ligand mediate receptor activation of PARs in detail: how they interact with the proteases, the complex structural rearrangement of the receptors upon activation, and the termination of the signaling. We also summarize the structural studies of the PARs and how single nucleotide polymorphisms impact the receptor reactivity. Finally, we review the current strategies for inhibiting PAR function with therapeutic targets for anti-thrombosis. The focus of this review is PAR1 and PAR4 as they are the thrombin signal mediators on human platelets and therapeutics targets. We also include the structural studies of PAR2 as it informs the mechanism of action for PARs in general.
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Affiliation(s)
- Xu Han
- Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA
| | - Marvin T Nieman
- Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA.
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23
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Chen X, Zhao X, Cooper M, Ma P. The Roles of GRKs in Hemostasis and Thrombosis. Int J Mol Sci 2020; 21:ijms21155345. [PMID: 32731360 PMCID: PMC7432802 DOI: 10.3390/ijms21155345] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Revised: 07/20/2020] [Accepted: 07/27/2020] [Indexed: 12/20/2022] Open
Abstract
Along with cancer, cardiovascular and cerebrovascular diseases remain by far the most common causes of death. Heart attacks and strokes are diseases in which platelets play a role, through activation on ruptured plaques and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which make these receptors ideal targets for many antiplatelet drugs. However, little is known about the mechanisms that provide feedback regulation on GPCRs to limit platelet activation. Emerging evidence from our group and others strongly suggests that GPCR kinases (GRKs) are critical negative regulators during platelet activation and thrombus formation. In this review, we will summarize recent findings on the role of GRKs in platelet biology and how one specific GRK, GRK6, regulates the hemostatic response to vascular injury. Furthermore, we will discuss the potential role of GRKs in thrombotic disorders, such as thrombotic events in COVID-19 patients. Studies on the function of GRKs during platelet activation and thrombus formation have just recently begun, and a better understanding of the role of GRKs in hemostasis and thrombosis will provide a fruitful avenue for understanding the hemostatic response to injury. It may also lead to new therapeutic options for the treatment of thrombotic and cardiovascular disorders.
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Affiliation(s)
- Xi Chen
- Cardeza Foundation for Hematologic Research, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA; (X.C.); (X.Z.); (M.C.)
| | - Xuefei Zhao
- Cardeza Foundation for Hematologic Research, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA; (X.C.); (X.Z.); (M.C.)
- Cyrus Tang Hematology Center, Soochow University, Suzhou 215123, China
| | - Matthew Cooper
- Cardeza Foundation for Hematologic Research, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA; (X.C.); (X.Z.); (M.C.)
| | - Peisong Ma
- Cardeza Foundation for Hematologic Research, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA; (X.C.); (X.Z.); (M.C.)
- Correspondence: ; Tel.: +1-215-955-3966
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24
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Kahremany S, Hofmann L, Gruzman A, Cohen G. Advances in Understanding the Initial Steps of Pruritoceptive Itch: How the Itch Hits the Switch. Int J Mol Sci 2020; 21:ijms21144883. [PMID: 32664385 PMCID: PMC7402353 DOI: 10.3390/ijms21144883] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Revised: 07/07/2020] [Accepted: 07/08/2020] [Indexed: 02/07/2023] Open
Abstract
Pruritoceptive (dermal) itch was long considered an accompanying symptom of diseases, a side effect of drug applications, or a temporary sensation induced by invading pruritogens, as produced by the stinging nettle. Due to extensive research in recent years, it was possible to provide detailed insights into the mechanism of itch mediation and modulation. Hence, it became apparent that pruritus is a complex symptom or disease in itself, which requires particular attention to improve patients’ health. Here, we summarize recent findings in pruritoceptive itch, including how this sensation is triggered and modulated by diverse endogenous and exogenous pruritogens and their receptors. A differentiation between mediating pruritogen and modulating pruritogen seems to be of great advantage to understand and decipher the molecular mechanism of itch perception. Only a comprehensive view on itch sensation will provide a solid basis for targeting this long-neglected adverse sensation accompanying numerous diseases and many drug side effects. Finally, we identify critical aspects of itch perception that require future investigation.
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Affiliation(s)
- Shirin Kahremany
- Department of Chemistry, Faculty of Exact Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel; (L.H.); (A.G.)
- The Skin Research Institute, The Dead Sea and Arava Science Center, Masada 86910, Israel;
- Correspondence:
| | - Lukas Hofmann
- Department of Chemistry, Faculty of Exact Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel; (L.H.); (A.G.)
| | - Arie Gruzman
- Department of Chemistry, Faculty of Exact Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel; (L.H.); (A.G.)
| | - Guy Cohen
- The Skin Research Institute, The Dead Sea and Arava Science Center, Masada 86910, Israel;
- Ben-Gurion University of the Negev, Eilat Campus, Eilat 8855630, Israel
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