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1
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Kaiser R, Gold C, Stark K. Recent Advances in Immunothrombosis and Thromboinflammation. Thromb Haemost 2025. [PMID: 40311639 DOI: 10.1055/a-2523-1821] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/03/2025]
Abstract
Inflammation and thrombosis are traditionally considered two separate entities of acute host responses to barrier breaks. While inciting inflammatory responses is a prerequisite to fighting invading pathogens and subsequent restoration of tissue homeostasis, thrombus formation is a crucial step of the hemostatic response to prevent blood loss following vascular injury. Though originally designed to protect the host, excessive induction of either inflammatory signaling or thrombus formation and their reciprocal activation contribute to a plethora of disorders, including cardiovascular, autoimmune, and malignant diseases. In this state-of-the-art review, we summarize recent insights into the intricate interplay of inflammation and thrombosis. We focus on the protective aspects of immunothrombosis as well as evidence of detrimental sequelae of thromboinflammation, specifically regarding recent studies that elucidate its pathophysiology beyond coronavirus disease 2019 (COVID-19). We introduce recently identified molecular aspects of key cellular players like neutrophils, monocytes, and platelets that contribute to both immunothrombosis and thromboinflammation. Further, we describe the underlying mechanisms of activation involving circulating plasma proteins and immune complexes. We then illustrate how these factors skew the inflammatory state toward detrimental thromboinflammation across cardiovascular as well as septic and autoimmune inflammatory diseases. Finally, we discuss how the advent of new technologies and the integration with clinical data have been used to investigate the mechanisms and signaling cascades underlying immunothrombosis and thromboinflammation. This review highlights open questions that will need to be addressed by the field to translate our mechanistic understanding into clinically meaningful therapeutic targeting.
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Affiliation(s)
- Rainer Kaiser
- Medizinische Klinik und Poliklinik I, University Hospital Ludwig-Maximilian University, Munich, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany
| | - Christoph Gold
- Medizinische Klinik und Poliklinik I, University Hospital Ludwig-Maximilian University, Munich, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany
| | - Konstantin Stark
- Medizinische Klinik und Poliklinik I, University Hospital Ludwig-Maximilian University, Munich, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany
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2
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Yuan J, Zhou S, Wu X, Li F, Lai Z, Ding Q, Wu W, Wang X, Dai J, Hu X, Lu Y. Asn384Ser Mutation in Protein C is Associated with Multiple-Site Thrombosis in a Young Heterozygous Male. Thromb Haemost 2025. [PMID: 40250449 DOI: 10.1055/a-2569-6439] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/20/2025]
Abstract
Protein C (PC) is an important physiological anticoagulant factor in humans. Activated protein C (APC) is generated from the PC zymogen through proteolytic activation by thrombin. APC inhibits thrombin generation by inactivating activated factors V and VIII via limited proteolysis. In addition to its anticoagulant function, APC also exhibits potent cytoprotective and anti-inflammatory properties. We have identified a young male with multiple-site thrombosis, who carries a heterozygous mutation c.1151A > G,p.Asn384Ser(N384S) in PC. Although this mutation has been previously documented, limited functional research has been conducted to elucidate its pathogenesis.To elucidate the functional alternations of the N384S mutant protein C and delineate the molecular mechanism underlying thrombosis in the patient carrying this mutation.We expressed the recombinant PC-N384S in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems.The expression level of the PC-N384S was reduced to approximately 7% of that observed for PC-WT. The activation of PC-N384S by thrombin or thrombin-thrombomodulin (TM) complex was significantly impaired, although the addition of TM exhibited a slight enhancement in the activation process. In terms of cleaving a chromogenic substrate, the catalytic efficiency reduced to approximately 50% of that observed in the wild type. In addition, in comparison with APC-WT, APC-N384S demonstrated a pronounced decline in amidolytic activity following an extended incubation period at 37°C. APC-N384S exhibited slightly impaired anticoagulant activity in either FVa inhibition assay or plasma-based assay systems. Furthermore, anti-inflammatory activity of APC-N384S was dramatically impaired as determined by evaluating the barrier-protective effect.The Asn384Ser mutation impairs both the anticoagulant and barrier-protective activities of protein C, thereby increasing the thrombosis risk in the heterozygous young male.
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Affiliation(s)
- Junwei Yuan
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- College of Health Science and Technology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Shijie Zhou
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xi Wu
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Fang Li
- State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
| | - Zhe Lai
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Qiulan Ding
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Wenman Wu
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xuefeng Wang
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Jing Dai
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xiaobo Hu
- Shanghai Center for Clinical Laboratory, Shanghai, China
| | - Yeling Lu
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
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3
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Chatterjee A, Paul S, Mukherjee T, Gupta S, Parashar D, Sahu B, Kumar U, Das K. Beyond coagulation: Coagulation protease factor VIIa in cytoprotective response. Int Immunopharmacol 2025; 150:114218. [PMID: 39955915 DOI: 10.1016/j.intimp.2025.114218] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2024] [Revised: 01/22/2025] [Accepted: 01/31/2025] [Indexed: 02/18/2025]
Abstract
Blood coagulation, the tightly regulated biological process prevents bleeding upon injury to the blood vessels. Vessel injury exposes the sub-endothelial tissue factor (TF) to the blood stream, thereby leading to the binding of coagulation protease, factor VII/activated VII with TF, and thus initiating the extrinsic pathway of blood coagulation. Apart from coagulation, FVIIa also promotes intracellular signaling via the activation of a unique class of G-protein-coupled receptor (GPCR) family protein, protease-activated receptor 1 (PAR1), thereby promoting anti-inflammation and endothelial barrier protection. Blood coagulation and inflammation are intrinsically connected, the activation of one process often leads to the activation of the other. The present review highlights the mechanisms by which FVIIa contributes to cytoprotective responses, either by direct action or through the release of extracellular vesicles (EVs) from vascular endothelium. FVIIa, due to its well-known ability to promote coagulation, is also used as a hemostatic agent in the treatment of several hyper bleeding disorders like hemophilia, thrombocytopenia etc. In addition to its hemostatic role, the topics discussed in the present review open a new therapeutic off-label effect of FVIIa, i.e., providing anti-inflammatory and vascular protective responses in several bleeding disorders and beyond.
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Affiliation(s)
- Akash Chatterjee
- School of Biological Sciences, Indian Association for the Cultivation of Science, Jadavpur 700032, West Bengal, India
| | - Subhojit Paul
- School of Biological Sciences, Indian Association for the Cultivation of Science, Jadavpur 700032, West Bengal, India
| | - Tanmoy Mukherjee
- Department of Cellular and Molecular Biology, The University of Texas at Tyler Health Science Center, Tyler, TX 75708, USA
| | - Saurabh Gupta
- Department of Biotechnology, GLA University, Mathura 281406, Uttar Pradesh, India
| | - Deepak Parashar
- Division of Hematology & Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Bhupender Sahu
- Centre for Molecular Biology, Central University of Jammu, Jammu 181143, Jammu and Kashmir, India
| | - Umesh Kumar
- Department of Biosciences, Institute of Management Studies Ghaziabad (University Courses Campus), NH09, Adhyatmik Nagar, Ghaziabad 201015, Uttar Pradesh, India.
| | - Kaushik Das
- Biotechnology Research and Innovation Council-National Institute of Biomedical Genomics, Kalyani 741251, West Bengal, India.
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4
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Leon G, Klavina PA, Rehill AM, Cooper SEJ, Dominik A, Basavarajappa SC, O'Donnell JS, Hussey S, Walsh PT, Preston RJS. Tissue factor-dependent colitogenic CD4 + T cell thrombogenicity is regulated by activated protein C signalling. Nat Commun 2025; 16:1677. [PMID: 39956825 PMCID: PMC11830781 DOI: 10.1038/s41467-025-57001-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Accepted: 02/07/2025] [Indexed: 02/18/2025] Open
Abstract
Patients with inflammatory bowel disease (IBD) have an increased risk of venous thromboembolism (VTE), but the underlying mechanistic basis remains poorly defined. Here, we find that colitogenic CD4+ T cells express tissue factor (TF) and promote rapid TF-dependent plasma thrombin generation. TF+CD3+CD4+ T cells are present in both the colons of mice with experimental colitis and blood and colonic tissue from patients with IBD. Expression of genes involved in regulating coagulation, including Protein C (PC; encoded by PROC) and its receptor (PROCR), are dysregulated in IBD patient gut biopsy tissues. Moreover, activated PC signalling reduces the procoagulant activity mediated by TF+CD4+ T cells. Our data thus identify TF-induced, colitogenic T cell-mediated thrombogenicity, and also demonstrate a new function for activated PC signalling in regulating T cell thrombo-inflammatory activity.
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Affiliation(s)
- Gemma Leon
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland
| | - Paula A Klavina
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland
| | - Aisling M Rehill
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland
| | - Sarah E J Cooper
- National Centre for Paediatric Gastroenterology, CHI-Crumlin, Dublin, Ireland
| | - Anna Dominik
- National Centre for Paediatric Gastroenterology, CHI-Crumlin, Dublin, Ireland
| | | | - James S O'Donnell
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland
| | - Seamus Hussey
- National Centre for Paediatric Gastroenterology, CHI-Crumlin, Dublin, Ireland
| | - Patrick T Walsh
- Department of Clinical Medicine, Trinity Translational Medicine Institute, Trinity College Dublin, Dublin, Ireland
| | - Roger J S Preston
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland.
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Chen J, Zhou C, Fang W, Yin J, Shi J, Ge J, Shen L, Liu SM, Liu SJ. Identification of endothelial protein C receptor as a novel druggable agonistic target for reendothelialization promotion and thrombosis prevention of eluting stent. Bioact Mater 2024; 41:485-498. [PMID: 39210965 PMCID: PMC11359769 DOI: 10.1016/j.bioactmat.2024.07.028] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Revised: 07/16/2024] [Accepted: 07/21/2024] [Indexed: 09/04/2024] Open
Abstract
The commercially available drug-eluting stent with limus (rapamycin, everolimus, etc.) or paclitaxel inhibits smooth muscle cell (SMC), reducing the in-stent restenosis, whereas damages endothelial cell (EC) and delays stent reendothelialization, increasing the risk of stent thrombosis (ST) and sudden cardiac death. Here we present a new strategy for promoting stent reendothelialization and preventing ST by exploring the application of precise molecular targets with EC specificity. Proteomics was used to investigate the molecular mechanism of EC injury caused by rapamycin. Endothelial protein C receptor (EPCR) was screened out as a crucial EC-specific effector. Limus and paclitaxel repressed the EPCR expression, while overexpression of EPCR protected EC from coating (eluting) drug-induced injury. Furthermore, the ligand activated protein C (APC), polypeptide TR47, and compound parmodulin 2, which activated the target EPCR, promoted EC functions and inhibited platelet or neutrophil adhesion, and enhanced rapamycin stent reendothelialization in the simulated stent environment and in vitro. In vivo, the APC/rapamycin-coating promoted reendothelialization rapidly and prevented ST more effectively than rapamycin-coating alone, in both traditional metal stents and biodegradable stents. Additionally, overexpression or activation of the target EPCR did not affect the cellular behavior of SMC or the inhibitory effect of rapamycin on SMC. In conclusion, EPCR is a promising therapeutical agonistic target for pro-reendothelialization and anti-thrombosis of eluting stent. Activation of EPCR protects against coating drugs-induced EC injury, inflammatory cell, or platelet adhesion onto the stent. The novel application formula for APC/rapamycin-combined eluting promotes stent reendothelialization and prevents ST.
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Affiliation(s)
- Jing Chen
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, PR China
- Department of Cardiology, The First Affliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510000, PR China
| | - Changyi Zhou
- Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, 200032, PR China
- Research Unit of Cardiovascular Techniques and Devices, Chinese Academy of Medical Sciences, Fudan University, Shanghai, 200433, PR China
| | - Weilun Fang
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, PR China
| | - Jiasheng Yin
- Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, 200032, PR China
- Research Unit of Cardiovascular Techniques and Devices, Chinese Academy of Medical Sciences, Fudan University, Shanghai, 200433, PR China
| | - Jian Shi
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, PR China
| | - Junbo Ge
- Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, 200032, PR China
- Research Unit of Cardiovascular Techniques and Devices, Chinese Academy of Medical Sciences, Fudan University, Shanghai, 200433, PR China
| | - Li Shen
- Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai, 200032, PR China
- Research Unit of Cardiovascular Techniques and Devices, Chinese Academy of Medical Sciences, Fudan University, Shanghai, 200433, PR China
| | - Shi-Ming Liu
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, PR China
- Department of Cardiology, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, PR China
| | - Shao-Jun Liu
- Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 510260, PR China
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6
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Wang Y, Thottappillil N, Gomez-Salazar M, Tower RJ, Qin Q, Del Rosario Alvia IC, Xu M, Cherief M, Cheng R, Archer M, Arondekar S, Reddy S, Broderick K, Péault B, James AW. Integrated transcriptomics of human blood vessels defines a spatially controlled niche for early mesenchymal progenitor cells. Dev Cell 2024; 59:2687-2703.e6. [PMID: 39025061 PMCID: PMC11496018 DOI: 10.1016/j.devcel.2024.06.015] [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: 11/01/2023] [Revised: 03/28/2024] [Accepted: 06/19/2024] [Indexed: 07/20/2024]
Abstract
Human blood vessel walls show concentric layers, with the outermost tunica adventitia harboring mesenchymal progenitor cells. These progenitor cells maintain vessel homeostasis and provide a robust cell source for cell-based therapies. However, human adventitial stem cell niche has not been studied in detail. Here, using spatial and single-cell transcriptomics, we characterized the phenotype, potential, and microanatomic distribution of human perivascular progenitors. Initially, spatial transcriptomics identified heterogeneity between perivascular layers of arteries and veins and delineated the tunica adventitia into inner and outer layers. From this spatial atlas, we inferred a hierarchy of mesenchymal progenitors dictated by a more primitive cell with a high surface expression of CD201 (PROCR). When isolated from humans and mice, CD201Low expression typified a mesodermal committed subset with higher osteogenesis and less proliferation than CD201High cells, with a downstream effect on canonical Wnt signaling through DACT2. CD201Low cells also displayed high translational potential for bone tissue generation.
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Affiliation(s)
- Yiyun Wang
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
| | | | | | - Robert J Tower
- Department of Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Qizhi Qin
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
| | | | - Mingxin Xu
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Masnsen Cherief
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Ray Cheng
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Mary Archer
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Shreya Arondekar
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Sashank Reddy
- Department of Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Kristen Broderick
- Department of Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Bruno Péault
- Department of Orthopedic Surgery and Orthopedic Hospital Research Center, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Aaron W James
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA.
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7
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Slotabec L, Seale B, Wang H, Wen C, Filho F, Rouhi N, Adenawoola MI, Li J. Platelets at the intersection of inflammation and coagulation in the APC-mediated response to myocardial ischemia/reperfusion injury. FASEB J 2024; 38:e23890. [PMID: 39143722 PMCID: PMC11373610 DOI: 10.1096/fj.202401128r] [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: 05/21/2024] [Revised: 07/23/2024] [Accepted: 08/05/2024] [Indexed: 08/16/2024]
Abstract
Thromboinflammation is a complex pathology associated with inflammation and coagulation. In cases of cardiovascular disease, in particular ischemia-reperfusion injury, thromboinflammation is a common complication. Increased understanding of thromboinflammation depends on an improved concept of the mechanisms of cells and proteins at the axis of coagulation and inflammation. Among these elements are activated protein C and platelets. This review summarizes the complex interactions of activated protein C and platelets regulating thromboinflammation in cardiovascular disease. By unraveling the pathways of platelets and APC in the inflammatory and coagulation cascades, this review summarizes the role of these vital mediators in the development and perpetuation of heart disease and the thromboinflammation-driven complications of cardiovascular disease. Furthermore, this review emphasizes the significance of the counteracting effects of platelets and APC and their combined role in disease states.
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Affiliation(s)
- Lily Slotabec
- Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, Mississippi, USA
- G.V. (Sonny) Montgomery VA Medical Center, Jackson, Mississippi, USA
| | - Blaise Seale
- Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, Mississippi, USA
| | - Hao Wang
- Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, Mississippi, USA
| | - Changhong Wen
- Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, Mississippi, USA
| | - Fernanda Filho
- Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, Mississippi, USA
| | - Nadiyeh Rouhi
- Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, Mississippi, USA
| | - Michael I Adenawoola
- Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, Mississippi, USA
| | - Ji Li
- Department of Physiology and Biophysics, Mississippi Center for Heart Research, University of Mississippi Medical Center, Jackson, Mississippi, USA
- G.V. (Sonny) Montgomery VA Medical Center, Jackson, Mississippi, USA
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8
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Chen Q, Hazra R, Crosby D, Lenhart D, Lenhart SC, Mondal P, Zhang Y, Nouraie SM, Tan RJ, Esmon CT, Rao LVM, Kim K, Ghosh S. Heme-induced loss of renovascular endothelial protein C receptor promotes chronic kidney disease in sickle mice. Blood 2024; 144:552-564. [PMID: 38820589 PMCID: PMC11307268 DOI: 10.1182/blood.2023023528] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Revised: 04/30/2024] [Accepted: 05/18/2024] [Indexed: 06/02/2024] Open
Abstract
ABSTRACT Chronic kidney disease (CKD) is a major contributor to morbidity and mortality in sickle cell disease (SCD). Anemia, induced by chronic persistent hemolysis, is associated with the progressive deterioration of renal health, resulting in CKD. Moreover, patients with SCD experience acute kidney injury (AKI), a risk factor for CKD, often during vaso-occlusive crisis associated with acute intravascular hemolysis. However, the mechanisms of hemolysis-driven pathogenesis of the AKI-to-CKD transition in SCD remain elusive. Here, we investigated the role of increased renovascular rarefaction and the resulting substantial loss of the vascular endothelial protein C receptor (EPCR) in the progressive deterioration of renal function in transgenic SCD mice. Multiple hemolytic events raised circulating levels of soluble EPCR (sEPCR), indicating loss of EPCR from the cell surface. Using bone marrow transplantation and super-resolution ultrasound imaging, we demonstrated that SCD mice overexpressing EPCR were protective against heme-induced CKD development. In a cohort of patients with SCD, plasma sEPCR was significantly higher in individuals with CKD than in those without CKD. This study concludes that multiple hemolytic events may trigger CKD in SCD through the gradual loss of renovascular EPCR. Thus, the restoration of EPCR may be a therapeutic target, and plasma sEPCR can be developed as a prognostic marker for sickle CKD.
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MESH Headings
- Animals
- Anemia, Sickle Cell/complications
- Anemia, Sickle Cell/pathology
- Anemia, Sickle Cell/metabolism
- Anemia, Sickle Cell/blood
- Renal Insufficiency, Chronic/pathology
- Renal Insufficiency, Chronic/metabolism
- Renal Insufficiency, Chronic/blood
- Renal Insufficiency, Chronic/complications
- Renal Insufficiency, Chronic/etiology
- Endothelial Protein C Receptor/metabolism
- Endothelial Protein C Receptor/genetics
- Mice
- Heme/metabolism
- Humans
- Mice, Transgenic
- Male
- Female
- Hemolysis
- Kidney/metabolism
- Kidney/pathology
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Affiliation(s)
- Qiyang Chen
- Division of Cardiology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA
| | - Rimi Hazra
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
| | - Danielle Crosby
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
| | - Diane Lenhart
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
| | - Shane C. Lenhart
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
| | - Paritosh Mondal
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
| | - Yingze Zhang
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
- Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA
| | - Seyed M. Nouraie
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
- Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA
| | - Roderick J. Tan
- Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, PA
| | - Charles T. Esmon
- Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, OK
| | - L. Vijay Mohan Rao
- Department of Cellular and Molecular Biology, The University of Texas Health Science Center at Tyler, Tyler, TX
| | - Kang Kim
- Division of Cardiology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
- Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA
| | - Samit Ghosh
- Department of Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA
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9
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Zhou S, Li F, Lai Z, Wu X, Yuan J, Wu W, Ding Q, Wang X, Dai J, Xu Q, Lu Y. Met343Val mutation disrupts the shuttling of Trp380 leading to a low-activity conformer of activated protein C and causes thrombosis. J Thromb Haemost 2024; 22:2270-2280. [PMID: 38788977 DOI: 10.1016/j.jtha.2024.05.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2024] [Revised: 05/11/2024] [Accepted: 05/15/2024] [Indexed: 05/26/2024]
Abstract
BACKGROUND Protein C (PC) pathway serves as a major defense mechanism against thrombosis by the activation of PC through the thrombin-thrombomodulin complex and subsequent inactivation of the activated factor (F)V (FVa) and FVIII (FVIIIa) with the assistance of protein S, thereby contributing to hemostatic balance. We identified 2 unrelated patients who suffered from recurrent thrombosis and carried the same heterozygous mutation c.1153A>G, p.Met343Val (M343V), in PROC gene. This mutation had not been previously reported. OBJECTIVES To explore the molecular basis underlying the anticoagulant defect in patients carrying the M343V mutation in PROC. METHODS We expressed PC-M343V variant in mammalian cells and characterized its properties through coagulation assays. RESULTS Our findings demonstrated that while activation of mutant zymogen by thrombin-thrombomodulin complex was slightly affected, cleavage of chromogenic substrate by APC-M343V was significantly impaired. However, Ca2+ increased the cleavage efficiency by approximately 50%. Additionally, there was a severe reduction in affinity between APC-M343V and Na+. Furthermore, the inhibitory ability of APC-M343V toward FVa was markedly impaired. Structural and simulation analyses suggested that Val343 might disrupt the potential hydrogen bonds with Trp380 and cause Trp380 to orient closer to His211, potentially interfering with substrate binding and destabilizing the catalytic triad of APC. CONCLUSION The M343V mutation in patients adversely affects the reactivity and/or folding of the active site as well as the binding of the physiological substrate to the protease, resulting in impaired protein C anticoagulant activity and ultimately leading to thrombosis.
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Affiliation(s)
- Shijie Zhou
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Fang Li
- State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
| | - Zhe Lai
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xi Wu
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Junwei Yuan
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; College of Health Science and Technology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Wenman Wu
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Qiulan Ding
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xuefeng Wang
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Jing Dai
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
| | - Qin Xu
- State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
| | - Yeling Lu
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
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Lin CH, Tang LY, Wang LY, Chang CP. Thrombomodulin Improves Cognitive Deficits in Heat-Stressed Mice. Int J Neuropsychopharmacol 2024; 27:pyae027. [PMID: 38938182 PMCID: PMC11259854 DOI: 10.1093/ijnp/pyae027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/20/2023] [Accepted: 06/24/2024] [Indexed: 06/29/2024] Open
Abstract
BACKGROUND Thrombomodulin (TM) exerts anticoagulant and anti-inflammatory effects to improve the survival of patients with septic shock. Heat stroke resembles septic shock in many aspects. We tested whether TM would improve cognitive deficits and related causative factors in heat-stressed (HS) mice. METHODS Adult male mice were exposed to HS (33°C for 2 hours daily for 7 consecutive days) to induce cognitive deficits. Recombinant human soluble TM (1 mg/kg, i.p.) was administered immediately after the first HS trial and then once daily for 7 consecutive days. We performed the Y-maze, novel objective recognition, and passive avoidance tests to evaluate cognitive function. Plasma levels of lipopolysaccharide (LPS), high-mobility group box 1 (HMGB1), coagulation parameters, and both plasma and tissue levels of inflammatory and oxidative stress markers were biochemically measured. The duodenum and hippocampus sections were immunohistochemically stained. The intestinal and blood-brain barrier permeability were determined. RESULTS Compared with controls, HS mice treated with TM had lesser extents of cognitive deficits, exacerbated stress reactions, gut barrier disruption, endotoxemia, blood-brain barrier disruption, and inflammatory, oxidative, and coagulatory injury to heart, duodenum, and hippocampal tissues, and increased plasma HMGB1. In addition to reducing cognitive deficits, TM therapy alleviated all the abovementioned complications in heat-stressed mice. CONCLUSIONS The findings suggest that HS can lead to exacerbated stress reactions, endotoxemia, gut barrier disruption, blood-brain barrier disruption, hippocampal inflammation, coagulopathy, and oxidative stress, which may act as causative factors for cognitive deficits. TM, an anti-inflammatory, antioxidant, and anti-coagulatory agent, inhibited heat stress-induced cognitive deficits in mice.
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Affiliation(s)
- Cheng-Hsien Lin
- Department of Medicine, Mackay Medical College, New Taipei City, Taiwan
| | | | - Lin-Yu Wang
- School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Center for General Education, Southern Taiwan University of Science and TechnologyTainan, Taiwan
- Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan
- Department of Pediatrics
- Chi Mei Medical Center, Tainan, Taiwan
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11
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Chen L, Dai X. Venous thromboembolism and severe COVID-19: a Mendelian randomization trial and transcriptomic analysis. Front Immunol 2024; 15:1363598. [PMID: 38742101 PMCID: PMC11089160 DOI: 10.3389/fimmu.2024.1363598] [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: 01/02/2024] [Accepted: 04/16/2024] [Indexed: 05/16/2024] Open
Abstract
Introduction Venous thromboembolism (VTE) is known to be intricately linked to severe COVID-19 (sCOVID-19) occurrence. Herein, we employed univariable Mendelian randomization (MR) and transcriptome analysis to predict the causal association and associated signaling networks between VTE and sCOVID-19. Methods Potential VTE and sCOVID-19 association was assessed using MR-Egger, weighted median, simple mode, weighted mode, and inverse variance weighted (IVW) regression. We conducted independent univariable analyses involving VTE and sCOVID-19. Using heterogeneity, pleiotropy, and the Leave-One-Out examinations, we performed sensitivity analyses. Thereafter, we performed transcriptome analysis of the GSE164805 dataset to identify differentially expressed genes (DEGs) linked to single nucleotide polymorphisms (SNPs). Lastly, we conducted immune analyses. Results Based on our univariable analysis, VTE was a strong indicator of sCOVID-19 development, and it was intricately linked to sCOVID-19. We further conducted sensitivity analysis to demonstrate the reliability of our results. Using differential analysis, we identified 15 major genes, namely, ACSS2, CEP250, CYP4V2, DDB2, EIF6, GBGT1, GSS, MADD, MAPK8IP1, MMP24, YBPC3, NT5DC3, PROCR, SURF6, and YIPF2, which were strongly connected to suppressive adaptive immune as well as augmented inflammatory cells. In addition, we uncovered strong associations with most differential immunologic gene sets, such as, the Major Histocompatibility Complex (MHC), immunoactivators, and immunosuppressors. Conclusion Herein, we demonstrated we strong association between VTE and enhanced sCOVID-19 risk. We also identified 15 DEGs which potentially contribute to the shared immunologic pathogenesis between VTE and sCOVID-19.
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Affiliation(s)
- Liang Chen
- Department of Infectious Diseases, Taikang Xianlin Drum Tower Hospital, Affiliated Hospital of Medical College of Nanjing University, Nanjing, China
| | - Xiaoting Dai
- Department of Infectious Diseases, Nanjing Lishui People’s Hospital, Zhongda Hospital Lishui Branch, Southeast University, Nanjing, China
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12
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Paul S, Mukherjee T, Das K. Coagulation Protease-Driven Cancer Immune Evasion: Potential Targets for Cancer Immunotherapy. Cancers (Basel) 2024; 16:1568. [PMID: 38672649 PMCID: PMC11048528 DOI: 10.3390/cancers16081568] [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: 03/13/2024] [Revised: 04/16/2024] [Accepted: 04/18/2024] [Indexed: 04/28/2024] Open
Abstract
Blood coagulation and cancer are intrinsically connected, hypercoagulation-associated thrombotic complications are commonly observed in certain types of cancer, often leading to decreased survival in cancer patients. Apart from the common role in coagulation, coagulation proteases often trigger intracellular signaling in various cancers via the activation of a G protein-coupled receptor superfamily protease: protease-activated receptors (PARs). Although the role of PARs is well-established in the development and progression of certain types of cancer, their impact on cancer immune response is only just emerging. The present review highlights how coagulation protease-driven PAR signaling plays a key role in modulating innate and adaptive immune responses. This is followed by a detailed discussion on the contribution of coagulation protease-induced signaling in cancer immune evasion, thereby supporting the growth and development of certain tumors. A special section of the review demonstrates the role of coagulation proteases, thrombin, factor VIIa, and factor Xa in cancer immune evasion. Targeting coagulation protease-induced signaling might be a potential therapeutic strategy to boost the immune surveillance mechanism of a host fighting against cancer, thereby augmenting the clinical consequences of targeted immunotherapeutic regimens.
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Affiliation(s)
- Subhojit Paul
- School of Biological Sciences, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, West Bengal, India;
| | - Tanmoy Mukherjee
- Department of Cellular and Molecular Biology, The University of Texas at Tyler Health Science Center, Tyler, TX 75708, USA;
| | - Kaushik Das
- Biotechnology Research and Innovation Council-National Institute of Biomedical Genomics, Kalyani 741251, West Bengal, India
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13
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O’Hehir ZD, Lynch T, O’Neill S, March L, Xue M. Endothelial Protein C Receptor and Its Impact on Rheumatic Disease. J Clin Med 2024; 13:2030. [PMID: 38610795 PMCID: PMC11012567 DOI: 10.3390/jcm13072030] [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: 03/12/2024] [Revised: 03/28/2024] [Accepted: 03/29/2024] [Indexed: 04/14/2024] Open
Abstract
Endothelial Protein C Receptor (EPCR) is a key regulator of the activated protein C anti-coagulation pathway due to its role in the binding and activation of this protein. EPCR also binds to other ligands such as Factor VII and X, γδ T-cells, plasmodium falciparum erythrocyte membrane protein 1, and Secretory group V Phospholipases A2, facilitating ligand-specific functions. The functions of EPCR can also be regulated by soluble (s)EPCR that competes for the binding sites of membrane-bound (m)EPCR. sEPCR is created when mEPCR is shed from the cell surface. The propensity of shedding alters depending on the genetic haplotype of the EPCR gene that an individual may possess. EPCR plays an active role in normal homeostasis, anti-coagulation pathways, inflammation, and cell stemness. Due to these properties, EPCR is considered a potential effector/mediator of inflammatory diseases. Rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus are autoimmune/inflammatory conditions that are associated with elevated EPCR levels and disease activity, potentially driven by EPCR. This review highlights the functions of EPCR and its contribution to rheumatic diseases.
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Affiliation(s)
- Zachary Daniel O’Hehir
- Sutton Arthritis Research Laboratory, Sydney Musculoskeletal Health, Kolling Institute, Faculty of Medicine and Health, The University of Sydney at Royal North Shore Hospital, Sydney, NSW 2065, Australia;
| | - Tom Lynch
- The Australian Arthritis and Autoimmune Biobank Collaborative (A3BC), Institute of Bone and Joint Research, Kolling Institute, Faculty of Medicine and Health, University of Sydney at Royal North Shore Hospital, St Leonards, NSW 2065, Australia; (T.L.); (L.M.)
| | - Sean O’Neill
- Department of Rheumatology, Royal North Shore Hospital, Syndey, NSW 2065, Australia;
| | - Lyn March
- The Australian Arthritis and Autoimmune Biobank Collaborative (A3BC), Institute of Bone and Joint Research, Kolling Institute, Faculty of Medicine and Health, University of Sydney at Royal North Shore Hospital, St Leonards, NSW 2065, Australia; (T.L.); (L.M.)
- Department of Rheumatology, Royal North Shore Hospital, Syndey, NSW 2065, Australia;
| | - Meilang Xue
- Sutton Arthritis Research Laboratory, Sydney Musculoskeletal Health, Kolling Institute, Faculty of Medicine and Health, The University of Sydney at Royal North Shore Hospital, Sydney, NSW 2065, Australia;
- The Australian Arthritis and Autoimmune Biobank Collaborative (A3BC), Institute of Bone and Joint Research, Kolling Institute, Faculty of Medicine and Health, University of Sydney at Royal North Shore Hospital, St Leonards, NSW 2065, Australia; (T.L.); (L.M.)
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14
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Biswas I, Giri H, Panicker SR, Rezaie AR. Thrombomodulin Switches Signaling and Protease-Activated Receptor 1 Cleavage Specificity of Thrombin. Arterioscler Thromb Vasc Biol 2024; 44:603-616. [PMID: 38174561 PMCID: PMC10922642 DOI: 10.1161/atvbaha.123.320185] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2023] [Accepted: 12/19/2023] [Indexed: 01/05/2024]
Abstract
BACKGROUND Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin. METHODS We prepared a PAR1 knockout (PAR1-/-) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1-/- cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags. RESULTS Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a β-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both β-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by β-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin. CONCLUSIONS These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.
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Affiliation(s)
- Indranil Biswas
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation
| | - Hemant Giri
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation
| | - Sumith R. Panicker
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation
| | - Alireza R. Rezaie
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation
- Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104
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15
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Hirano K. New Mechanistic Insight Into Biased Signaling of Proteinase-Activated Receptor 1. Arterioscler Thromb Vasc Biol 2024; 44:617-619. [PMID: 38269587 DOI: 10.1161/atvbaha.123.320616] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2024]
Affiliation(s)
- Katsuya Hirano
- Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Japan
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16
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Rehill AM, Leon G, McCluskey S, Schoen I, Hernandez-Santana Y, Annett S, Klavina P, Robson T, Curtis AM, Renné T, Hussey S, O'Donnell JS, Walsh PT, Preston RJS. Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability. J Thromb Haemost 2024; 22:394-409. [PMID: 37865288 DOI: 10.1016/j.jtha.2023.10.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Revised: 10/06/2023] [Accepted: 10/06/2023] [Indexed: 10/23/2023]
Abstract
BACKGROUND Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood. OBJECTIVES We aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. METHODS We used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease. RESULTS Glycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and activated protein C generation compared with macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+ innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. CONCLUSION Collectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thromboinflammatory disease.
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Affiliation(s)
- Aisling M Rehill
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland; National Children's Research Centre, Children's Health Ireland Crumlin, Dublin, Ireland. https://twitter.com/aislingrehill
| | - Gemma Leon
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland; National Children's Research Centre, Children's Health Ireland Crumlin, Dublin, Ireland
| | - Sean McCluskey
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland; National Children's Research Centre, Children's Health Ireland Crumlin, Dublin, Ireland
| | - Ingmar Schoen
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland
| | - Yasmina Hernandez-Santana
- National Children's Research Centre, Children's Health Ireland Crumlin, Dublin, Ireland; Department of Clinical Medicine, Trinity Translational Medicine Institute, Trinity College Dublin, Ireland
| | - Stephanie Annett
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland
| | - Paula Klavina
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland
| | - Tracy Robson
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland
| | - Annie M Curtis
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland
| | - Thomas Renné
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland; Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Seamus Hussey
- National Children's Research Centre, Children's Health Ireland Crumlin, Dublin, Ireland; Department of Paediatrics, University College Dublin and Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland
| | - James S O'Donnell
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland
| | - Patrick T Walsh
- National Children's Research Centre, Children's Health Ireland Crumlin, Dublin, Ireland; Department of Clinical Medicine, Trinity Translational Medicine Institute, Trinity College Dublin, Ireland
| | - Roger J S Preston
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland; National Children's Research Centre, Children's Health Ireland Crumlin, Dublin, Ireland.
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Dai C, Lin X, Qi Y, Wang Y, Lv Z, Zhao F, Deng Z, Feng X, Zhang T, Pu X. Vitamin D3 improved hypoxia-induced lung injury by inhibiting the complement and coagulation cascade and autophagy pathway. BMC Pulm Med 2024; 24:9. [PMID: 38166725 PMCID: PMC10759436 DOI: 10.1186/s12890-023-02784-y] [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: 03/02/2023] [Accepted: 11/23/2023] [Indexed: 01/05/2024] Open
Abstract
BACKGROUND Pulmonary metabolic dysfunction can cause lung tissue injury. There is still no ideal drug to protect against hypoxia-induced lung injury, therefore, the development of new drugs to prevent and treat hypoxia-induced lung injury is urgently needed. We aimed to explore the ameliorative effects and molecular mechanisms of vitamin D3 (VD3) on hypoxia-induced lung tissue injury. METHODS Sprague-Dawley (SD) rats were randomly divided into three groups: normoxia, hypoxia, and hypoxia + VD3. The rat model of hypoxia was established by placing the rats in a hypobaric chamber. The degree of lung injury was determined using hematoxylin and eosin (H&E) staining, lung water content, and lung permeability index. Transcriptome data were subjected to differential gene expression and pathway analyses. In vitro, type II alveolar epithelial cells were co-cultured with hepatocytes and then exposed to hypoxic conditions for 24 h. For VD3 treatment, the cells were treated with low and high concentrations of VD3. RESULTS Transcriptome and KEGG analyses revealed that VD3 affects the complement and coagulation cascade pathways in hypoxia-induced rats, and the genes enriched in this pathway were Fgb/Fga/LOC100910418. Hypoxia can cause increases in lung edema, inflammation, and lung permeability disruption, which are attenuated by VD3 treatment. VD3 weakened the complement and coagulation cascade in the lung and liver of hypoxia-induced rats, characterized by lower expression of fibrinogen alpha chain (Fga), fibrinogen beta chain (Fgb), protease-activated receptor 1 (PAR1), protease-activated receptor 3 (PAR3), protease-activated receptor 4 (PAR4), complement (C) 3, C3a, and C5. In addition, VD3 improved hypoxic-induced type II alveolar epithelial cell damage and inflammation by inhibiting the complement and coagulation cascades. Furthermore, VD3 inhibited hypoxia-induced autophagy in vivo and in vitro, which was abolished by the mitophagy inducer, carbonyl cyanide-m-chlorophenylhydrazone (CCCP). CONCLUSION VD3 alleviated hypoxia-induced pulmonary edema by inhibiting the complement and coagulation cascades and autophagy pathways.
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Affiliation(s)
- Chongyang Dai
- Qinghai University, Xining, Qinghai Province, 810016, People's Republic of China
| | - Xue Lin
- West China Hospital, Sichuan University, Chengdu, Sichuan Province, 610000, People's Republic of China
| | - Yinglian Qi
- Qinghai Normal University, Xining, Qinghai Province, 810008, People's Republic of China
| | - Yaxuan Wang
- Qinghai University, Xining, Qinghai Province, 810016, People's Republic of China
| | - Zhongkui Lv
- Qinghai University, Xining, Qinghai Province, 810016, People's Republic of China
| | - Fubang Zhao
- Qinghai University, Xining, Qinghai Province, 810016, People's Republic of China
| | - Zhangchang Deng
- Qinghai University, Xining, Qinghai Province, 810016, People's Republic of China
| | - Xiaokai Feng
- Department of Pulmonary and Critical Care Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, 100020, People's Republic of China.
- Department of Respiratory and Critical Care Medicine, Qinghai Provincial People's Hospital, Qinghai University, Xining, Qinghai Province, 810007, People's Republic of China.
| | - Tongzuo Zhang
- Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, Qinghai Province, 810001, People's Republic of China.
| | - Xiaoyan Pu
- Qinghai University, Xining, Qinghai Province, 810016, People's Republic of China.
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Thielen O, Mitra S, Debot M, Schaid T, Hallas W, Gallagher LT, Erickson C, Cralley A, Stafford P, Silliman C, D'Alessandro A, Hansen K, Sauaia A, Moore E, Mosnier L, Griffin J, Cohen M. Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation. J Trauma Acute Care Surg 2024; 96:116-122. [PMID: 37733304 PMCID: PMC10841096 DOI: 10.1097/ta.0000000000004142] [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] [Indexed: 09/22/2023]
Abstract
BACKGROUND Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability. METHODS We used electric cell-substrate impedance sensing to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 μg/mL solution of 3K3A-aPC at 180 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma [TP]). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey's multiple comparison's test. RESULTS Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pretreatment group was higher than TP alone (mean diff. 22.12 95% CI [13.75, 30.49], p < 0.0001) (Figure). Moreover, the AUC of the 60-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval [CI], 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01). CONCLUSION Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function. LEVEL OF EVIDENCE Prognostic and Epidemiological; Level III.
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Affiliation(s)
- Otto Thielen
- From the Department of Gastrointestinal, Trauma, and Endocrine Surgery (O.T., S.M., M.D., T.S., W.H., L.T.G., C.E., A.C., P.S., C.S., A.D'A., K.H., A.S., E.M., M.C.), University of Colorado, Denver, Colorado; Department of Surgery (A.S., E.M.), Denver Health Medical Center, Denver, Colorado; and Department of Molecular Medicine (L.M., J.G.), Scripps Research, La Jolla, California
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Alshaya DS, Alzamil LA, Alghamdi AS, Alshammari LS, Alhowaiti SA, Bohol MFF, Abudouleh E, Alhamlan F, Al-Qahtani AA, Al Hazzani A, Owaidah T, Al-Qahtani AA. Frequency and Association of Polymorphisms in F2, F7, and PROS1 Coagulation Genes with Disease Severity in Coronavirus Disease 2019. Clin Appl Thromb Hemost 2024; 30:10760296241295731. [PMID: 39497411 PMCID: PMC11536613 DOI: 10.1177/10760296241295731] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2024] [Revised: 09/24/2024] [Accepted: 10/14/2024] [Indexed: 11/07/2024] Open
Abstract
Abnormal transcriptomic profiles of coagulation genes have been linked to coagulopathies in patients with coronavirus disease 2019 (COVID-19). The objective of the present study was to explore the frequency of genotypes and potential association of polymorphisms in genes encoding coagulation factors with the disease severity in COVID-19 patients.The patients were clinically categorized into four groups of COVID-19 disease severity (asymptomatic, mild, moderate, and severe). Three variants of genes, involving the coagulation genes rs3136516 (F2 gene), rs6042 (F7 gene), and rs6123 (PROS1 gene), were studied. Polymorphisms were genotyped by Sanger DNA sequencing.Most of the subjects had moderate COVID-19 infection (n = 53, 62.4%), followed by mild (n = 16, 18.8%), and severe infections (n = 15, 17.6%). The frequency of the rs3136516 AG genotype was considerably higher in non-ICU patients compared to ICU patients (51.3% vs 34.1%, OR 3.167, 95% CI 1.094-9.170, P = .031). Furthermore, the dominant genetic model (AA + AG vs GG) was significantly associated with a decreased probability of admission to the ICU in COVID-19 patients (OR 0.340, 95% CI 0.127 - 0.905, and P = .028). No other variants of the coagulation genes studied were found to be associated with the severity of COVID-19 disease, admission to the ICU, and mortality (P > .05).The rs3136516 AG genotype could predispose COVID-19 patients to increased disease severity and therefore admission to the ICU, while the dominant genetic model (AA + AG vs GG) of rs3136516 exerts a protective role.
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Affiliation(s)
- Dalal Sulaiman Alshaya
- Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
| | - Lama A. Alzamil
- Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
| | - Alanoud S. Alghamdi
- Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
| | - Lolwah S. Alshammari
- Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
| | - Saltana A. Alhowaiti
- Department of Infection and Immunity, Research Centre, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia
| | - Marie Fe F. Bohol
- Department of Infection and Immunity, Research Centre, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia
| | - Esra’a Abudouleh
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia
| | - Fatimah Alhamlan
- Department of Infection and Immunity, Research Centre, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia
- Department of Microbiology and Immunology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia
| | - Arwa A. Al-Qahtani
- Department of Family Medicine, College of Medicine, Al-Imam Mohammad Ibn Saud Islamic University, Riyadh, Saudi Arabia
| | - Amal Al Hazzani
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia
| | - Tarek Owaidah
- Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
- Department of Pathology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia
| | - Ahmed A. Al-Qahtani
- Department of Infection and Immunity, Research Centre, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia
- Department of Microbiology and Immunology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia
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20
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Zhao R, Xue M, Lin H, Smith M, Liang H, Weiler H, Griffin JH, Jackson CJ. A recombinant signalling-selective activated protein C that lacks anticoagulant activity is efficacious and safe in cutaneous wound preclinical models. Wound Repair Regen 2024; 32:90-103. [PMID: 38155595 DOI: 10.1111/wrr.13148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2023] [Revised: 12/09/2023] [Accepted: 12/18/2023] [Indexed: 12/30/2023]
Abstract
Various preclinical and clinical studies have demonstrated the robust wound healing capacity of the natural anticoagulant activated protein C (APC). A bioengineered APC variant designated 3K3A-APC retains APC's cytoprotective cell signalling actions with <10% anticoagulant activity. This study was aimed to provide preclinical evidence that 3K3A-APC is efficacious and safe as a wound healing agent. 3K3A-APC, like wild-type APC, demonstrated positive effects on proliferation of human skin cells (keratinocytes, endothelial cells and fibroblasts). Similarly it also increased matrix metollaproteinase-2 activation in keratinocytes and fibroblasts. Topical 3K3A-APC treatment at 10 or 30 μg both accelerated mouse wound healing when culled on Day 11. And at 10 μg, it was superior to APC and had half the dermal wound gape compared to control. Further testing was conducted in excisional porcine wounds due to their congruence to human skin. Here, 3K3A-APC advanced macroscopic healing in a dose-dependent manner (100, 250 and 500 μg) when culled on Day 21. This was histologically corroborated by greater collagen maturity, suggesting more advanced remodelling. A non-interference arm of this study found no evidence that topical 3K3A-APC caused either any significant systemic side-effects or any significant leakage into the circulation. However the female pigs exhibited transient and mild local reactions after treatments in week three, which did not impact healing. Overall these preclinical studies support the hypothesis that 3K3A-APC merits future human wound studies.
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Affiliation(s)
- Ruilong Zhao
- Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
| | - Meilang Xue
- Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
| | - Haiyan Lin
- Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
| | - Margaret Smith
- Raymond Purves Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
| | - Helena Liang
- Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
| | - Hartmut Weiler
- Department of Physiology, Blood Research Institute, Milwaukee, Wisconsin, USA
| | - John H Griffin
- Department of Molecular Medicine, The Scripps Research Institute, San Diego, California, USA
| | - Christopher J Jackson
- Sutton Laboratory, Kolling Institute of Medical Research, Sydney, New South Wales, Australia
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21
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Julovi SM, McKelvey K, Minhas N, Chan YKA, Xue M, Jackson CJ. Involvement of PAR-2 in the Induction of Cell-Specific Matrix Metalloproteinase-2 by Activated Protein C in Cutaneous Wound Healing. Int J Mol Sci 2023; 25:370. [PMID: 38203540 PMCID: PMC10779272 DOI: 10.3390/ijms25010370] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2023] [Revised: 12/23/2023] [Accepted: 12/23/2023] [Indexed: 01/12/2024] Open
Abstract
We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.
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Affiliation(s)
- Sohel M. Julovi
- Sutton Arthritis Research Laboratory, Kolling Institute of Medical Research, University of Sydney, Pacific Highway, St. Leonards, NSW 2065, Australia; (K.M.); (N.M.); (Y.-K.A.C.); (M.X.); (C.J.J.)
- Kidney Injury Group, Centre for Transplant and Renal Research, Westmead Institute for Medical Research, Westmead, NSW 2045, Australia
| | - Kelly McKelvey
- Sutton Arthritis Research Laboratory, Kolling Institute of Medical Research, University of Sydney, Pacific Highway, St. Leonards, NSW 2065, Australia; (K.M.); (N.M.); (Y.-K.A.C.); (M.X.); (C.J.J.)
| | - Nikita Minhas
- Sutton Arthritis Research Laboratory, Kolling Institute of Medical Research, University of Sydney, Pacific Highway, St. Leonards, NSW 2065, Australia; (K.M.); (N.M.); (Y.-K.A.C.); (M.X.); (C.J.J.)
| | - Yee-Ka Agnes Chan
- Sutton Arthritis Research Laboratory, Kolling Institute of Medical Research, University of Sydney, Pacific Highway, St. Leonards, NSW 2065, Australia; (K.M.); (N.M.); (Y.-K.A.C.); (M.X.); (C.J.J.)
| | - Meilang Xue
- Sutton Arthritis Research Laboratory, Kolling Institute of Medical Research, University of Sydney, Pacific Highway, St. Leonards, NSW 2065, Australia; (K.M.); (N.M.); (Y.-K.A.C.); (M.X.); (C.J.J.)
| | - Christopher J. Jackson
- Sutton Arthritis Research Laboratory, Kolling Institute of Medical Research, University of Sydney, Pacific Highway, St. Leonards, NSW 2065, Australia; (K.M.); (N.M.); (Y.-K.A.C.); (M.X.); (C.J.J.)
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22
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O'Donnell JS, Fleming H, Noone D, Preston RJS. Unraveling coagulation factor-mediated cellular signaling. J Thromb Haemost 2023; 21:3342-3353. [PMID: 37391097 DOI: 10.1016/j.jtha.2023.06.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 05/15/2023] [Accepted: 06/12/2023] [Indexed: 07/02/2023]
Abstract
Blood coagulation is initiated in response to blood vessel injury or proinflammatory stimuli, which activate coagulation factors to coordinate complex biochemical and cellular responses necessary for clot formation. In addition to these critical physiologic functions, plasma protein factors activated during coagulation mediate a spectrum of signaling responses via receptor-binding interactions on different cell types. In this review, we describe examples and mechanisms of coagulation factor signaling. We detail the molecular basis for cell signaling mediated by coagulation factor proteases via the protease-activated receptor family, considering new insights into the role of protease-specific cleavage sites, cofactor and coreceptor interactions, and distinct signaling intermediate interactions in shaping protease-activated receptor signaling diversity. Moreover, we discuss examples of how injury-dependent conformational activation of other coagulation proteins, such as fibrin(ogen) and von Willebrand factor, decrypts their signaling potential, unlocking their capacity to contribute to aberrant proinflammatory signaling. Finally, we consider the role of coagulation factor signaling in disease development and the status of pharmacologic approaches to either attenuate or enhance coagulation factor signaling for therapeutic benefit, emphasizing new approaches to inhibit deleterious coagulation factor signaling without impacting hemostatic activity.
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Affiliation(s)
- James S O'Donnell
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland; National Children's Research Centre, Children's Health Ireland, Crumlin, Dublin, Ireland. https://twitter.com/profJSOdonnell
| | - Harry Fleming
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland. https://www.twitter.com/PrestonLab_RCSI
| | - David Noone
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland. https://www.twitter.com/PrestonLab_RCSI
| | - Roger J S Preston
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland; National Children's Research Centre, Children's Health Ireland, Crumlin, Dublin, Ireland.
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23
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Zoungrana LI, Krause-Hauch M, Wang H, Fatmi MK, Li Z, Slotabec L, James AS, Didik S, Li J. Activated protein C signaling mediates neuroinflammation in seizure induced by pilocarpine. Biochem Biophys Rep 2023; 35:101550. [PMID: 37823005 PMCID: PMC10562752 DOI: 10.1016/j.bbrep.2023.101550] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2023] [Revised: 09/18/2023] [Accepted: 09/25/2023] [Indexed: 10/13/2023] Open
Abstract
Epilepsy is one of the most common and oldest neurological disorders, characterized by periodic seizures that affect millions globally. Despite its long history, its pathophysiology is not fully understood. Additionally, the current treatment methods have their limitations. Finding a new alternative is necessary. Activated Protein C (APC) has been proven to have neurological protection in other neurological disorders; however, there is no study that focuses on the role of APC in seizures. We propose that APC's protective effect could be associated with seizures through inflammation and apoptosis regulation. The results demonstrated that APC's pathway proteins are involved in neuroprotection mechanisms in seizure-induced models by acting on certain inflammatory factors, such as NF-κB and apoptosis proteins.
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Affiliation(s)
- Linda Ines Zoungrana
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, USA
| | - Meredith Krause-Hauch
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, USA
| | - Hao Wang
- Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS, 39216, USA
| | - Mohammad Kasim Fatmi
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, USA
| | - Zehui Li
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, USA
| | - Lily Slotabec
- Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS, 39216, USA
| | - Adewale Segun James
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, USA
| | - Steven Didik
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, USA
| | - Ji Li
- Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS, 39216, USA
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24
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Lin DS, Trumpp A. Differential expression of endothelial protein C receptor (EPCR) in hematopoietic stem and multipotent progenitor cells in young and old mice. Cells Dev 2023; 174:203843. [PMID: 37080459 DOI: 10.1016/j.cdev.2023.203843] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 03/26/2023] [Accepted: 04/13/2023] [Indexed: 04/22/2023]
Abstract
Endothelial protein C receptor (EPCR) has emerged as one of the most conserved and reliable surface markers for the prospective identification and isolation of hematopoietic stem cells (HSCs). Prior studies have consistently demonstrated that EPCR expression enriches HSCs capable of long-term multilineage repopulation in both mouse and human across different hematopoietic tissues, including bone marrow (BM), fetal liver and ex vivo HSC expansion cultures. However, little is known about the expression profiles of EPCR in multipotent progenitor (MPP) populations located immediately downstream of HSCs in the hematopoietic hierarchy and which play a major role in sustaining lifelong blood cell production. Here, we incorporate EPCR antibody detection into a multi-parameter flow cytometric panel, which allows accurate identification of HSCs and five MPP subsets (MPP1-5) in mouse BM. Our data reveal that all MPP populations contain EPCR-expressing cells. Multipotent MPP1 and MPP5 contain higher proportion of EPCR+ cells compared to the more lineage-biased MPP2-4. Notably, high expression of EPCR enriches phenotypic HSC and MPP5, but not MPP1. Comparison of EPCR expression profiles between young and old BM reveals ageing mediated expansion of EPCR-expressing cells only in HSCs, but not in any of the MPP populations. Collectively, our study provides a comprehensive characterization of the surface expression pattern of EPCR in mouse HSC and MPP1-5 cells during normal and aged hematopoiesis.
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Affiliation(s)
- Dawn S Lin
- Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany; Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany
| | - Andreas Trumpp
- Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany; Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany.
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25
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Giri H, Biswas I, Rezaie AR. Activated protein C inhibits mesothelial-to-mesenchymal transition in experimental peritoneal fibrosis. J Thromb Haemost 2023; 21:133-144. [PMID: 36695376 PMCID: PMC10726528 DOI: 10.1016/j.jtha.2022.10.012] [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: 07/28/2022] [Revised: 10/14/2022] [Accepted: 10/25/2022] [Indexed: 01/11/2023]
Abstract
BACKGROUND In addition to its anticoagulant function in downregulating thrombin generation, activated protein C (APC) evokes pleiotropic cytoprotective signaling activities when it binds to endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) in endothelial cells. OBJECTIVES To investigate the protective effect of APC in a chlorhexidine gluconate (CG)-induced peritoneal fibrosis model. METHODS Peritoneal fibrosis was induced in wild-type as well as EPCR- and PAR1-deficient mice via daily injection of CG (0.2 mL of 0.1% CG in 15% ethanol and 85% saline) for 21 days with or without concomitant injection of recombinant human APC derivatives (50 μg/kg of bodyweight). The expression of proinflammatory cytokines and profibrotic markers as well as collagen deposition were analyzed using established methods. RESULTS CG significantly upregulated the expression of transforming growth factor-β1 in peritoneal tissues, which culminated in the deposition of excessive extracellular matrix proteins, thickening of the peritoneal membrane, and mesothelial-to-mesenchymal transition in damaged tissues. APC potently inhibited CG-induced peritoneal fibrosis and downregulated the expression of proinflammatory cytokines, collagen deposition, Smad3 phosphorylation, and markers of mesothelial-to-mesenchymal transition (α-smooth muscle actin, vimentin, and N-cadherin). APC also inhibited transforming growth factor-β1-mediated upregulation of α-smooth muscle actin, Smad3, and fibronectin in human primary mesothelial cells. Employing signaling-selective and anticoagulant-selective variants of APC and mutant mice deficient for either EPCR or PAR1, we demonstrated that the EPCR-dependent signaling function of APC through PAR1 activation was primarily responsible for its antifibrotic activity in the CG-induced peritoneal fibrosis model. CONCLUSION APC and signaling-selective variants of APC may have therapeutic potential for preventing or treating pathologies associated with peritoneal fibrosis.
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Affiliation(s)
- Hemant Giri
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA
| | - Indranil Biswas
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA
| | - Alireza R Rezaie
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
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26
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Schwarz N, Müller J, Yadegari H, McRae HL, Reda S, Hamedani NS, Oldenburg J, Pötzsch B, Rühl H. Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach. Arterioscler Thromb Vasc Biol 2023; 43:109-119. [PMID: 36353988 DOI: 10.1161/atvbaha.122.318433] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
BACKGROUND The endothelial cell-dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation. METHODS Endothelial colony-forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC (activated PC) formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of TM (thrombomodulin) expression, ECFCs were stimulated with IL-1β (interleukin 1β). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII). RESULTS The median peak APC concentration was 1.12 nmol/L in experiments with IL-1β stimulated ECFCs and 3.66 nmol/L without IL-1β. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031, P=0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo. CONCLUSIONS Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.
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Affiliation(s)
- Nadine Schwarz
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
| | - Jens Müller
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
| | - Hamideh Yadegari
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
| | - Hannah L McRae
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
| | - Sara Reda
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
| | - Nasim Shahidi Hamedani
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
| | - Johannes Oldenburg
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
| | - Bernd Pötzsch
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
| | - Heiko Rühl
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Germany
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27
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Peach CJ, Edgington-Mitchell LE, Bunnett NW, Schmidt BL. Protease-activated receptors in health and disease. Physiol Rev 2023; 103:717-785. [PMID: 35901239 PMCID: PMC9662810 DOI: 10.1152/physrev.00044.2021] [Citation(s) in RCA: 46] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Revised: 07/08/2022] [Accepted: 07/10/2022] [Indexed: 11/22/2022] Open
Abstract
Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.
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Affiliation(s)
- Chloe J Peach
- Department of Molecular Pathobiology, College of Dentistry, New York University, New York, New York
- Department of Neuroscience and Physiology and Neuroscience Institute, Grossman School of Medicine, New York University, New York, New York
| | - Laura E Edgington-Mitchell
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, New York, New York
| | - Nigel W Bunnett
- Department of Molecular Pathobiology, College of Dentistry, New York University, New York, New York
- Department of Neuroscience and Physiology and Neuroscience Institute, Grossman School of Medicine, New York University, New York, New York
| | - Brian L Schmidt
- Department of Molecular Pathobiology, College of Dentistry, New York University, New York, New York
- Bluestone Center for Clinical Research, Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, New York, New York
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28
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Dysregulated haemostasis in thrombo-inflammatory disease. Clin Sci (Lond) 2022; 136:1809-1829. [PMID: 36524413 PMCID: PMC9760580 DOI: 10.1042/cs20220208] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2022] [Revised: 11/17/2022] [Accepted: 11/25/2022] [Indexed: 12/23/2022]
Abstract
Inflammatory disease is often associated with an increased incidence of venous thromboembolism in affected patients, although in most instances, the mechanistic basis for this increased thrombogenicity remains poorly understood. Acute infection, as exemplified by sepsis, malaria and most recently, COVID-19, drives 'immunothrombosis', where the immune defence response to capture and neutralise invading pathogens causes concurrent activation of deleterious prothrombotic cellular and biological responses. Moreover, dysregulated innate and adaptive immune responses in patients with chronic inflammatory conditions, such as inflammatory bowel disease, allergies, and neurodegenerative disorders, are now recognised to occur in parallel with activation of coagulation. In this review, we describe the detailed cellular and biochemical mechanisms that cause inflammation-driven haemostatic dysregulation, including aberrant contact pathway activation, increased tissue factor activity and release, innate immune cell activation and programmed cell death, and T cell-mediated changes in thrombus resolution. In addition, we consider how lifestyle changes increasingly associated with modern life, such as circadian rhythm disruption, chronic stress and old age, are increasingly implicated in unbalancing haemostasis. Finally, we describe the emergence of potential therapies with broad-ranging immunothrombotic functions, and how drug development in this area is challenged by our nascent understanding of the key molecular and cellular parameters that control the shared nodes of proinflammatory and procoagulant pathways. Despite the increasing recognition and understanding of the prothrombotic nature of inflammatory disease, significant challenges remain in effectively managing affected patients, and new therapeutic approaches to curtail the key pathogenic steps in immune response-driven thrombosis are urgently required.
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29
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Miceli G, Basso MG, Rizzo G, Pintus C, Tuttolomondo A. The Role of the Coagulation System in Peripheral Arterial Disease: Interactions with the Arterial Wall and Its Vascular Microenvironment and Implications for Rational Therapies. Int J Mol Sci 2022; 23:14914. [PMID: 36499242 PMCID: PMC9739112 DOI: 10.3390/ijms232314914] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Revised: 11/19/2022] [Accepted: 11/23/2022] [Indexed: 11/30/2022] Open
Abstract
Peripheral artery disease (PAD) is a clinical manifestation of atherosclerotic disease with a large-scale impact on the economy and global health. Despite the role played by platelets in the process of atherogenesis being well recognized, evidence has been increasing on the contribution of the coagulation system to the atherosclerosis formation and PAD development, with important repercussions for the therapeutic approach. Histopathological analysis and some clinical studies conducted on atherosclerotic plaques testify to the existence of different types of plaques. Likely, the role of coagulation in each specific type of plaque can be an important determinant in the histopathological composition of atherosclerosis and in its future stability. In this review, we analyze the molecular contribution of inflammation and the coagulation system on PAD pathogenesis, focusing on molecular similarities and differences between atherogenesis in PAD and coronary artery disease (CAD) and discussing the possible implications for current therapeutic strategies and future perspectives accounting for molecular inflammatory and coagulation targets. Understanding the role of cross-talking between coagulation and inflammation in atherosclerosis genesis and progression could help in choosing the right patients for future dual pathway inhibition strategies, where an antiplatelet agent is combined with an anticoagulant, whose role, despite pathophysiological premises and trials' results, is still under debate.
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Affiliation(s)
- Giuseppe Miceli
- Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties (ProMISE), Università degli Studi di Palermo, Piazza delle Cliniche 2, 90127 Palermo, Italy
- Internal Medicine and Stroke Care Ward, University Hospital Policlinico “P. Giaccone”, 90100 Palermo, Italy
| | - Maria Grazia Basso
- Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties (ProMISE), Università degli Studi di Palermo, Piazza delle Cliniche 2, 90127 Palermo, Italy
- Internal Medicine and Stroke Care Ward, University Hospital Policlinico “P. Giaccone”, 90100 Palermo, Italy
| | - Giuliana Rizzo
- Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties (ProMISE), Università degli Studi di Palermo, Piazza delle Cliniche 2, 90127 Palermo, Italy
- Internal Medicine and Stroke Care Ward, University Hospital Policlinico “P. Giaccone”, 90100 Palermo, Italy
| | - Chiara Pintus
- Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties (ProMISE), Università degli Studi di Palermo, Piazza delle Cliniche 2, 90127 Palermo, Italy
- Internal Medicine and Stroke Care Ward, University Hospital Policlinico “P. Giaccone”, 90100 Palermo, Italy
| | - Antonino Tuttolomondo
- Department of Health Promotion, Mother and Child Care, Internal Medicine and Medical Specialties (ProMISE), Università degli Studi di Palermo, Piazza delle Cliniche 2, 90127 Palermo, Italy
- Internal Medicine and Stroke Care Ward, University Hospital Policlinico “P. Giaccone”, 90100 Palermo, Italy
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30
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Rix B, Maduro AH, Bridge KS, Grey W. Markers for human haematopoietic stem cells: The disconnect between an identification marker and its function. Front Physiol 2022; 13:1009160. [PMID: 36246104 PMCID: PMC9564379 DOI: 10.3389/fphys.2022.1009160] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2022] [Accepted: 08/22/2022] [Indexed: 11/13/2022] Open
Abstract
The haematopoietic system is a classical stem cell hierarchy that maintains all the blood cells in the body. Haematopoietic stem cells (HSCs) are rare, highly potent cells that reside at the apex of this hierarchy and are historically some of the most well studied stem cells in humans and laboratory models, with haematopoiesis being the original system to define functional cell types by cell surface markers. Whilst it is possible to isolate HSCs to near purity, we know very little about the functional activity of markers to purify HSCs. This review will focus on the historical efforts to purify HSCs in humans based on cell surface markers, their putative functions and recent advances in finding functional markers on HSCs.
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Affiliation(s)
| | | | | | - William Grey
- *Correspondence: Katherine S. Bridge, ; William Grey,
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Erausquin E, Morán-Garrido M, Sáiz J, Barbas C, Dichiara-Rodríguez G, Urdiciain A, López-Sagaseta J. Identification of a broad lipid repertoire associated to the endothelial cell protein C receptor (EPCR). Sci Rep 2022; 12:15127. [PMID: 36068249 PMCID: PMC9448719 DOI: 10.1038/s41598-022-18844-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2022] [Accepted: 08/22/2022] [Indexed: 11/09/2022] Open
Abstract
Evidence is mounting that the nature of the lipid bound to the endothelial cell protein C receptor (EPCR) has an impact on its biological roles, as observed in anticoagulation and more recently, in autoimmune disease. Phosphatidylethanolamine and phosphatidylcholine species dominate the EPCR lipid cargo, yet, the extent of diversity in the EPCR-associated lipid repertoire is still unknown and remains to be uncovered. We undertook mass spectrometry analyses to decipher the EPCR lipidome, and identified species not yet described as EPCR ligands, such as phosphatidylinositols and phosphatidylserines. Remarkably, we found further, more structurally divergent lipids classes, represented by ceramides and sphingomyelins, both in less abundant quantities. In support of our mass spectrometry results and previous studies, high-resolution crystal structures of EPCR in three different space groups point to a prevalent diacyl phospholipid moiety in EPCR’s pocket but a mobile and ambiguous lipid polar head group. In sum, these studies indicate that EPCR can associate with varied lipid classes, which might impact its properties in anticoagulation and the onset of autoimmune disease.
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Affiliation(s)
- Elena Erausquin
- Unit of Protein Crystallography and Structural Immunology, Navarrabiomed, 31008, Pamplona, Navarra, Spain.,Public University of Navarra (UPNA), 31008, Pamplona, Navarra, Spain.,Navarra University Hospital, 31008, Pamplona, Navarra, Spain
| | - María Morán-Garrido
- Centre of Metabolomics and Bioanalysis (CEMBIO), Department of Chemistry and Biochemistry, School of Pharmacy, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660, Boadilla del Monte, Spain
| | - Jorge Sáiz
- Centre of Metabolomics and Bioanalysis (CEMBIO), Department of Chemistry and Biochemistry, School of Pharmacy, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660, Boadilla del Monte, Spain
| | - Coral Barbas
- Centre of Metabolomics and Bioanalysis (CEMBIO), Department of Chemistry and Biochemistry, School of Pharmacy, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660, Boadilla del Monte, Spain
| | - Gilda Dichiara-Rodríguez
- Unit of Protein Crystallography and Structural Immunology, Navarrabiomed, 31008, Pamplona, Navarra, Spain.,Public University of Navarra (UPNA), 31008, Pamplona, Navarra, Spain.,Navarra University Hospital, 31008, Pamplona, Navarra, Spain
| | - Alejandro Urdiciain
- Unit of Protein Crystallography and Structural Immunology, Navarrabiomed, 31008, Pamplona, Navarra, Spain.,Public University of Navarra (UPNA), 31008, Pamplona, Navarra, Spain.,Navarra University Hospital, 31008, Pamplona, Navarra, Spain
| | - Jacinto López-Sagaseta
- Unit of Protein Crystallography and Structural Immunology, Navarrabiomed, 31008, Pamplona, Navarra, Spain. .,Public University of Navarra (UPNA), 31008, Pamplona, Navarra, Spain. .,Navarra University Hospital, 31008, Pamplona, Navarra, Spain.
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Abstract
PURPOSE OF REVIEW To provide an overview of the state-of-the-art in protein C (PC) pathway research. RECENT FINDINGS The PC pathway is crucial for maintaining hemostasis to prevent venous thromboembolism. This is evident from genetic mutations that result in impaired PC pathway activity and contribute to increased venous thromboembolism risk in affected individuals. In addition to its anticoagulant role, activated PC (APC) also mediates a complex, pleiotropic role in the maintenance of vascular cell health, which it achieves via anti-inflammatory and antiapoptotic cell signaling on endothelial cells. Emerging data have demonstrated that cell signaling by APC, mediated by multiple receptor interactions on different cell types, also confers cytoprotective and anti-inflammatory benefits. Defects in both arms of the PC pathway are associated with increased susceptibility to thrombo-inflammatory disease in various preclinical thrombotic, proinflammatory and neurological disease models. Moreover, recent studies have identified attenuation of anticoagulant PC pathway activity as an exciting therapeutic opportunity to promote hemostasis in patients with inherited or acquired bleeding disorders. SUMMARY In this review, we provide an overview of some recent developments in our understanding of the PC pathways.
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Affiliation(s)
- Gemma Leon
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland
- National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin 12, Ireland
| | - Aisling M Rehill
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland
| | - Roger J S Preston
- Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland
- National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Crumlin, Dublin 12, Ireland
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Bochenek ML, Gogiraju R, Großmann S, Krug J, Orth J, Reyda S, Georgiadis GS, Spronk H, Konstantinides S, Münzel T, Griffin JH, Wild PS, Espinola-Klein C, Ruf W, Schäfer K. EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia. JCI Insight 2022; 7:157701. [PMID: 35700057 PMCID: PMC9431695 DOI: 10.1172/jci.insight.157701] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Accepted: 06/08/2022] [Indexed: 11/17/2022] Open
Abstract
Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation, as well as immune and vascular signaling, by protease activated receptors (PARs). Here, we show that endothelial EPCR-PAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCR-PAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR-PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.
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Affiliation(s)
- Magdalena L Bochenek
- Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
| | | | - Stefanie Großmann
- Department of Cardiology, University Medical Center Mainz, Mainz, Germany
| | - Janina Krug
- Department of Cardiology, University Medical Center Mainz, Mainz, Germany
| | - Jennifer Orth
- Department of Cardiology, University Medical Center Mainz, Mainz, Germany
| | - Sabine Reyda
- Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
| | - George S Georgiadis
- Department of Vascular Surgery, University Hospital of Alexandroupolis, Alexandroupolis, Greece
| | - Henri Spronk
- CARIM School for Cardiovascular Disease, Maastricht University, Maastricht, Netherlands
| | | | - Thomas Münzel
- Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
| | - John H Griffin
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, United States of America
| | - Philipp S Wild
- Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
| | | | - Wolfram Ruf
- Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
| | - Katrin Schäfer
- Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
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Luxen M, van Meurs M, Molema G. Unlocking the Untapped Potential of Endothelial Kinase and Phosphatase Involvement in Sepsis for Drug Treatment Design. Front Immunol 2022; 13:867625. [PMID: 35634305 PMCID: PMC9136877 DOI: 10.3389/fimmu.2022.867625] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2022] [Accepted: 03/28/2022] [Indexed: 11/29/2022] Open
Abstract
Sepsis is a devastating clinical condition that can lead to multiple organ failure and death. Despite advancements in our understanding of molecular mechanisms underlying sepsis and sepsis-associated multiple organ failure, no effective therapeutic treatment to directly counteract it has yet been established. The endothelium is considered to play an important role in sepsis. This review highlights a number of signal transduction pathways involved in endothelial inflammatory activation and dysregulated endothelial barrier function in response to sepsis conditions. Within these pathways – NF-κB, Rac1/RhoA GTPases, AP-1, APC/S1P, Angpt/Tie2, and VEGF/VEGFR2 – we focus on the role of kinases and phosphatases as potential druggable targets for therapeutic intervention. Animal studies and clinical trials that have been conducted for this purpose are discussed, highlighting reasons why they might not have resulted in the expected outcomes, and which lessons can be learned from this. Lastly, opportunities and challenges that sepsis and sepsis-associated multiple organ failure research are currently facing are presented, including recommendations on improved experimental design to increase the translational power of preclinical research to the clinic.
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Affiliation(s)
- Matthijs Luxen
- Department of Pathology and Medical Biology, Medical Biology Section, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
- Department of Critical Care, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
- *Correspondence: Matthijs Luxen,
| | - Matijs van Meurs
- Department of Pathology and Medical Biology, Medical Biology Section, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
- Department of Critical Care, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
| | - Grietje Molema
- Department of Pathology and Medical Biology, Medical Biology Section, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
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Regulation of the Homeostatic Unfolded Protein Response in Diabetic Nephropathy. Pharmaceuticals (Basel) 2022; 15:ph15040401. [PMID: 35455399 PMCID: PMC9030951 DOI: 10.3390/ph15040401] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2022] [Revised: 03/09/2022] [Accepted: 03/23/2022] [Indexed: 02/06/2023] Open
Abstract
A growing body of scientific evidence indicates that protein homeostasis, also designated as proteostasis, is causatively linked to chronic diabetic nephropathy (DN). Experimental studies have demonstrated that the insulin signaling in podocytes maintain the homeostatic unfolded protein response (UPR). Insulin signaling via the insulin receptor non-canonically activates the spliced X-box binding protein-1 (sXBP1), a highly conserved endoplasmic reticulum (ER) transcription factor, which regulates the expression of genes that control proteostasis. Defective insulin signaling in mouse models of diabetes or the genetic disruption of the insulin signaling pathway in podocytes propagates hyperglycemia induced maladaptive UPR and DN. Insulin resistance in podocytes specifically promotes activating transcription factor 6 (ATF6) dependent pathogenic UPR. Akin to insulin, recent studies have identified that the cytoprotective effect of anticoagulant serine protease-activated protein C (aPC) in DN is mediated by sXBP1. In mouse models of DN, treatment with chemical chaperones that improve protein folding provides an additional benefit on top of currently used ACE inhibitors. Understanding the molecular mechanisms that transmute renal cell specific adaptive responses and that deteriorate renal function in diabetes will enable researchers to develop new therapeutic regimens for DN. Within this review, we focus on the current understanding of homeostatic mechanisms by which UPR is regulated in DN.
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Liu C, Lin C, Wang D, Wang J, Tao Y, Li Y, Chen X, Bai L, Jia Y, Chen J, Zeng YA. Procr functions as a signaling receptor and is essential for the maintenance and self-renewal of mammary stem cells. Cell Rep 2022; 38:110548. [PMID: 35320720 DOI: 10.1016/j.celrep.2022.110548] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 01/23/2022] [Accepted: 03/01/2022] [Indexed: 11/18/2022] Open
Abstract
The protein C receptor (Procr) has been implicated as a stem cell surface marker in several tissues. It is unknown whether Procr acts as a functional signaling receptor in stem cells. Here, by conditional knockout in mammary stem cells (MaSCs), we demonstrate that Procr is essential for mammary gland development and homeostasis. Through proteomics profiling, we identify that, upon stimulation by the ligand protein C, Procr interacts with heat shock protein 90 (HSP90AA1) via its short cytoplasmic tail, recruiting Src and IGF1R to the complex at the plasma membrane. We show that Procr acts as a signaling receptor of protein C in regulation of MaSCs through HSP90, Src, and IGF1R in vitro. In vivo, IGF1R deletion in MaSCs displays similar phenotypes to Procr deletion. These findings illustrate the essential role of Procr signaling in MaSC maintenance, shedding light onto the molecular regulation by Procr in tissue stem cells.
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Affiliation(s)
- Chunye Liu
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Changdong Lin
- Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Daisong Wang
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Jingqiang Wang
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Yu Tao
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Yue Li
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Xinyi Chen
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Lanyue Bai
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
| | - Yingying Jia
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
| | - Jianfeng Chen
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China.
| | - Yi Arial Zeng
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China.
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Takaki W, Konishi H, Matsubara D, Shoda K, Arita T, Kataoka S, Shibamoto J, Furuke H, Takabatake K, Shimizu H, Komatsu S, Shiozaki A, Kubota T, Okamoto K, Otsuji E. Role of Extracellular High-Mobility Group Box-1 as a Therapeutic Target of Gastric Cancer. Int J Mol Sci 2022; 23:ijms23063264. [PMID: 35328684 PMCID: PMC8953630 DOI: 10.3390/ijms23063264] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 03/15/2022] [Accepted: 03/16/2022] [Indexed: 02/04/2023] Open
Abstract
Background: High-mobility group box-1 (HMGB1) is involved in the tumorigenesis and metastasis of various cancers. The present study investigated the roles of extracellular HMGB1 in the progression of gastric cancer (GC) and the therapeutic effects of recombinant human soluble thrombomodulin (rTM) targeting HMGB1. Methods: The effects of extracellular HMGB1 and rTM on GC cells were assessed using proliferation and Transwell assays. Their effects on local tumor growth and metastasis were evaluated using subcutaneous tumor and liver metastasis mouse models, respectively. Plasma HMGB1 concentrations in GC patients were measured using ELISA. The relationships between plasma HMGB1 concentrations and the prognosis and clinicopathological factors of patients were also investigated. Results: GC proliferation, migration, and invasion abilities were promoted by increases in extracellular HMGB1 concentrations and alleviated by rTM. In the subcutaneous tumor model, local tumor growth was promoted by the addition of rhHMGB1 and alleviated by rTM. Similar changes occurred in the liver metastasis model. Recurrence-free survival (p < 0.01) and overall survival (p = 0.01) were significantly worse in patients with high plasma HMGB1 concentrations. Conclusion: Plasma HMGB1 concentrations are a prognostic marker in GC patients. Extracellular HMGB1 promotes cancer progression and has potential as a novel treatment target in GC cells for rTM.
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Affiliation(s)
- Wataru Takaki
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Hirotaka Konishi
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
- Correspondence: ; Tel.: +81-75-251-5527; Fax: +81-75-251-5522
| | - Daiki Matsubara
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Katsutoshi Shoda
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
- First Department of Surgery, Faculty of Medicine, University of Yamanashi, Kofu 400-8510, Japan
| | - Tomohiro Arita
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Satoshi Kataoka
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Jun Shibamoto
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Hirotaka Furuke
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Kazuya Takabatake
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Hiroki Shimizu
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Shuhei Komatsu
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Atsushi Shiozaki
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Takeshi Kubota
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Kazuma Okamoto
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
| | - Eigo Otsuji
- Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; (W.T.); (D.M.); (K.S.); (T.A.); (S.K.); (J.S.); (H.F.); (K.T.); (H.S.); (S.K.); (A.S.); (T.K.); (K.O.); (E.O.)
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Protein C receptor maintains cancer stem cell properties via activating lipid synthesis in nasopharyngeal carcinoma. Signal Transduct Target Ther 2022; 7:46. [PMID: 35169126 PMCID: PMC8847456 DOI: 10.1038/s41392-021-00866-z] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Revised: 11/12/2021] [Accepted: 11/30/2021] [Indexed: 11/29/2022] Open
Abstract
Metastasis and recurrence account for 95% of deaths from nasopharyngeal carcinoma (NPC). Cancer stem cells (CSCs) are regarded as one of the main reasons for tumor cell resistance to clinical therapy, and cancer metastasis or recurrence, while little is known about CSCs in NPC. The present study uncovers a subpopulation of cells labeled as CD45−EPCAM+PROCR+ in NPC biopsy samples that exhibit stem cell-like characteristics. A relatively low number of these cells initiate xenograft tumors in mice. Functional analysis reveals that protein C receptor (PROCR) not only serves as a stem cell marker in NPC, but also maintains tumor cells’ stemness potential through regulating lipid metabolism and mitochondrial fission. Epistatic studies reveal that cAMP-protein kinase A stimulates Ca2+ release to manipulate lipid metabolism related genes’ expression. Finally, in a cohort of 207 NPC samples, PROCR expression is correlated with tumor metastasis or recurrence, and predicts poor prognosis. These novel findings link PROCR labeled CSCs with lipid metabolism and mitochondrial plasticity, and provides new clinical target against metastatic or recurrent NPC.
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Ren D, Fedorova J, Davitt K, Van Le TN, Griffin JH, Liaw PC, Esmon CT, Rezaie AR, Li J. Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging. Circ Res 2022; 130:252-272. [PMID: 34930019 PMCID: PMC8882057 DOI: 10.1161/circresaha.121.319044] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
BACKGROUND APC (activated protein C) is a plasma serine protease with anticoagulant and anti-inflammatory activities. EPCR (Endothelial protein C receptor) is associated with APC's activity and mediates its downstream signaling events. APC exerts cardioprotective effects during ischemia and reperfusion (I/R). This study aims to characterize the role of the APC-EPCR axis in ischemic insults in aging. METHODS Young (3-4 months) and aged (24-26 months) wild-type C57BL/6J mice, as well as EPCR point mutation (EPCRR84A/R84A) knockin C57BL/6J mice incapable of interaction with APC and its wild type of littermate C57BL/6J mice, were subjected to I/R. Wild-type APC, signaling-selective APC-2Cys, or anticoagulant-selective APC-E170A were administrated before reperfusion. RESULTS The results demonstrated that cardiac I/R reduces APC activity, and the APC activity was impaired in the aged versus young hearts possibly attributable to the declined EPCR level with aging. Serum EPCR measurement showed that I/R triggered the shedding of membrane EPCR into circulation, while administration of APC attenuated the I/R-induced EPCR shedding in both young and aged hearts. Subsequent echocardiography showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction during I/R in both young and aged mice. Importantly, APC elevated the resistance of the aged heart to ischemic insults through stabilizing EPCR. However, all these cardioprotective effects of APC were blunted in the EPCRR84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMPK (AMP-activated protein kinase) mediates acute adaptive response while AKT (protein kinase B) is involved in chronic metabolic programming in the hearts with APC treatment. CONCLUSIONS I/R stress causes shedding of the membrane EPCR in the heart, and administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.
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Affiliation(s)
- Di Ren
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL 33612
| | - Julia Fedorova
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL 33612
| | - Kayla Davitt
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL 33612
| | - Tran Ngoc Van Le
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL 33612
| | - John H. Griffin
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037
| | - Patricia C. Liaw
- Thrombosis and Atherosclerosis Research Institute, Department of Medicine, McMaster University, Hamilton, ON, Canada
| | - Charles T. Esmon
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104
| | - Alireza R. Rezaie
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104
| | - Ji Li
- Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL 33612
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Activated Protein C Protects against Murine Contact Dermatitis by Suppressing Protease-Activated Receptor 2. Int J Mol Sci 2022; 23:ijms23010516. [PMID: 35008942 PMCID: PMC8745259 DOI: 10.3390/ijms23010516] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Revised: 12/31/2021] [Accepted: 12/31/2021] [Indexed: 12/12/2022] Open
Abstract
Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1β, IL-6 and TGF-β1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.
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Friebel J, Moritz E, Witkowski M, Jakobs K, Strässler E, Dörner A, Steffens D, Puccini M, Lammel S, Glauben R, Nowak F, Kränkel N, Haghikia A, Moos V, Schutheiss HP, Felix SB, Landmesser U, Rauch BH, Rauch U. Pleiotropic Effects of the Protease-Activated Receptor 1 (PAR1) Inhibitor, Vorapaxar, on Atherosclerosis and Vascular Inflammation. Cells 2021; 10:cells10123517. [PMID: 34944024 PMCID: PMC8700178 DOI: 10.3390/cells10123517] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Revised: 12/08/2021] [Accepted: 12/09/2021] [Indexed: 12/28/2022] Open
Abstract
BACKGROUND Protease-activated receptor 1 (PAR1) and toll-like receptors (TLRs) are inflammatory mediators contributing to atherogenesis and atherothrombosis. Vorapaxar, which selectively antagonizes PAR1-signaling, is an approved, add-on antiplatelet therapy for secondary prevention. The non-hemostatic, platelet-independent, pleiotropic effects of vorapaxar have not yet been studied. METHODS AND RESULTS Cellular targets of PAR1 signaling in the vasculature were identified in three patient cohorts with atherosclerotic disease. Evaluation of plasma biomarkers (n = 190) and gene expression in endomyocardial biopsies (EMBs) (n = 12) revealed that PAR1 expression correlated with endothelial activation and vascular inflammation. PAR1 colocalized with TLR2/4 in human carotid plaques and was associated with TLR2/4 gene transcription in EMBs. In addition, vorapaxar reduced atherosclerotic lesion size in apolipoprotein E-knock out (ApoEko) mice. This reduction was associated with reduced expression of vascular adhesion molecules and TLR2/4 presence, both in isolated murine endothelial cells and the aorta. Thrombin-induced uptake of oxLDL was augmented by additional TLR2/4 stimulation and abrogated by vorapaxar. Plaque-infiltrating pro-inflammatory cells were reduced in vorapaxar-treated ApoEko mice. A shift toward M2 macrophages paralleled a decreased transcription of pro-inflammatory cytokines and chemokines. CONCLUSIONS PAR1 inhibition with vorapaxar may be effective in reducing residual thrombo-inflammatory event risk in patients with atherosclerosis independent of its effect on platelets.
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Affiliation(s)
- Julian Friebel
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
- Berlin Institute of Health, 10178 Berlin, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10785 Berlin, Germany
| | - Eileen Moritz
- Center of Drug Absorption and Transport, Institute of Pharmacology, University Medicine Greifswald, 17489 Greifswald, Germany; (E.M.); (B.H.R.)
- DZHK (German Centre for Cardiovascular Research), Partner Site Greifswald, 17475 Greifswald, Germany;
| | - Marco Witkowski
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
- Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Kai Jakobs
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
| | - Elisabeth Strässler
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
- DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10785 Berlin, Germany
| | - Andrea Dörner
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
- Berlin Institute of Health, 10178 Berlin, Germany
| | - Daniel Steffens
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
| | - Marianna Puccini
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
| | - Stella Lammel
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
| | - Rainer Glauben
- Medical Department I, Gastroenterology, Infectious Diseases and Rheumatology, Charité—University Medicine, 12203 Berlin, Germany; (R.G.); (F.N.); (V.M.)
| | - Franziska Nowak
- Medical Department I, Gastroenterology, Infectious Diseases and Rheumatology, Charité—University Medicine, 12203 Berlin, Germany; (R.G.); (F.N.); (V.M.)
| | - Nicolle Kränkel
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
- DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10785 Berlin, Germany
| | - Arash Haghikia
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
- Berlin Institute of Health, 10178 Berlin, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10785 Berlin, Germany
| | - Verena Moos
- Medical Department I, Gastroenterology, Infectious Diseases and Rheumatology, Charité—University Medicine, 12203 Berlin, Germany; (R.G.); (F.N.); (V.M.)
| | | | - Stephan B. Felix
- DZHK (German Centre for Cardiovascular Research), Partner Site Greifswald, 17475 Greifswald, Germany;
- Department of Internal Medicine B, Cardiology, University Medicine Greifswald, 17489 Greifswald, Germany
| | - Ulf Landmesser
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
- Berlin Institute of Health, 10178 Berlin, Germany
- DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10785 Berlin, Germany
| | - Bernhard H. Rauch
- Center of Drug Absorption and Transport, Institute of Pharmacology, University Medicine Greifswald, 17489 Greifswald, Germany; (E.M.); (B.H.R.)
- DZHK (German Centre for Cardiovascular Research), Partner Site Greifswald, 17475 Greifswald, Germany;
- Department of Human Medicine, Section of Pharmacology and Toxicology, Carl von Ossietzky Universität, 26129 Oldenburg, Germany
| | - Ursula Rauch
- Charité Center 11—Department of Cardiology, Charité—University Medicine, 12203 Berlin, Germany; (J.F.); (M.W.); (K.J.); (E.S.); (A.D.); (D.S.); (M.P.); (S.L.); (N.K.); (A.H.); (U.L.)
- DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 10785 Berlin, Germany
- Correspondence: ; Tel.: +49-30-450-513794
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Which proteinase-activated receptor-1 antagonist is better?: Evaluation of vorapaxar and parmodulin-2 effects on human left internal mammary artery endothelial function. Life Sci 2021; 286:120045. [PMID: 34653426 DOI: 10.1016/j.lfs.2021.120045] [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: 08/05/2021] [Revised: 09/26/2021] [Accepted: 10/06/2021] [Indexed: 11/20/2022]
Abstract
OBJECTIVE Endothelial dysfunction occurs as an early event in cardiovascular disease. Previously, vorapaxar, a proteinase-activated receptor-1 antagonist, was shown to cause endothelial damage in a cell culture study. Therefore, our study aimed to compare the effects of vorapaxar and parmodulin-2, proteinase-activated receptor-1 biased agonist, on human left internal mammary artery endothelial function in vitro. METHOD Isolated arteries were hung in the organ baths. Acetylcholine responses (10-11-10-6 M) were obtained in endothelium-intact tissues the following incubation with vorapaxar/parmodulin-2 (10-6 M) to determine the effects of these molecules on the endothelium-dependent relaxation. Subsequently, endothelium-dependent relaxation responses of tissues were investigated in the presence of L-NAME (10-4 M), L-arginine (10-5 M), indomethacin (10-5 M), and charybdotoxin-apamin (10-7 M) in addition to vorapaxar/parmodulin-2 incubation. Besides, the effect of these molecules on endothelium-independent relaxation response was evaluated with sodium nitroprusside (10-11-10-6 M). Finally, the sections of human arteries were imaged using a transmission electron microscope, and the integrity of the endothelial layer was evaluated. RESULTS We found that vorapaxar caused significant endothelial dysfunction by disrupting nitric oxide and endothelium-derived hyperpolarizing factor-dependent relaxation mechanisms. Parmodulin-2 did not cause endothelial damage. Neither vorapaxar nor parmodulin-2 disrupted endothelium-independent relaxation responses. The effect of vorapaxar on the endothelial layer was supported by the transmission electron microscope images. CONCLUSION Parmodulin-2 may be a better option than vorapaxar in treating cardiovascular diseases since it can inhibit PAR-1 without caused endothelial dysfunction.
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Vassiliou AG, Keskinidou C, Jahaj E, Gallos P, Zacharis A, Athanasiou N, Tsipilis S, Mastora Z, Dimopoulou I, Kotanidou A, Orfanos SE. Could Soluble Endothelial Protein C Receptor Levels Recognize SARS-CoV2-Positive Patients Requiring Hospitalization? Shock 2021; 56:733-736. [PMID: 33756504 PMCID: PMC8518207 DOI: 10.1097/shk.0000000000001780] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Accepted: 03/08/2021] [Indexed: 11/25/2022]
Abstract
INTRODUCTION The endothelial protein C receptor (EPCR) is a protein that regulates the protein C anticoagulant and anti-inflammatory pathways. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits activated protein C (APC) activities. The clinical impact of sEPCR and its involvement in COVID-19 has not been explored. In this study, we investigated whether sEPCR levels were related to COVID-19 patients' requirement for hospitalization. METHODS Plasma sEPCR levels were measured on hospital admission in 84 COVID-19 patients, and in 11 non-hospitalized SARS-CoV2-positive patients approximately 6 days after reported manifestation of their symptoms. Multiple logistic regression analysis was performed to identify potential risk factors for hospitalization and receiver operating characteristic (ROC) curves were generated to assess their value. RESULTS In our cohort, hospitalized patients had considerably higher sEPCR levels upon admission compared with outpatients [107.5 (76.7-156.3) vs. 44.6 (12.1-84.4) ng/mL; P < 0.0001)]. The ROC curve using hospitalization as the classification variable and sEPCR levels as the prognostic variable generated an area under the curve at 0.845 (95% CI = 0.710-0.981, P < 0.001). Additionally, we investigated the predictive value of sEPCR combined with BMI, age, or D-dimers. CONCLUSIONS In our cohort, sEPCR levels in COVID-19 patients upon hospital admission appear considerably elevated compared with outpatients; this could lead to impaired APC activities and might contribute to the pro-coagulant phenotype reported in such patients. sEPCR measurement might be useful as a point-of-care test in SARS-CoV2-positive patients.
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Affiliation(s)
- Alice G. Vassiliou
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Chrysi Keskinidou
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Edison Jahaj
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Parisis Gallos
- Health Informatics Laboratory, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece
| | - Alexandros Zacharis
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Nikolaos Athanasiou
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Stamatios Tsipilis
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Zafeiria Mastora
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Ioanna Dimopoulou
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Anastasia Kotanidou
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
| | - Stylianos E. Orfanos
- First Department of Critical Care Medicine and Pulmonary Services, School of Medicine, National and Kapodistrian University of Athens, Evangelismos Hospital, Athens, Greece
- Second Department of Critical Care, School of Medicine, National and Kapodistrian University of Athens, “Attikon” Hospital, Haidari Athens, Greece
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Reda S, Rühl H, Witkowski J, Müller J, Pavlova A, Oldenburg J, Pötzsch B. PC Deficiency Testing: Thrombin-Thrombomodulin as PC Activator and Aptamer-Based Enzyme Capturing Increase Diagnostic Accuracy. Front Cardiovasc Med 2021; 8:755281. [PMID: 34708097 PMCID: PMC8542722 DOI: 10.3389/fcvm.2021.755281] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Accepted: 09/12/2021] [Indexed: 11/30/2022] Open
Abstract
Protein C (PC) activity tests are routinely performed in a thrombophilia workup to screen for PC deficiency. Currently used tests combine conversion of PC to activated PC (APC) by the snake venom Protac with subsequent APC detection through hydrolysis of a chromogenic peptide substrate or prolongation of a clotting time. In this prospective cohort study, we analyzed how different modes of PC activation and subsequent APC determination influence the diagnostic accuracy of PC activity testing in a cohort of 31 patients with genetically confirmed PC deficiency. In addition to chromogenic and clot-based measurement, an oligonucleotide-based enzyme capture assay utilizing a basic exosite-targeting aptamer was used for APC detection. To study the influence of the PC activation step on diagnostic sensitivity, PC activation through Protac and through the thrombin-thrombomodulin (TM) complex were compared. Twenty-six (84%) and 24 (77%) PC deficient patients were identified as true-positive using the chromogenic and the clot-based PC activity assay, respectively. True-positive results increased to 27 (87%) when the basic exosite-targeting aptamer approach was used for APC measurement. Additional replacement of the PC activator Protac by thrombin-TM gave true-positive results in all patients. These data indicate that the mode of PC activation is crucial in determining the accuracy of PC activity testing and that diagnostic sensitivity can be significantly improved by replacing the PC activator Protac with thrombin-TM. APC detection using a basic exosite-targeting aptamer achieves high sensitivity toward mutations outside the active center while being less subject to interfering factors than clot-based PC activity assays.
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Affiliation(s)
- Sara Reda
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Heiko Rühl
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Jana Witkowski
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Jens Müller
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Anna Pavlova
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Johannes Oldenburg
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Bernd Pötzsch
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
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Ebermeyer T, Cognasse F, Berthelot P, Mismetti P, Garraud O, Hamzeh-Cognasse H. Platelet Innate Immune Receptors and TLRs: A Double-Edged Sword. Int J Mol Sci 2021; 22:ijms22157894. [PMID: 34360659 PMCID: PMC8347377 DOI: 10.3390/ijms22157894] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2021] [Revised: 07/14/2021] [Accepted: 07/20/2021] [Indexed: 12/17/2022] Open
Abstract
Platelets are hematopoietic cells whose main function has for a long time been considered to be the maintenance of vascular integrity. They have an essential role in the hemostatic response, but they also have functional capabilities that go far beyond it. This review will provide an overview of platelet functions. Indeed, stress signals may induce platelet apoptosis through proapoptotis or hemostasis receptors, necrosis, and even autophagy. Platelets also interact with immune cells and modulate immune responses in terms of activation, maturation, recruitment and cytokine secretion. This review will also show that platelets, thanks to their wide range of innate immune receptors, and in particular toll-like receptors, and can be considered sentinels actively participating in the immuno-surveillance of the body. We will discuss the diversity of platelet responses following the engagement of these receptors as well as the signaling pathways involved. Finally, we will show that while platelets contribute significantly, via their TLRs, to immune response and inflammation, these receptors also participate in the pathophysiological processes associated with various pathogens and diseases, including cancer and atherosclerosis.
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Affiliation(s)
- Théo Ebermeyer
- INSERM U1059-SAINBIOSE, Université de Lyon, F-42023 Saint-Etienne, France; (T.E.); (F.C.); (P.M.); (O.G.)
| | - Fabrice Cognasse
- INSERM U1059-SAINBIOSE, Université de Lyon, F-42023 Saint-Etienne, France; (T.E.); (F.C.); (P.M.); (O.G.)
- Etablissement Français du Sang Auvergne-Rhône-Alpes, 25 bd Pasteur, F-42100 Saint-Étienne, France
| | - Philippe Berthelot
- Team GIMAP, CIRI—Centre International de Recherche en Infectiologie, Université de Lyon, U1111, UMR5308, F-69007 Lyon, France;
- Infectious Diseases Department, CHU de St-Etienne, F-42055 Saint-Etienne, France
| | - Patrick Mismetti
- INSERM U1059-SAINBIOSE, Université de Lyon, F-42023 Saint-Etienne, France; (T.E.); (F.C.); (P.M.); (O.G.)
- Department of Vascular Medicine and Therapeutics, INNOVTE, CHU de St-Etienne, F-42055 Saint-Etienne, France
| | - Olivier Garraud
- INSERM U1059-SAINBIOSE, Université de Lyon, F-42023 Saint-Etienne, France; (T.E.); (F.C.); (P.M.); (O.G.)
| | - Hind Hamzeh-Cognasse
- INSERM U1059-SAINBIOSE, Université de Lyon, F-42023 Saint-Etienne, France; (T.E.); (F.C.); (P.M.); (O.G.)
- Correspondence:
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Lu Y, Villoutreix BO, Biswas I, Ding Q, Wang X, Rezaie AR. Antithrombin Resistance Rescues Clotting Defect of Homozygous Prothrombin-Y510N Dysprothrombinemia. Thromb Haemost 2021; 122:679-691. [PMID: 34256393 PMCID: PMC8755856 DOI: 10.1055/a-1549-6407] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
A patient with hematuria in our clinic was diagnosed with urolithiasis. Analysis of the patient's plasma clotting time indicated that both activated partial thromboplastin time (52.6 seconds) and prothrombin time (19.4 seconds) are prolonged and prothrombin activity is reduced to 12.4% of normal, though the patient exhibited no abnormal bleeding phenotype and a prothrombin antigen level of 87.9%. Genetic analysis revealed the patient is homozygous for prothrombin Y510N mutation. We expressed and characterized the prothrombin-Y510N variant in appropriate coagulation assays and found that the specificity constant for activation of the mutant zymogen by factor Xa is impaired approximately fivefold. Thrombin generation assay using patient's plasma and prothrombin-deficient plasma supplemented with either wild-type or prothrombin-Y510N revealed that both peak height and time to peak for the prothrombin mutant are decreased; however, the endogenous thrombin generation potential is increased. Further analysis indicated that the thrombin mutant exhibits resistance to antithrombin and is inhibited by the serpin with approximately 12-fold slower rate constant. Protein C activation by thrombin-Y510N was also decreased by approximately 10-fold; however, thrombomodulin overcame the catalytic defect. The Na+-concentration-dependence of the amidolytic activities revealed that the dissociation constant for the interaction of Na+ with the mutant has been elevated approximately 20-fold. These results suggest that Y510 (Y184a in chymotrypsin numbering) belongs to network of residues involved in binding Na+. A normal protein C activation by thrombin-Y510N suggests that thrombomodulin modulates the conformation of the Na+-binding loop of thrombin. The clotting defect of thrombin-Y510N appears to be compensated by its markedly lower reactivity with antithrombin, explaining patient's normal hemostatic phenotype.
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Affiliation(s)
- Yeling Lu
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States.,Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Bruno O Villoutreix
- INSERM 1141, NeuroDiderot, Université de Paris, Hôpital Robert-Debré, Paris, France
| | - Indranil Biswas
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States
| | - Qiulan Ding
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xuefeng Wang
- Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Alireza R Rezaie
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States.,Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
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47
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Jeon MT, Kim KS, Kim ES, Lee S, Kim J, Hoe HS, Kim DG. Emerging pathogenic role of peripheral blood factors following BBB disruption in neurodegenerative disease. Ageing Res Rev 2021; 68:101333. [PMID: 33774194 DOI: 10.1016/j.arr.2021.101333] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2020] [Revised: 03/03/2021] [Accepted: 03/19/2021] [Indexed: 12/15/2022]
Abstract
The responses of central nervous system (CNS) cells such as neurons and glia in neurodegenerative diseases (NDs) suggest that regulation of neuronal and glial functions could be a strategy for ND prevention and/or treatment. However, attempts to develop such therapeutics for NDs have been hindered by the challenge of blood-brain barrier (BBB) permeability and continued constitutive neuronal loss. These limitations indicate the need for additional perspectives for the prevention/treatment of NDs. In particular, the disruption of the blood-brain barrier (BBB) that accompanies NDs allows brain infiltration by peripheral factors, which may stimulate innate immune responses involved in the progression of neurodegeneration. The accumulation of blood factors like thrombin, fibrinogen, c-reactive protein (CRP) and complement components in the brain has been observed in NDs and may activate the innate immune system in the CNS. Thus, strengthening the integrity of the BBB may enhance its protective role to attenuate ND progression and functional loss. In this review, we describe the innate immune system in the CNS and the contribution of blood factors to the role of the CNS immune system in neurodegeneration and neuroprotection.
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Affiliation(s)
- Min-Tae Jeon
- Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41062, Republic of Korea
| | - Kyu-Sung Kim
- Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41062, Republic of Korea; Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science & Technology (DGIST), 333, Techno jungang-daero, Hyeonpung-eup, Dalseong-gun, Daegu, 42988, Republic of Korea
| | - Eun Seon Kim
- Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41062, Republic of Korea; Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science & Technology (DGIST), 333, Techno jungang-daero, Hyeonpung-eup, Dalseong-gun, Daegu, 42988, Republic of Korea
| | - Suji Lee
- Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41062, Republic of Korea; Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology, and Neuroscience, King's College London, 16 De Crespigny Park, London, SE5 8AF, UK
| | - Jieun Kim
- Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41062, Republic of Korea
| | - Hyang-Sook Hoe
- Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41062, Republic of Korea; Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science & Technology (DGIST), 333, Techno jungang-daero, Hyeonpung-eup, Dalseong-gun, Daegu, 42988, Republic of Korea.
| | - Do-Geun Kim
- Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41062, Republic of Korea.
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48
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Fang XZ, Wang YX, Xu JQ, He YJ, Peng ZK, Shang Y. Immunothrombosis in Acute Respiratory Dysfunction of COVID-19. Front Immunol 2021; 12:651545. [PMID: 34149692 PMCID: PMC8207198 DOI: 10.3389/fimmu.2021.651545] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2021] [Accepted: 05/12/2021] [Indexed: 01/10/2023] Open
Abstract
COVID-19 is an acute, complex disorder that was caused by a new β-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on current reports, it was surprising that the characteristics of many patients with COVID-19, who fulfil the Berlin criteria for acute respiratory distress syndrome (ARDS), are not always like those of patients with typical ARDS and can change over time. While the mechanisms of COVID-19–related respiratory dysfunction in COVID-19 have not yet been fully elucidated, pulmonary microvascular thrombosis is speculated to be involved. Considering that thrombosis is highly related to other inflammatory lung diseases, immunothrombosis, a two-way process that links coagulation and inflammation, seems to be involved in the pathophysiology of COVID-19, including respiratory dysfunction. Thus, the current manuscript will describe the proinflammatory milieu in COVID-19, summarize current evidence of thrombosis in COVID-19, and discuss possible interactions between these two.
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Affiliation(s)
- Xiang-Zhi Fang
- Department of Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Ya-Xin Wang
- Department of Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Ji-Qain Xu
- Department of Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Ya-Jun He
- Department of Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Zhe-Kang Peng
- Department of Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - You Shang
- Department of Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Institute of Anesthesiology and Critical Care Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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49
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Brünnert D, Kumar V, Kaushik V, Ehrhardt J, Chahar KR, Sharma PK, Zygmunt M, Goyal P. Thrombin impairs the angiogenic activity of extravillous trophoblast cells via monocyte chemotactic protein-1 (MCP-1): A possible link with preeclampsia. Reprod Biol 2021; 21:100516. [PMID: 34058707 DOI: 10.1016/j.repbio.2021.100516] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 05/05/2021] [Accepted: 05/19/2021] [Indexed: 11/18/2022]
Abstract
Cytokines' secretion from the decidua and trophoblast cells has been known to regulate trophoblast cell functions, such as Extravillous trophoblasts (EVTs) cell migration and invasion and remodeling of spiral arteries. Defective angiogenesis and spiral arteries transformation are mainly caused by proinflammatory cytokines and excessive thrombin generation during preeclampsia. Monocyte chemotactic protein-1 (MCP-1), a crucial cytokine, has a role in maintaining normal pregnancy. In this study, we explored whether thrombin regulates the secretion of MCP-1 in HTR-8/SVneo cells; if yes, what is its function? We used HTR-8/SVneo cells, developed from first trimester villous explants of early pregnancy, as the model of EVTs. MCP-1 gene silencing was performed using gene-specific siRNA. qPCR and ELISA were performed to estimate the expression and secretion of MCP-1. Here, we found that thrombin enhanced the secretion of MCP-1 in HTR-8/SVneo cells. Proteinase-activated receptor-1 (PAR-1) was found as the primary receptor, regulating MCP-1 secretion in these cells. Furthermore, MCP-1 secretion is modulated via protein kinase C (PKC) α, β, and Rho/Rho-kinase-dependent pathways. Thrombin negatively regulates HTR-8/SVneo cells' ability to mimic tube formation in an MCP-1 dependent manner. In conclusion, we propose that thrombin-controlled MCP-1 secretion may play an essential role in normal placental development and successful pregnancy maintenance. Improper thrombin production and MCP-1 secretion during pregnancy might cause inadequate vascular formation and transformation of spiral arteries, which may contribute to pregnancy disorders, such as preeclampsia.
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Affiliation(s)
- Daniela Brünnert
- Experimental Tumor Immunology, Department of Obstetrics and Gynecology, University of Würzburg Medical School, D-97080, Würzburg, Germany; Department of Obstetrics and Gynecology, University of Greifswald, Ferdinand-Sauerbruchstrasse, D-17489, Greifswald, Germany.
| | - Vijay Kumar
- Department of Biotechnology, School of Life Sciences, Central University of Rajasthan, Bandarsindri, Kishangarh, Rajasthan, 305817, India
| | - Vibha Kaushik
- Department of Biotechnology, School of Life Sciences, Central University of Rajasthan, Bandarsindri, Kishangarh, Rajasthan, 305817, India
| | - Jens Ehrhardt
- Department of Obstetrics and Gynecology, University of Greifswald, Ferdinand-Sauerbruchstrasse, D-17489, Greifswald, Germany
| | - Kirti Raj Chahar
- Department of Biotechnology, School of Life Sciences, Central University of Rajasthan, Bandarsindri, Kishangarh, Rajasthan, 305817, India
| | - Phulwanti Kumari Sharma
- Department of Biotechnology, School of Life Sciences, Central University of Rajasthan, Bandarsindri, Kishangarh, Rajasthan, 305817, India
| | - Marek Zygmunt
- Department of Obstetrics and Gynecology, University of Greifswald, Ferdinand-Sauerbruchstrasse, D-17489, Greifswald, Germany
| | - Pankaj Goyal
- Department of Biotechnology, School of Life Sciences, Central University of Rajasthan, Bandarsindri, Kishangarh, Rajasthan, 305817, India.
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50
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Apolipoprotein A-I enhances activated protein C cytoprotective activity. Blood Adv 2021; 4:2404-2408. [PMID: 32484855 DOI: 10.1182/bloodadvances.2019001316] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2019] [Accepted: 05/09/2020] [Indexed: 12/12/2022] Open
Abstract
Key Points
High-density lipoprotein and apolipoprotein A-I enhance activated protein C cytoprotective activity. High-density lipoprotein and apolipoprotein A-I significantly increase the rate at which activated protein C degrades cytotoxic extracellular histones.
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