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Fan D, Liu X, Chen H. Endothelium-Mimicking Materials: A "Rising Star" for Antithrombosis. ACS APPLIED MATERIALS & INTERFACES 2024; 16:53343-53371. [PMID: 39344055 DOI: 10.1021/acsami.4c12117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/01/2024]
Abstract
The advancement of antithrombotic materials has significantly mitigated the thrombosis issue in clinical applications involving various medical implants. Extensive research has been dedicated over the past few decades to developing blood-contacting materials with complete resistance to thrombosis. However, despite these advancements, the risk of thrombosis and other complications persists when these materials are implanted in the human body. Consequently, the modification and enhancement of antithrombotic materials remain pivotal in 21st-century hemocompatibility studies. Previous research indicates that the healthy endothelial cells (ECs) layer is uniquely compatible with blood. Inspired by bionics, scientists have initiated the development of materials that emulate the hemocompatible properties of ECs by replicating their diverse antithrombotic mechanisms. This review elucidates the antithrombotic mechanisms of ECs and examines the endothelium-mimicking materials developed through single, dual-functional and multifunctional strategies, focusing on nitric oxide release, fibrinolytic function, glycosaminoglycan modification, and surface topography modification. These materials have demonstrated outstanding antithrombotic performance. Finally, the review outlines potential future research directions in this dynamic field, aiming to advance the development of antithrombotic materials.
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Affiliation(s)
- Duanqi Fan
- State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China
| | - Xiaoli Liu
- State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China
| | - Hong Chen
- State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China
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Therapeutic Strategies for Disseminated Intravascular Coagulation Associated with Aortic Aneurysm. Int J Mol Sci 2022; 23:ijms23031296. [PMID: 35163216 PMCID: PMC8836167 DOI: 10.3390/ijms23031296] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2021] [Revised: 01/05/2022] [Accepted: 01/22/2022] [Indexed: 01/22/2023] Open
Abstract
Aortic aneurysms are sometimes associated with enhanced-fibrinolytic-type disseminated intravascular coagulation (DIC). In enhanced-fibrinolytic-type DIC, both coagulation and fibrinolysis are markedly activated. Typical cases show decreased platelet counts and fibrinogen levels, increased concentrations of fibrin/fibrinogen degradation products (FDP) and D-dimer, and increased FDP/D-dimer ratios. Thrombin-antithrombin complex or prothrombin fragment 1 + 2, as markers of coagulation activation, and plasmin-α2 plasmin inhibitor complex, a marker of fibrinolytic activation, are all markedly increased. Prolongation of prothrombin time (PT) is not so obvious, and the activated partial thromboplastin time (APTT) is rather shortened in some cases. As a result, DIC can be neither diagnosed nor excluded based on PT and APTT alone. Many of the factors involved in coagulation and fibrinolysis activation are serine proteases. Treatment of enhanced-fibrinolytic-type DIC requires consideration of how to control the function of these serine proteases. The cornerstone of DIC treatment is treatment of the underlying pathology. However, in some cases surgery is either not possible or exacerbates the DIC associated with aortic aneurysm. In such cases, pharmacotherapy becomes even more important. Unfractionated heparin, other heparins, synthetic protease inhibitors, recombinant thrombomodulin, and direct oral anticoagulants (DOACs) are agents that inhibit serine proteases, and all are effective against DIC. Inhibition of activated coagulation factors by anticoagulants is key to the treatment of DIC. Among them, DOACs can be taken orally and is useful for outpatient treatment. Combination therapy of heparin and nafamostat allows fine-adjustment of anticoagulant and antifibrinolytic effects. While warfarin is an anticoagulant, this agent is ineffective in the treatment of DIC because it inhibits the production of coagulation factors as substrates without inhibiting activated coagulation factors. In addition, monotherapy using tranexamic acid in cases of enhanced-fibrinolytic-type DIC may induce fatal thrombosis. If tranexamic acid is needed for DIC, combination with anticoagulant therapy is of critical importance.
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Yamato K, Nakajo Y, Yamamoto-Imoto H, Kokame K, Miyata T, Takahashi JC, Kataoka H, Yanamoto H. Low-Dose Activated Protein C Suppresses the Development of Cerebral Infarction and Neurological Deficits in Mice. NEUROSURGERY OPEN 2020. [DOI: 10.1093/neuopn/okaa014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Abstract
BACKGROUND
A large prospective study previously reported that a higher plasma level of protein C (PC) was associated with a lower incidence of ischemic stroke.
OBJECTIVE
To investigate the neuroprotective properties of activated PC (APC) against acute ischemic stroke using the 3-vessel occlusion model.
METHODS
Male C57BL/6J mice received APC (human APC) at 0.25, 0.5, or 1.0 (low dose) or 2.0, 4.0, or 8.0 mg/kg (high dose). Edaravone (Eda) (1.0, 3.0, or 10 mg/kg, a neuroprotectant approved for use in Japan), albumin (2.0 mg/kg), heparin (100 or 600 U/kg), or saline was used as the control. The drug or control was administered intravenously twice in the initial 24 h or 5 times in 3 d, starting 5 min after the induction of ischemia.
RESULTS
Low-dose APC significantly reduced lesion volumes, not affecting the depth of ischemia. High-dose APC did not significantly reduce lesion volumes, causing hemorrhagic transformation in some cases. In the chronic phase, lesion volumes were significantly suppressed in the APC or Eda group, and only the APC group showed a significant attenuation of neurological deficits. The protease-activated receptor (PAR)-1 antagonist SCH79797, administered during preischemia, completely abolished APC-induced neuroprotection. The overshoot-like abrupt recovery in regional cerebral blood flow observed in the control in the initial reperfusion phase was significantly suppressed by the APC treatment, indicating that the cerebral autoregulation system, consisting of endothelial cells and blood-brain barrier functions, was preserved.
CONCLUSION
Low-dose APC, potentially via the PAR-1-dependent anti-inflammatory cascade, protects the brain against ischemic stroke without increasing the risk of hemorrhagic transformation or death.
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Affiliation(s)
- Keiko Yamato
- Laboratory of Neurology and Neurosurgery, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Yukako Nakajo
- Laboratory of Neurology and Neurosurgery, National Cerebral and Cardiovascular Center, Suita, Japan
- Research Laboratories, Rakuwa-kai Otowa Hospital, Kyoto, Japan
| | - Hitomi Yamamoto-Imoto
- Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Koichi Kokame
- Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Toshiyuki Miyata
- Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Jun C Takahashi
- Department of Neurosurgery, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Hiroharu Kataoka
- Department of Neurosurgery, National Cerebral and Cardiovascular Center, Suita, Japan
| | - Hiroji Yanamoto
- Laboratory of Neurology and Neurosurgery, National Cerebral and Cardiovascular Center, Suita, Japan
- Department of Cardiovascular Science, Division of Surgical Medicine, Osaka University Graduate School of Medicine, Suita, Japan
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Sokolovskaya I, Kryachok I, Chorna I, Semeniv I, Kotuza A, Hryhorieva N, Plakhotnik O, Zarytska V, Zhuravel V, Kmetyuk Y, Kondratiuk A. Influence of exogenous, physiological and factors of chronic inflammatory process on indicators of disturbances of adaptive processes of the person. FRENCH-UKRAINIAN JOURNAL OF CHEMISTRY 2020. [DOI: 10.17721/fujcv8i2p183-202] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Changes in laboratory parameters can be caused by physical, chemical, biological and other external factors, depending on physiological conditions, in most cases they have an adaptive nature, and it is not always possible to distinguish them from physiological fluctuations. At the same time, obvious shifts in laboratory parameters may indicate a violation of adaptation processes and damage of tissues and organs, despite the absence of clinical manifestations of a disease.
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Affiliation(s)
| | | | | | - Igor Semeniv
- Hospital "Feofania" the State Management of Affairs
| | | | - Nataliia Hryhorieva
- State Institution "DF Chebotareva Institute of Gerontology National Academy of Medical Sciences of Ukraine"
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Comment on "Endothelial Protein C Receptor (EPCR), Protease Activated Receptor-1 (PAR-1) and Their Interplay in Cancer Growth and Metastatic Dissemination" Cancers 2019, 11, 51. Cancers (Basel) 2019; 11:cancers11030374. [PMID: 30884838 PMCID: PMC6468748 DOI: 10.3390/cancers11030374] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2019] [Revised: 02/25/2019] [Accepted: 03/14/2019] [Indexed: 01/12/2023] Open
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Li Y, Hu C, Wang P, Liu Y, Wang L, Pi Q, Gong Z, Yang X, Mak M, Wu Y. Indoor nanoscale particulate matter-induced coagulation abnormality based on a human 3D microvascular model on a microfluidic chip. J Nanobiotechnology 2019; 17:20. [PMID: 30709410 PMCID: PMC6357445 DOI: 10.1186/s12951-019-0458-2] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Accepted: 01/21/2019] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND A growing body of evidence shows that indoor concentrations of airborne particles are often higher than is typically encountered outdoors. Since exposure to indoor PM2.5 is thought to be associated with cardiovascular disease, the health impacts of indoor air pollution need to be explored. Based on animal models, ambient particulate matter has been proved to promote coagulation which is very likely involved in the pathogenic development of cardiovascular disease. However, animal models are insufficient to predict what will happen with any certainty in humans. For this reason, the precise pathogenic mechanisms behind the development of cardiovascular disease in humans have not yet been determined. RESULTS We generated a 3D functional human microvascular network in a microfluidic device. This model enables human vascular endothelial cells to form tissue-like microvessels that behave very similarly to human blood vessels. The perfusable microvasculature allows the delivery of particles introduced into these generated human-like microvessels to follow the fluid flow. This exposure path effectively simulates the dynamic movement of airborne nanoscale particles (ANPs) within human vessels. In this study, we first identified the existence of ANPs in indoor air pollution. We then showed that ANPs could activate endothelial cells via ROS induced inflammation, and further resulted in abnormal expression of the coagulation factors (TF, TM and t-PA) involved in coagulation cascades. In addition, we found that a protein could cover ANPs, and this biointeraction could interfere with heparan sulfate (HS). Human organotypic 3D microvessel models provide a bridge for how research outcomes can translate to humans. CONCLUSIONS The 3D human microvessel model was used to determine the physiological responses of human vessels to ANP stimulation. Based on the obtained data, we concluded that ANPs not only disrupts normal coagulation functions, but also act directly on anticoagulant factors in human vessels. These experimental observations provide a potential biological explanation for the epidemiologically established link between ANPs and coagulation abnormality. This organ-on-chip model may provide a bridge from in vitro results to human responses.
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Affiliation(s)
- Yan Li
- Key Laboratory for Deep Processing of Major Grain and Oil (Wuhan Polytechnic University), Ministry of Education, College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023 People’s Republic of China
- Department of Biomedical Engineering, School of Engineering & Applied Science, Yale University, New Haven, 06520 USA
| | - Chuanlin Hu
- State Key Laboratory of Silicate Materials for Architectures, Wuhan University of Technology, Wuhan, 430070 People’s Republic of China
| | - Pengcheng Wang
- Key Laboratory for Deep Processing of Major Grain and Oil (Wuhan Polytechnic University), Ministry of Education, College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023 People’s Republic of China
| | - Yan Liu
- Key Laboratory for Deep Processing of Major Grain and Oil (Wuhan Polytechnic University), Ministry of Education, College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023 People’s Republic of China
| | - Luyang Wang
- Department of Building Science, Tsinghua University, Beijing, 100084 People’s Republic of China
| | - Qingmeng Pi
- Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA
- Department of Plastic and Reconstructive Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200129 People’s Republic of China
| | - Zhiyong Gong
- Key Laboratory for Deep Processing of Major Grain and Oil (Wuhan Polytechnic University), Ministry of Education, College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023 People’s Republic of China
| | - Xu Yang
- Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan, 430079 People’s Republic of China
| | - Michael Mak
- Department of Biomedical Engineering, School of Engineering & Applied Science, Yale University, New Haven, 06520 USA
| | - Yang Wu
- Hubei Key Laboratory for Processing and Transformation of Agricultural Products (Wuhan Polytechnic University), College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023 People’s Republic of China
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Li P, Qin C. Recurrent cerebellar infarction associated with hereditary heterozygous protein C deficiency in a 35-year-old woman: A case report and genetic study on the pedigree. Exp Ther Med 2018; 16:2677-2681. [PMID: 30210609 DOI: 10.3892/etm.2018.6518] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2017] [Accepted: 05/17/2018] [Indexed: 12/16/2022] Open
Abstract
Deficiency of protein C may cause deep venous thrombosis and pulmonary embolism, leading to ischemic stroke. The present study reports on a case of a young adult with recurrent cerebellar infarction due to hereditary heterozygous protein C deficiency and performs a literature review. A 35-year-old Asian woman was admitted t o the Department of Neurology of The First Affiliated Hospital of Guangxi Medical University (Nanning, China) due to right limb paralysis and vomiting. The diagnosis of stroke was confirmed by computed tomography and magnetic resonance imaging, which indicated acute cerebral infarction of the right cerebellar hemisphere and cerebellar vermis, as well as a previous cerebral infarction on the left cerebellar hemisphere. This patient had taken aspirin orally for 4 years following surgical therapy for small intestine thrombosis and was regularly taking hydroxychloroquine sulfate to treat systemic lupus erythematosus. The protein C (PROC) levels were 57.6%, while protein S and antithrombin levels were normal. Gene sequencing analysis of the patient and the patient's pedigree revealed a heterozygous mutation, c.565C>T, on the PROC gene in the patient and the patient's father. In conclusion, the clinical manifestations of hereditary PROC deficiency may vary between individuals. The heterozygous mutation locus c.565C>T on the PROC gene is associated with thrombophilia. Awareness of the association between natural anticoagulants and thrombophilia may promote the prevention and therapy of stroke.
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Affiliation(s)
- Pingping Li
- Department of Neurology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
| | - Chao Qin
- Department of Neurology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
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Iverson N, Abildgaard U. State-of-the-Art Review : Role of Antithrombin and Tissue Factor Pathway Inhibitor in the Control of Thrombosis and Mediation of Heparin Action. Clin Appl Thromb Hemost 2016. [DOI: 10.1177/107602969600200101] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Deficiency of any of the two coagulation in hibitors antithrombin (AT) and tissue factor pathway in hibitor (TFPI) lowers the resistance to thrombosis. He reditary deficiency of AT leads to a high risk of throm bosis, which occasionally responds poorly to heparin therapy. Experimental deficiency of TFPI lowers the re sistance to infusion of both tissue factor and endotoxin, both regarding microvascular thrombosis and fatality. Administration of either AT or TFPI protects against mi cro- and macrovascular thrombosis. Injection of heparin and some other glycosaminoglycans releases intima bound TFPI to the blood. Heparin accelerates the inhib itory effects of both inhibitors, in particular the effect of AT. The influence of the two inhibitors on the various anticoagulant reactions have been studied using blocking antibodies. It is suggested that the anticoagulant and an tithrombotic effects of heparin are mainly mediated by the accelerated inactivation of thrombin, factor IXa and factor X by AT, and augmented inactivation of tissue factor-factor VIIa by TFPI released to the blood.
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Affiliation(s)
- Nina Iverson
- Haematological Research Laboratory, Aker University Hospital, Oslo, Norway
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Swanepoel AC, Nielsen VG, Pretorius E. Viscoelasticity and Ultrastructure in Coagulation and Inflammation: Two Diverse Techniques, One Conclusion. Inflammation 2015; 38:1707-26. [PMID: 25772112 DOI: 10.1007/s10753-015-0148-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The process of blood clotting has been studied for centuries. A synopsis of current knowledge pertaining to haemostasis and the blood components, including platelets and fibrin networks which are closely involved in coagulation, are discussed. Special emphasis is placed on tissue factor (TF), calcium and thrombin since these components have been implicated in both the coagulation process and inflammation. Analysis of platelets and fibrin morphology indicate that calcium, tissue factor and thrombin at concentrations used during viscoelastic analysis (with thromboelastography or TEG) bring about alterations in platelet and fibrin network ultrastructure, which is similar to that seen in inflammation. Scanning electron microscopy indicated that, when investigating platelet structure in disease, addition of TF, calcium or thrombin will mask disease-induced alterations associated with platelet activation. Therefore, washed platelets without any additives is preferred for morphological analysis. Furthermore, morphological and viscoelastic analysis confirmed that thrombin activation is the preferred method of fibrin activation when investigating fibrin network ultrastructure.
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Affiliation(s)
- Albe C Swanepoel
- Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Private Bag x323, Arcadia, 0007, South Africa,
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McKelvey K, Jackson CJ, Xue M. Activated protein C: A regulator of human skin epidermal keratinocyte function. World J Biol Chem 2014; 5:169-179. [PMID: 24921007 PMCID: PMC4050111 DOI: 10.4331/wjbc.v5.i2.169] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2013] [Accepted: 04/03/2014] [Indexed: 02/05/2023] Open
Abstract
Activated protein C (APC) is a physiological anticoagulant, derived from its precursor protein C (PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor (EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC’s function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.
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Foley JH, Orfeo T, Undas A, McLean KC, Bernstein IM, Rivard GE, Mann KG, Everse SJ, Brummel-Ziedins KE. From principle to practice: bridging the gap in patient profiling. PLoS One 2013; 8:e54728. [PMID: 23372761 PMCID: PMC3556038 DOI: 10.1371/journal.pone.0054728] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2012] [Accepted: 12/14/2012] [Indexed: 12/20/2022] Open
Abstract
The standard clinical coagulation assays, activated partial thromboplastin time (aPTT) and prothrombin time (PT) cannot predict thrombotic or bleeding risk. Since thrombin generation is central to haemorrhage control and when unregulated, is the likely cause of thrombosis, thrombin generation assays (TGA) have gained acceptance as "global assays" of haemostasis. These assays generate an enormous amount of data including four key thrombin parameters (lag time, maximum rate, peak and total thrombin) that may change to varying degrees over time in longitudinal studies. Currently, each thrombin parameter is averaged and presented individually in a table, bar graph or box plot; no method exists to visualize comprehensive thrombin generation data over time. To address this need, we have created a method that visualizes all four thrombin parameters simultaneously and can be animated to evaluate how thrombin generation changes over time. This method uses all thrombin parameters to intrinsically rank individuals based on their haemostatic status. The thrombin generation parameters can be derived empirically using TGA or simulated using computational models (CM). To establish the utility and diverse applicability of our method we demonstrate how warfarin therapy (CM), factor VIII prophylaxis for haemophilia A (CM), and pregnancy (TGA) affects thrombin generation over time. The method is especially suited to evaluate an individual's thrombotic and bleeding risk during "normal" processes (e.g pregnancy or aging) or during therapeutic challenges to the haemostatic system. Ultimately, our method is designed to visualize individualized patient profiles which are becoming evermore important as personalized medicine strategies become routine clinical practice.
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Affiliation(s)
- Jonathan H. Foley
- Department of Biochemistry, University of Vermont, Burlington, Vermont, United States of America
| | - Thomas Orfeo
- Department of Biochemistry, University of Vermont, Burlington, Vermont, United States of America
| | - Anetta Undas
- Institute of Cardiology, Jagiellonian University School of Medicine, Krakow, Poland
| | - Kelley C. McLean
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Vermont, Burlington, Vermont, United States of America
| | - Ira M. Bernstein
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Vermont, Burlington, Vermont, United States of America
| | - Georges-Etienne Rivard
- Department of Hematology-Oncology, Centre Hospitalier Universitaire Sainte-Justine, Montréal, Canada
| | - Kenneth G. Mann
- Department of Biochemistry, University of Vermont, Burlington, Vermont, United States of America
| | - Stephen J. Everse
- Department of Biochemistry, University of Vermont, Burlington, Vermont, United States of America
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Nature’s “Silver Bullet” for Anticoagulation: Mechanism of Zymogen Protein C to Activated Protein C. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 765:15-21. [DOI: 10.1007/978-1-4614-4989-8_3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
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Pancreaticoduodenectomy Using Perioperative Zymogen Protein C to Help Prevent Blood Clotting: A Trilogy on Increased Patient Safety. OXYGEN TRANSPORT TO TISSUE XXXV 2013; 789:299-307. [DOI: 10.1007/978-1-4614-7411-1_40] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
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Abstract
Protein C, the zymogen form of the anticoagulant protein, activated protein C, is a member of the vitamin K-dependent class of proteins that function in generation and control of formation of blood clots. This plasma protein consists of a series of domain regions that are qualitatively similar to those present in human coagulation factors VII, IX, and X, and that appear to be related to specific properties of these proteins. With the recent advent of rigorous application of genetic engineering strategies to this system, and the continuing discoveries and characterization of genotypes and phenotypes of protein C in patients, great progress has been made in understanding structure-function relationships of protein C and activated protein C. This review is a summary and synthesis of recent pertinent studies with an emphasis on these topics.
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Sopel MJ, Rosin NL, Falkenham AG, Bezuhly M, Esmon CT, Lee TDG, Liwski RS, Légaré JF. Treatment with activated protein C (aPC) is protective during the development of myocardial fibrosis: an angiotensin II infusion model in mice. PLoS One 2012; 7:e45663. [PMID: 23029168 PMCID: PMC3446915 DOI: 10.1371/journal.pone.0045663] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2012] [Accepted: 08/20/2012] [Indexed: 12/31/2022] Open
Abstract
Aims Myocardial fibrosis contributes to the development of heart failure. Activated Protein C (aPC) is a circulating anticoagulant with anti-inflammatory and cytoprotective properties. Using a model of myocardial fibrosis second to Angiotensin II (AngII) infusion, we investigated the novel therapeutic function aPC in the development of fibrosis. Methods and Results C57Bl/6 and Tie2-EPCR mice were infused with AngII (2.0 µg/kg/min), AngII and aPC (0.4 µg/kg/min) or saline for 3d. Hearts were harvested and processed for analysis or used for cellular isolation. Basic histology and collagen deposition were assessed using histologic stains. Transcript levels of molecular mediators were analyzed by quantitative RT-PCR. Mice infused with AngII exhibited multifocal areas of myocardial cellular infiltration associated with significant collagen deposition compared to saline control animals (p<0.01). AngII-aPC infusion inhibited this cellular infiltration and the corresponding collagen deposition. AngII-aPC infusion also inhibited significant expression of the pro-fibrotic cytokines TGF-β1, CTGF and PDGF found in AngII only infused animals (p<0.05). aPC signals through its receptor, EPCR. Using Tie2-EPCR animals, where endothelial cells over-express EPCR and exhibit enhanced aPC-EPCR signaling, no significant reduction in cellular infiltration or fibrosis was evident with AngII infusion suggesting aPC-mediate protection is endothelial cell independent. Isolated infiltrating cells expressed significant EPCR transcripts suggesting a direct effect on infiltrating cells. Conclusions This data indicates that aPC treatment abrogates the fibrogenic response to AngII. aPC does not appear to confer protection by stimulating the endothelium but by acting directly on the infiltrating cells, potentially inhibiting migration or activation.
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Affiliation(s)
- Mryanda J. Sopel
- Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
| | - Nicole L. Rosin
- Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
| | - Alec G. Falkenham
- Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
| | - Michael Bezuhly
- Department of Surgery, Dalhousie University, Halifax, Nova Scotia, Canada
| | - Charles T. Esmon
- Howard Hughes Medical Institute and Cardiovascular Biology Research Program, Oklahoma City, Oklahoma, United States of America
- Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America
| | - Timothy D. G. Lee
- Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Surgery, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
| | - Robert S. Liwski
- Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
- * E-mail: (RSL); (J-FL)
| | - Jean-Francois Légaré
- Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Surgery, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
- * E-mail: (RSL); (J-FL)
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Bravo MC, Orfeo T, Mann KG, Everse SJ. Modeling of human factor Va inactivation by activated protein C. BMC SYSTEMS BIOLOGY 2012; 6:45. [PMID: 22607732 PMCID: PMC3403913 DOI: 10.1186/1752-0509-6-45] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/15/2012] [Accepted: 05/20/2012] [Indexed: 11/10/2022]
Abstract
BACKGROUND Because understanding of the inventory, connectivity and dynamics of the components characterizing the process of coagulation is relatively mature, it has become an attractive target for physiochemical modeling. Such models can potentially improve the design of therapeutics. The prothrombinase complex (composed of the protease factor (F)Xa and its cofactor FVa) plays a central role in this network as the main producer of thrombin, which catalyses both the activation of platelets and the conversion of fibrinogen to fibrin, the main substances of a clot. A key negative feedback loop that prevents clot propagation beyond the site of injury is the thrombin-dependent generation of activated protein C (APC), an enzyme that inactivates FVa, thus neutralizing the prothrombinase complex. APC inactivation of FVa is complex, involving the production of partially active intermediates and "protection" of FVa from APC by both FXa and prothrombin. An empirically validated mathematical model of this process would be useful in advancing the predictive capacity of comprehensive models of coagulation. RESULTS A model of human APC inactivation of prothrombinase was constructed in a stepwise fashion by analyzing time courses of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin. Model predictions were assessed against empirical data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with plasma proteins derived from multiple preparations. Our work integrates previously published findings and through the cooperative analysis of in vitro experiments and mathematical constructs we are able to produce a final validated model that includes 24 chemical reactions and interactions with 14 unique rate constants which describe the flux in concentrations of 24 species. CONCLUSION This study highlights the complexity of the inactivation process and provides a module of equations describing the Protein C pathway that can be integrated into existing comprehensive mathematical models describing tissue factor initiated coagulation.
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Affiliation(s)
- Maria Cristina Bravo
- Cell and Molecular Biology Program, University of Vermont, 89 Beaumont Ave, Burlington, VT 05405, USA
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17
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Kubier A, O'Brien M. Endogenous Anticoagulants. Top Companion Anim Med 2012; 27:81-7. [DOI: 10.1053/j.tcam.2012.07.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2012] [Accepted: 07/17/2012] [Indexed: 12/21/2022]
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18
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Weiler H. Multiple receptor-mediated functions of activated protein C. Hamostaseologie 2012; 31:185-95. [PMID: 21826371 DOI: 10.5482/ha-1166] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2011] [Accepted: 06/17/2011] [Indexed: 01/08/2023] Open
Abstract
The central effector protease of the protein C pathway, activated protein C (APC), interacts with the endothelial cell protein C receptor, with protease activated receptors (PAR), the apolipoprotein E2 receptor, and integrins to exert multiple effects on haemostasis and immune cell function. Such receptor interactions modify the activation of PC and determine the biological response to endogenous and therapeutically administered APC. This review summarizes the current knowledge about interactions of APC with cell surface-associated receptors, novel substrates such as histones and tissue factor pathway inhibitor, and their implications for the biologic function of APC in the control of coagulation and inflammation.
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Affiliation(s)
- H Weiler
- Blood Research Institute, BloodCenter of Wisconsin, Milwaukee WI 53226, USA.
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19
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Oh H, Smith CL. Evolving methods for single nucleotide polymorphism detection: Factor V Leiden mutation detection. J Clin Lab Anal 2012; 25:259-88. [PMID: 21786330 DOI: 10.1002/jcla.20470] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
Abstract
BACKGROUND The many techniques used to diagnose the Factor V Leiden (FVL) mutation, the most common hereditary hypercoagulation disorder in Eurasians, and the most frequently requested genetic test reflect the evolving strategies in protein and DNA diagnosis. METHODS Here, molecular methods to diagnose the FVL mutation are discussed. RESULTS Protein-based detection assays include the conventional functional activated protein C resistance coagulation test and the recently reported antibody-mediated sensor detection; and DNA-based assays include approaches that use electrophoretic fractionation e.g., restriction fragment length polymorphism, denaturing gradient gel electrophoresis, and single-stranded conformational PCR analysis, DNA hybridization (e.g., microarrays), DNA polymerase-based assays, e.g., extension reactions, fluorescence polarization template-directed dye-terminator incorporation, PCR assays (e.g., amplification-refractory mutation system, melting curve analysis using real-time quantitative PCR, and helicase-dependent amplification), DNA sequencing (e.g., direct sequencing, pyrosequencing), cleavase-based Invader assay and ligase-based assays (e.g., oligonucleotide ligation assay and ligase-mediated rolling circle amplification). CONCLUSION The method chosen by a laboratory to diagnose FVL not only depends on the available technical expertise and equipment, but also the type, variety, and extent of other genetic disorders being diagnosed.
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Affiliation(s)
- Herin Oh
- Molecular Biotechnology Research Laboratory, Boston University, Boston, MA, USA.
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20
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Conway EM. Thrombomodulin and its role in inflammation. Semin Immunopathol 2012; 34:107-25. [PMID: 21805323 DOI: 10.1007/s00281-011-0282-8] [Citation(s) in RCA: 220] [Impact Index Per Article: 16.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2011] [Accepted: 07/20/2011] [Indexed: 12/30/2022]
Abstract
The goal is to provide an extensive review of the physiologic role of thrombomodulin (TM) in maintaining vascular homeostasis, with a focus on its anti-inflammatory properties. Data were collected from published research. TM is a transmembrane glycoprotein expressed on the surface of all vascular endothelial cells. Expression of TM is tightly regulated to maintain homeostasis and to ensure a rapid and localized hemostatic and inflammatory response to injury. By virtue of its strategic location, its multidomain structure and complex interactions with thrombin, protein C (PC), thrombin activatable fibrinolysis inhibitor (TAFI), complement components, the Lewis Y antigen, and the cytokine HMGB1, TM exhibits a range of physiologically important anti-inflammatory, anti-coagulant, and anti-fibrinolytic properties. TM is an essential cofactor that impacts on multiple biologic processes. Alterations in expression of TM and its partner proteins may be manifest by inflammatory and thrombotic disorders. Administration of soluble forms of TM holds promise as effective therapies for inflammatory diseases, and infections and malignancies that are complicated by disseminated intravascular coagulation.
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Affiliation(s)
- Edward M Conway
- Division of Hematology-Oncology, Department of Medicine, Centre for Blood Research (CBR), University of British Columbia, Vancouver, BC, Canada.
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21
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Abstract
The mechanisms of vascular control of thrombotic events remain unclear. The vasculature possesses essential anticoagulant factors that regulate coagulation. Because the endothelium-to-blood ratios are much higher in the microcirculation, it is likely that stasis contributes to thrombotic risk, due in large part to failure to rapidly access the microcirculation and to gain access to this highly anticoagulant environment. Inflammation can potentiate thrombosis in part through downregulation of the vascular anticoagulants, a process that appears to be exacerbated in aging, a well-known risk factor for thrombosis. Surgery and trauma, two major risk factors for thrombosis, result in the release of a variety of cellular components that trigger coagulation through separate mechanisms.
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Affiliation(s)
- Charles T Esmon
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, 73104, USA.
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22
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Kim PY, Nesheim ME. Down regulation of prothrombinase by activated protein C during prothrombin activation. Thromb Haemost 2010; 104:61-70. [PMID: 20390226 PMCID: PMC3152479 DOI: 10.1160/th09-09-0650] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2009] [Accepted: 02/11/2010] [Indexed: 01/27/2023]
Abstract
Activated protein C (aPC) proteolytically inactivates factor Va (FVa) and thereby downregulates prothrombinase. Although FVa inactivation by aPC has been studied extensively, the inactivation of prothrombinase during prothrombin activation has not. Therefore, prothrombin activation initiated both without and with aPC (5.0, 7.5 or 10.0 nM) was monitored over time by fluorescence. The experiments were performed with 0.075 nM FVa and 1.0 nM FXa, and with these concentrations reversed. The time courses of the residual prothrombinase activity with aPC, determined from the slopes of fluorescence over time, were pseudo first order with both limiting and excess FVa. With FVa limiting or in excess, the second rate constants for inactivation of prothrombinase were 1.98 +/- 0.09 x 10(5) M(-1)s(-1) and 2.54 +/- 0.13 x 10(5) M(-1)s(-1), respectively. The former value is 101-fold smaller than that for FVa inactivation by aPC alone. Since with limiting FVa the second order rate constants for prothrombinase inactivation and FVa inactivation are equal, FVa is protected 101-fold, presumably by both FXa and prothrombin. In contrast, with excess FVa, the calculated rate constant for FVa inactivation exceeds that for prothrombinase inactivation 17.3-fold, which reflects a loss of protection by FXa. Since the protective effects of the two proteins are theoretically multiplicative, FXa protected 17.3-fold and prothrombin protected 5.8-fold. With 150 nM protein S and limiting FVa, prothrombinase inactivation was two-fold faster, yet it was still protected 91-fold. These studies show that FVa is down-regulated by aPC during prothrombin activation, but both FXa and prothrombin protect FVa in a multiplicative way, with or without protein S.
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Affiliation(s)
- Paul Y. Kim
- Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6
| | - Michael E. Nesheim
- Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6
- Department of Medicine, Queen’s University, Kingston, Ontario, Canada K7L 3N6
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23
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Danese S, Vetrano S, Zhang L, Poplis VA, Castellino FJ. The protein C pathway in tissue inflammation and injury: pathogenic role and therapeutic implications. Blood 2010. [PMID: 20018912 DOI: 10.1182/blood-2009-09- 201616] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Inflammation and coagulation are closely linked interdependent processes. Under physiologic conditions, the tissue microcirculation functions in anticoagulant and anti-inflammatory fashions. However, when inflammation occurs, coagulation is also set in motion and actively participates in enhancing inflammation. Recently, novel and unexpected roles of hemostasis in the humoral and cellular components of innate immunity have been described. In particular, the protein C system, besides its well-recognized role in anticoagulation, plays a crucial role in inflammation. Indeed, the protein C system is now emerging as a novel participant in the pathogenesis of acute and chronic inflammatory diseases, such as sepsis, asthma, inflammatory bowel disease, atherosclerosis, and lung and heart inflammation, and may emerge as unexpected therapeutic targets for intervention.
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Affiliation(s)
- Silvio Danese
- IBD Unit, Division of Gastroenterology, Istituto Clinico Humanitas IRCCS, Rozzano, Italy.
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24
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Rong Y, Zhang M, Zhang L, Wang XL, Shen YH. JNK-ATF-2 inhibits thrombomodulin (TM) expression by recruiting histone deacetylase4 (HDAC4) and forming a transcriptional repression complex in the TM promoter. FEBS Lett 2010; 584:852-8. [PMID: 20116378 DOI: 10.1016/j.febslet.2010.01.048] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2009] [Revised: 01/18/2010] [Accepted: 01/21/2010] [Indexed: 10/19/2022]
Abstract
Thrombomodulin (TM) is an important vascular protective molecule that has anticoagulant, anti-inflammatory and anti-apoptotic properties. TM is downregulated in many thrombotic and vascular diseases. However, the mechanisms responsible for TM suppression are not completely understood. In this study, we investigated the mechanism involved in fatty acid-induced suppression of TM expression in human aortic endothelial cells. We found that palmitic acid inhibited TM expression through the JNK and p38 pathways. ATF-2, a JNK and p38 target transcription factor, was involved in the suppression. ATF-2 can bind to the TM promoter, recruit HDAC4 and form a transcriptional repression complex in the promoter, which may lead to chromatin condensation and transcriptional arrest. This study provides novel insight into TM down-regulation by stress signaling pathways.
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Affiliation(s)
- Yuanyuan Rong
- The key laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan, Shandong, China
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25
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Veldman A, Fischer D, Wong FY, Kreuz W, Sasse M, Eberspächer B, Mansmann U, Schosser R. Human protein C concentrate in the treatment of purpura fulminans: a retrospective analysis of safety and outcome in 94 pediatric patients. Crit Care 2010; 14:R156. [PMID: 20723255 PMCID: PMC2945140 DOI: 10.1186/cc9226] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2010] [Revised: 07/15/2010] [Accepted: 08/19/2010] [Indexed: 11/10/2022] Open
Abstract
INTRODUCTION Purpura fulminans (PF) is a devastating complication of uncontrolled systemic inflammation, associated with high incidence of amputations, skin grafts and death. In this study, we aimed to clarify the clinical profile of pediatric patients with PF who improved with protein C (PC) treatment, explore treatment effects and safety, and to refine the prognostic significance of protein C plasma levels. METHODS In Germany, patients receiving protein C concentrate (Ceprotin, Baxter AG, Vienna, Austria) are registered. The database was used to locate all pediatric patients with PF treated with PC from 2002 to 2005 for this national, retrospective, multi-centered study. RESULTS Complete datasets were acquired in 94 patients, treated in 46 centers with human, non-activated protein C concentrate for purpura fulminans. PC was given for 2 days (median, range 1-24 days) with a median daily dose of 100 IU/kg. Plasma protein C levels increased from a median of 27% to a median of 71% under treatment. 22.3% of patients died, 77.7% survived to discharge. Skin grafts were required in 9.6%, amputations in 5.3%. PF recovered or improved in 79.8%, remained unchanged in 13.8% and deteriorated in 6.4%. Four adverse events occurred in 3 patients, none classified as severe. Non-survivors had lower protein C plasma levels (P < 0.05) and higher prevalence of coagulopathy at admission (P < 0.01). Time between admission and start of PC substitution was longer in patients who died compared to survivors (P = 0.03). CONCLUSIONS This retrospective dataset shows that, compared to historic controls, only few pediatric patients with PF under PC substitution needed dermatoplasty and/or amputations. Apart from epistaxis, no bleeding was observed. Although the data comes from a retrospective study, the evidence we present suggests that PC had a beneficial impact on the need for dermatoplasty and amputations, pointing to the potential value of carrying out a prospective randomised controlled trial.
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Affiliation(s)
- Alex Veldman
- Monash Newborn, Monash Medical Centre; The Ritchie Centre, Monash Institute for Medical Research and Department of Pediatrics, Monash University, 246 Clayton RD, Clayton 3168, Melbourne, Australia
| | - Doris Fischer
- Department of Pediatrics, J.W. Goethe University Hospital, Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany
| | - Flora Y Wong
- Monash Newborn, Monash Medical Centre; The Ritchie Centre, Monash Institute for Medical Research and Department of Pediatrics, Monash University, 246 Clayton RD, Clayton 3168, Melbourne, Australia
| | - Wolfhart Kreuz
- Department of Pediatrics, J.W. Goethe University Hospital, Theodor Stern Kai 7, 60590 Frankfurt/Main, Germany
| | - Michael Sasse
- Department of Pediatric Cardiology and Pediatric Intensive Care, University Childrens Hospital Hannover, Carl Neuberg Str. 1, 30625 Hannover, Germany
| | | | - Ulrich Mansmann
- Department of Medical Informatics, Biometry, and Epidemiology, L. Maximilian University, Marchioninistr. 15, 81377 Munich, Germany
| | - Rudolf Schosser
- Baxter BioScience, EdisonStr. 4, 85716 Unterschleißheim, Germany
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26
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Bruley DF, Mears SC, Streiff MB. Safer Surgery Using Zymogen Protein C Concentrate. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2010; 662:439-45. [DOI: 10.1007/978-1-4419-1241-1_63] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
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27
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The protein C pathway in tissue inflammation and injury: pathogenic role and therapeutic implications. Blood 2009; 115:1121-30. [PMID: 20018912 DOI: 10.1182/blood-2009-09-201616] [Citation(s) in RCA: 97] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Inflammation and coagulation are closely linked interdependent processes. Under physiologic conditions, the tissue microcirculation functions in anticoagulant and anti-inflammatory fashions. However, when inflammation occurs, coagulation is also set in motion and actively participates in enhancing inflammation. Recently, novel and unexpected roles of hemostasis in the humoral and cellular components of innate immunity have been described. In particular, the protein C system, besides its well-recognized role in anticoagulation, plays a crucial role in inflammation. Indeed, the protein C system is now emerging as a novel participant in the pathogenesis of acute and chronic inflammatory diseases, such as sepsis, asthma, inflammatory bowel disease, atherosclerosis, and lung and heart inflammation, and may emerge as unexpected therapeutic targets for intervention.
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28
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Gramling MW, Beaulieu LM, Church FC. Activated protein C enhances cell motility of endothelial cells and MDA-MB-231 breast cancer cells by intracellular signal transduction. Exp Cell Res 2009; 316:314-28. [PMID: 19891966 DOI: 10.1016/j.yexcr.2009.10.024] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2008] [Revised: 10/27/2009] [Accepted: 10/28/2009] [Indexed: 01/06/2023]
Abstract
Activated protein C (APC), an anticoagulant serine protease, has been shown to have non-hemostatic functions related to inflammation, cell survival, and cell migration. In this study we investigate the mechanism by which APC promotes angiogenesis and breast cancer invasion using ex vivo and in vitro methods. When proteolytically active, APC promotes cell motility/invasion and tube formation of endothelial cells. Ex vivo aortic ring assays verify the role of APC in promoting angiogenesis, which was determined to be dependent on EGFR and MMP activation. Given the capacity of APC to promote angiogenesis and the importance of this process in cancer pathology, we investigated whether the mechanisms by which APC promotes angiogenesis can also promote motility and invasion in the MDA-MB-231 breast cancer cell line. Our results indicate that, extracellularly, APC engages EPCR, PAR-1, and EGFR in order to increase the invasiveness of MDA-MB-231 cells. APC activation of matrix metalloprotease (MMP) -2 and/or -9 is necessary but not sufficient to increase invasion, and APC does not utilize the endogenous plasminogen activation system to increase invasion. Intracellularly, APC activates ERK, Akt, and NFkappaB, but not the JNK pathway to promote MDA-MB-231 cell motility. Similar to the hemostatic protease thrombin, APC has the ability to enhance both endothelial cell motility/angiogenesis and breast cancer cell migration.
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Affiliation(s)
- Mark W Gramling
- Departments of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599-7035, USA
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29
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Abstract
Alterations in expression of protein C (PC) pathway components have been identified in patients with active inflammatory disease states. While the PC pathway plays a pivotal role in regulating coagulation and fibrinolysis, activated PC (aPC) also exhibits cytoprotective properties. For example, PC-deficient mice challenged in septic/endotoxemic models exhibit phenotypes that include hypotension, disseminated intravascular coagulation, elevated inflammatory mediators, neutrophil adhesion to the microvascular endothelium, and loss of protective endothelial and epithelial cell barriers. Further, inflammatory bowel disease has been correlated with diminished endothelial PC receptor and thrombomodulin levels in the intestinal mucosa. Downregulated expression of the cofactor, protein S, as well as PC, is also associated with ischemic stroke. Studies to elucidate further the structural elements that differentiate the various functions of PC will serve to identify novel therapeutic approaches toward regulating these and other diseases.
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Affiliation(s)
- F J Castellino
- W. M. Keck Center for Transgene Research, and Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
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30
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Neyrinck AP, Liu KD, Howard JP, Matthay MA. Protective mechanisms of activated protein C in severe inflammatory disorders. Br J Pharmacol 2009; 158:1034-47. [PMID: 19466992 DOI: 10.1111/j.1476-5381.2009.00251.x] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
The protein C system is an important natural anticoagulant mechanism mediated by activated protein C (APC) that regulates the activity of factors VIIIa and Va. Besides well-defined anticoagulant properties, APC also demonstrates anti-inflammatory, anti-apoptotic and endothelial barrier-stabilizing effects that are collectively referred to as the cytoprotective effects of APC. Many of these beneficial effects are mediated through its co-receptor endothelial protein C receptor, and the protease-activated receptor 1, although exact mechanisms remain unclear and are likely pleiotropic in nature. Increased insight into the structure-function relationships of APC facilitated design of APC variants that conserve cytoprotective effects and reduce anticoagulant features, thereby attenuating the risk of severe bleeding with APC therapy. Impairment of the protein C system plays an important role in acute lung injury/acute respiratory distress syndrome and severe sepsis. The pathophysiology of both diseases states involves uncontrolled inflammation, enhanced coagulation and compromised fibrinolysis. This leads to microvascular thrombosis and organ injury. Administration of recombinant human APC to correct the dysregulated protein C system reduced mortality in severe sepsis patients (PROWESS trial), which stimulated further research into its mechanisms of action. Several other clinical trials evaluating recombinant human APC have been completed, including studies in children and less severely ill adults with sepsis as well as a study in acute lung injury. On the whole, these studies have not supported the use of APC in these populations and challenge the field of APC research to search for additional answers.
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Affiliation(s)
- Arne P Neyrinck
- University of California San Francisco, Cardiovascular Research Institute, San Francisco, CA, USA
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31
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Recombinant human activated protein C inhibits integrin-mediated neutrophil migration. Blood 2009; 113:4078-85. [PMID: 19244161 DOI: 10.1182/blood-2008-09-180968] [Citation(s) in RCA: 72] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Integrin-mediated cell migration is central to many biologic and pathologic processes. During inflammation, tissue injury results from excessive infiltration and sequestration of activated leukocytes. Recombinant human activated protein C (rhAPC) has been shown to protect patients with severe sepsis, although the mechanism underlying this protective effect remains unclear. Here, we show that rhAPC directly binds to beta(1) and beta(3) integrins and inhibits neutrophil migration, both in vitro and in vivo. We found that human APC possesses an Arg-Gly-Asp (RGD) sequence, which is critical for the inhibition. Mutation of this sequence abolished both integrin binding and inhibition of neutrophil migration. In addition, treatment of septic mice with a RGD peptide recapitulated the beneficial effects of rhAPC on survival. Thus, we conclude that leukocyte integrins are novel cellular receptors for rhAPC and the interaction decreases neutrophil recruitment into tissues, providing a potential mechanism by which rhAPC may protect against sepsis.
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Abstract
Venous thromboembolism is a major medical problem, annually affecting 1 in 1000 individuals. It is a typical multifactorial disease, involving both genetic and circumstantial risk factors that affect a delicate balance between procoagulant and anticoagulant forces. In the last 50 years, the molecular basis of blood coagulation and the anticoagulant systems that control it have been elucidated. This has laid the foundation for discoveries of both common and rare genetic traits that tip the natural balance in favor of coagulation, with a resulting lifelong increased risk of venous thrombosis. Multiple mutations in the genes for anticoagulant proteins such as antithrombin, protein C, and protein S have been identified and constitute important risk factors. Two single mutations in the genes for coagulation factor V (FV Leiden) and prothrombin (20210G>A), resulting from approximately 20,000-year-old mutations with subsequent founder effects, are common in the general population and constitute major genetic risk factors for thrombosis. In celebration of the 50-year anniversary of the American Society of Hematology, this invited review highlights discoveries that have contributed to our present understanding of the systems that control blood coagulation and the genetic factors that are involved in the pathogenesis of venous thrombosis.
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Severe congenital protein C deficiency: description of a new mutation and prophylactic protein C therapy and in vivo pharmacokinetics. J Pediatr Hematol Oncol 2008; 30:166-71. [PMID: 18376272 DOI: 10.1097/mph.0b013e31815d8943] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Severe congenital protein C deficiency is a rare life-threatening disorder that presents with purpura fulminans, disseminated intravascular coagulation, and thrombotic complications during the neonatal period. Affected children require acute replacement therapy with fresh frozen plasma or protein C concentrate, for example, Ceprotin (Baxter AG, Vienna). Long-term management and outcome is dependent on effective anticoagulation with warfarin, low-molecular weight heparin, or protein C concentrate. We describe the successful use of intravenous protein C concentrate for thrombotic prophylaxis in 2 sisters with severe type I protein C deficiency. Individualized long-term prophylactic regimens were developed based on clinical response. In vivo pharmacokinetic analyses of protein C concentrate were performed in each patient. Analysis of the protein C gene coding sequences identified 2 mutations in both patients, the previously described Arg169 to Trp mutation, and a novel mutation that changes Cys17 into a stop codon.
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35
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Majluf-Cruz A, Moreno-Hernández M, Ruiz-de-Chávez-Ochoa A, Monroy-García R, Majluf-Cruz K, Guardado-Mendoza R, Molina-Ávila I, Isordia-Salas I, Corona-de la Peña N, Vargas-Vorackova F, Vela-Ojeda J, García-Chávez J. Activated Protein C Resistance and Factor V Leiden in Mexico. Clin Appl Thromb Hemost 2007; 14:428-37. [DOI: 10.1177/1076029607306807] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
A common cause of hereditary thrombophilia is activated protein C resistance (APCR), and most cases result from factor V Leiden mutation. An APCR phenotype without association with factor V Leiden has been described. This transversal, observational, nonrandomized study evaluated these 2 phenomena in healthy indigenous and mestizo Mexican subjects (n = 4345), including 600 Mexican natives. No indigenous subjects had APCR, but 82 mestizo subjects did. After retesting, 50 subjects had a negative test. The remaining 32 subjects had factor V Leiden, giving a 0.85% prevalence of factor V Leiden in the mestizo Mexican population. Only 31% of APCR carriers had factor V Leiden. These results show a very low prevalence of APCR and factor V Leiden in Mexico. Except for factor V Leiden, there are no other mutations in the factor V gene responsible for the APCR phenotype. Acquired APCR is nearly twice as prevalent as the inherited variant.
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Affiliation(s)
- Abraham Majluf-Cruz
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico, , amajlufc@ gmail.com
| | - Manuel Moreno-Hernández
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Adriana Ruiz-de-Chávez-Ochoa
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Rosario Monroy-García
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Karim Majluf-Cruz
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Rodolfo Guardado-Mendoza
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Irma Molina-Ávila
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Irma Isordia-Salas
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Norma Corona-de la Peña
- Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico
| | - Florencia Vargas-Vorackova
- Departamento de Gastroenterología, Instituto Nacional de la Nutrición Salvador Zubirán, Mexico City, Mexico
| | - Jorge Vela-Ojeda
- Departamento de Hematología, Hospital de Especialidades, Centro Médico La Raza, IMSS, Mexico City, Mexico
| | - Jaime García-Chávez
- Departamento de Hematología, Hospital de Especialidades, Centro Médico La Raza, IMSS, Mexico City, Mexico
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Altinisik J, Ates O, Ulutin T, Cengiz M, Buyru N. Factor V Leiden, Prothrombin G20210A, and Protein C Mutation Frequency in Turkish Venous Thrombosis Patients. Clin Appl Thromb Hemost 2007; 14:415-20. [DOI: 10.1177/1076029607306404] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Several inherited polymorphisms are associated with risk of venous thrombosis, including mutation at codon 506 of the factor V gene, mutation at position 20210 of the prothrombin gene, and mutations in the protein C gene. In this study, genotyping for factor V, prothrombin, and protein C mutations was performed in 50 patients and 25 control subjects by polymerase chain reaction—based analysis. The prevalence of factor V and prothrombin mutations was not significantly different from that in the general population. Nine of the patients had heterozygous protein C mutation. There was a high prevalence of the mutated protein C allele in the pulmonary emboli group (42.8%). Protein C mutation incidence was higher in the pulmonary emboli group than in the deep vein thrombosis (8.33%) and cerebral vein thrombosis (16.1%) groups. These results indicate that patients with protein C deficiency have a greater risk of thrombosis than patients with factor V or prothrombin G20210A mutation.
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Affiliation(s)
- Julide Altinisik
- Cerrahpasa Medical Faculty, Department of Medical Biology, Istanbul University, Istanbul, Turkey
| | - Omer Ates
- Cerrahpasa Medical Faculty, Department of Medical Biology, Istanbul University, Istanbul, Turkey
| | - Turgut Ulutin
- Cerrahpasa Medical Faculty, Department of Medical Biology, Istanbul University, Istanbul, Turkey
| | - Mujgan Cengiz
- Cerrahpasa Medical Faculty, Department of Medical Biology, Istanbul University, Istanbul, Turkey
| | - Nur Buyru
- Cerrahpasa Medical Faculty, Department of Medical Biology, Istanbul University, Istanbul, Turkey,
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Mangan S, Clancy P, Golledge J. Modulation of endothelial cell thrombomodulin by PPAR ligands--variation according to environment. Thromb Res 2007; 121:827-34. [PMID: 17869327 PMCID: PMC2577783 DOI: 10.1016/j.thromres.2007.08.007] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2007] [Revised: 07/19/2007] [Accepted: 08/09/2007] [Indexed: 11/21/2022]
Abstract
INTRODUCTION Thrombomodulin (TM) is an important anti-coagulant protein that is down-regulated on endothelial cells overlying atherosclerotic plaques. We investigated the effects of the peroxisome proliferator-activated receptor (PPAR) ligands, fenofibrate and rosiglitazone, on the expression of TM ex vivo by advanced carotid atheromas, and in vitro by endothelial cells. METHODS Adjacent carotid atheroma biopsies were incubated in vehicle control or PPAR ligand in explant culture for 4 days. Human aortic endothelial cells were incubated with PPAR ligands in vitro. TM expression was measured by Western blotting and flow cytometry. TM activity was assessed by generation of activated protein C. RESULTS The PPAR-alpha activator, fenofibrate, up-regulated total TM expression within carotid explants by 1.7-fold (P<0.001) with no effect on activity. Rosiglitazone treatment had no effect on protein levels but reduced activity by 73% of the control (P<0.05). We noted disparate effects of PPAR ligands in atheroma samples from different patients and postulated that the response of endothelial cells to medication was influenced by the atheromatous environment. Incubation of human aortic endothelial cells with fenofibrate alone led to a dose-dependent increase in TM expression (P<0.05), however, in the presence of oxidized LDL a dose-dependent reduction in TM expression was induced by fenofibrate (P<0.05). CONCLUSIONS The ability of fenofibrate to increase endothelial cell and carotid atheroma TM protein expression suggests a potential therapeutic role for this medication. The response to PPAR ligands likely varies depending on the exact constituents of individual atherosclerotic plaques, such as the relative amount of oxidized LDL.
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Affiliation(s)
- Simone Mangan
- Vascular Biology Unit, School of Medicine, James Cook University, Townsville, Queensland, 4811, Australia
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38
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Chuansumrit A, Kanogsunthornrat N, Sirachainan N, Porapakpenjun S, Chantarukha R, Warrasak S, Wongwerawattanakoon P. Negative pressure dressing for promoting wound healing of purpura fulminans in a newborn with homozygous protein C deficiency. Blood Coagul Fibrinolysis 2007; 18:77-9. [PMID: 17179832 DOI: 10.1097/mbc.0b013e3280120e16] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
A negative pressure dressing to promote wound healing of purpura fulminans in a girl aged 35 days with homozygous protein C deficiency is reported. Two wounds of 11 x 11 cm2 at the abdominal wall and 14 x 14 cm2 at the left trunk were covered with sterile sponges embedded with a multiple-hole drain tube and transparent plastic film. The exposed end of the drain was then connected to the wall suction apparatus to create negative pressure at -120 mmHg. The dressing was changed every 2 days. Within 4-6 weeks, the wounds were completely healed and skin grafting was not required.
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Affiliation(s)
- Ampaiwan Chuansumrit
- Department of Pediatrics, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
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Beaulieu LM, Church FC. Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1. Exp Cell Res 2006; 313:677-87. [PMID: 17254565 PMCID: PMC4055951 DOI: 10.1016/j.yexcr.2006.11.019] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2006] [Revised: 10/27/2006] [Accepted: 11/13/2006] [Indexed: 11/17/2022]
Abstract
Activated protein C (APC) is a serine protease that regulates thrombin (IIa) production through inactivation of blood coagulation factors Va and VIIIa. APC also has non-hemostatic functions related to inflammation, proliferation, and apoptosis through various mechanisms. Using two breast cancer cell lines, MDA-MB-231 and MDA-MB-435, we investigated the role of APC in cell chemotaxis and invasion. Treatment of cells with increasing APC concentrations (1-50 microg/ml) increased invasion and chemotaxis in a concentration-dependent manner. Only the active form of APC increased invasion and chemotaxis of the MDA-MB-231 cells when compared to 3 inactive APC derivatives. Using a modified "checkerboard" analysis, APC was shown to only affect migration when plated with the cells; therefore, APC is not a chemoattractant. Blocking antibodies to endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) attenuated the effects of APC on chemotaxis in the MDA-MB-231 cells. Finally, treatment of the MDA-MB-231 cells with the proliferation inhibitor, Na butyrate, showed that APC did not increase migration by increasing cell number. Therefore, APC increases invasion and chemotaxis of cells by binding to the cell surface and activating specific signaling pathways through EPCR and PAR-1.
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Affiliation(s)
- Lea M. Beaulieu
- Department of Pathology and Laboratory Medicine, University of North Carolina, School of Medicine, Chapel Hill, NC 27599, USA
| | - Frank C. Church
- Department of Pathology and Laboratory Medicine, University of North Carolina, School of Medicine, Chapel Hill, NC 27599, USA
- Departments of Medicine and Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, NC 27599, USA
- Corresponding author. Department of Pathology and Laboratory Medicine, University of North Carolina-Chapel Hill, 932 Mary Ellen Jones Building, CB#7035, Chapel Hill, NC 27599-7035, USA. Fax: +1 919 966 7639 (F.C. Church)
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40
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Butenas S, Orfeo T, Kalafatis M, Mann KG. Peptidomimetic inhibitors for activated protein C: implications for hemophilia management. J Thromb Haemost 2006; 4:2411-6. [PMID: 17059471 DOI: 10.1111/j.1538-7836.2006.02226.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
BACKGROUND Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.
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Affiliation(s)
- S Butenas
- Department of Biochemistry, University of Vermont, Colchester, VT 05446, USA.
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Lay AJ, Donahue D, Tsai MJ, Castellino FJ. Acute inflammation is exacerbated in mice genetically predisposed to a severe protein C deficiency. Blood 2006; 109:1984-91. [PMID: 17047151 PMCID: PMC1801051 DOI: 10.1182/blood-2006-07-037945] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The anticoagulant, activated protein C (aPC), possesses antithrombotic, profibrinolytic, anti-inflammatory, and antiapoptotic properties, and the level of this protein is an important marker of acute inflammatory responses. Although infusion of aPC improves survival in a subset of patients with severe sepsis, evidence as to how aPC decreases mortality in these cases is limited. Because a total deficiency of PC shows complete neonatal lethality, no animal model currently exists to address the mechanistic relationships between very low endogenous aPC levels and inflammatory diseases. Here, we show for the first time that novel genetic dosing of PC strongly correlates with survival outcomes following endotoxin (LPS) challenge in mice. The data provide evidence that very low endogenous levels of PC predispose mice to early-onset disseminated intravascular coagulation, thrombocytopenia, hypotension, organ damage, and reduced survival after LPS challenge. Furthermore, evidence of an exacerbated inflammatory response is observed in very low PC mice but is greatly reduced in wild-type cohorts. Reconstitution of low-PC mice with recombinant human aPC improves hypotension and extends survival after LPS challenge. This study directly links host endogenous levels of PC with various coagulation, inflammation, and hemodynamic end points following a severe acute inflammatory challenge.
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Affiliation(s)
- Angelina J Lay
- W. M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IN 46556, USA
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Tseng PY, Rele SS, Sun XL, Chaikof EL. Membrane-mimetic films containing thrombomodulin and heparin inhibit tissue factor-induced thrombin generation in a flow model. Biomaterials 2006; 27:2637-50. [PMID: 16376423 DOI: 10.1016/j.biomaterials.2005.10.025] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2005] [Accepted: 10/29/2005] [Indexed: 11/23/2022]
Abstract
Membrane-mimetic thin films containing thrombomodulin (TM) and/or heparin were produced and their capacity to inhibit thrombin generation evaluated in a continuous flow system. Tissue factor (TF) along with TM and heparin were immobilized in spatially restricted zones as components of a membrane-mimetic film. Specifically, TF was positioned as an upstream trigger for thrombin generation and TM and/or heparin positioned over the remaining downstream portion of test films. Peak and steady-state levels of thrombin were decreased by antithrombin III (ATIII), as well as by surface bound heparin and TM. Although physiologic concentrations of ATIII have the capacity to significantly inhibit thrombin activity, surface bound TM and heparin nearly abolished steady-state thrombin responses. In particular, surface bound TM appears to be superior to heparin in reducing local thrombin concentrations. These studies are the first to demonstrate the additive effect of surface bound heparin and TM as a combined interactive strategy to limit TF-induced thrombin formation.
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Affiliation(s)
- Po-Yuan Tseng
- School of Chemical Engineering, Georgia Institute of Technology, Atlanta, 30320, USA
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Tsuneyoshi N, Fukudome K, Higuchi K, Kimoto M. Preparation and Characterization of Monoclonal Antibodies to Thrombomodulin. Hybridoma (Larchmt) 2005; 24:189-96. [PMID: 16120024 DOI: 10.1089/hyb.2005.24.189] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Thrombomodulin (TM) is an endothelial cell surface molecule, capable of specific binding for thrombin. The thrombin/TM complex promotes activation of plasma anticoagulant protein C (PC) and negatively regulates blood coagulation. Along with anticoagulant function, TM has been shown to have additional physiological functions such as regulation of fibrinolysis, cell adhesion, tumor growth, and embryonic development. The extracellular region of TM contains a lectin domain and six epidermal growth factor (EGF)-like domains, which are required for the various functions. To analyze the functions, we established a panel of monoclonal antibodies (MAbs) reactive to each functional domain. We obtained MAbs that react to the lectin domain or the front half of EGF domains from the first to the third using the antigen of a transfected cell line expressing full-length TM. We also obtained MAbs that reacted to the bottom half of the EGF domain from the fourth to the sixth using the antigen of a transfected cell line expressing truncated form of TM lacking the lectin domain and the EGF domains from the first to the third. All obtained MAbs could be used for Western blotting. Endothelial cell function for PC activation can be mimicked by transfected cells positive for TM and the endothelial cell protein C receptor (EPCR). Effects of the established MAbs on thrombin-dependent PC activation on the transfected cells were examined. Strong inhibition was demonstrated by three MAbs, which reacted to the fourth or fifth EGF domain, but not by MAbs to the other domains. The fourth EGF domain is known as the interaction site for PC, and the fifth domain is known to be required for thrombin binding. The sixth EGF domain also has been shown to be required for thrombin binding. An MAb against the domain strongly inhibited thrombin-binding. However, the MAb demonstrated little effect on thrombin dependent PC activation. The contradictory results demonstrated with the MAb to the sixth EGF domain suggest an unknown molecular mechanism for PC activation on the cell surface. A panel of MAbs reactive to each domain could be useful for analyzing the multifunctional molecule thrombomodulin.
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Lay AJ, Liang Z, Rosen ED, Castellino FJ. Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities. J Clin Invest 2005; 115:1552-61. [PMID: 15902301 PMCID: PMC1089798 DOI: 10.1172/jci24030] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2004] [Accepted: 03/29/2005] [Indexed: 12/11/2022] Open
Abstract
Anticoagulant protein C (PC) is important not only for maintenance of normal hemostasis, but also for regulating the host immune response during inflammation. Because mice with a designed total genetic deficiency in PC (PC-/- mice) die soon after birth, attempts to dissect PC function in various coagulation/inflammation-based pathologies through use of mice with less than 50% of normal PC levels have not been successful to date. In the current investigation, we have used a novel transgenic strategy to generate different mouse models expressing 1-18% of normal PC levels. In contrast to PC-/- mice, mice with only partial PC deficiency survived beyond birth and also developed thrombosis and inflammation. The onset and severity of these phenotypes vary significantly and are strongly dependent on plasma PC levels. Our findings additionally provide the first evidence that maternal PC is vital for sustaining pregnancy beyond 7.5 days postcoitum, likely by regulating the balance of coagulation and inflammation during trophoblast invasion. These low PC-expressing transgenic mouse lines provide novel animal models that can be used to elucidate the importance of PC in maintenance of the organism and in disease.
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Affiliation(s)
- Angelina J Lay
- W.M. Keck Center for Transgene Research, University of Notre Dame, Notre Dame, Indiana 46556, USA
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Obayashi K, Hanyu O, Miida T, Ando Y, Nakamura M, Zhu Y, Okada M. Circadian rhythm of protein C in human plasma--useful marker of autonomic function in liver. Clin Auton Res 2005; 15:57-8. [PMID: 15768205 DOI: 10.1007/s10286-005-0219-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2004] [Accepted: 07/20/2004] [Indexed: 10/25/2022]
Abstract
We have demonstrated changes in the circadian rhythm of plasma protein C levels in patients with autonomic dysfunction and liver-transplanted patients, compared with that in healthy volunteers. The circadian rhythm of protein C serves as a useful marker to screen for autonomic dysfunction in the liver.
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46
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Preston RJS, Villegas-Mendez A, Sun YH, Hermida J, Simioni P, Philippou H, Dahlbäck B, Lane DA. Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation. FEBS J 2004; 272:97-108. [PMID: 15634335 DOI: 10.1111/j.1432-1033.2004.04401.x] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23 x 10(5) m(-1).s(-1), a dissociation rate constant of 7.61 x 10(-2) s(-1) and equilibrium binding constant (K(D)) of 147 nm. It was activated by thrombin over endothelial cells with a K(m) of 213 nm and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R-1L, R-1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (< twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased K(m) (358 nm for QGNSEDY) for endothelial cell surface activation by thrombin. Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa. Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation.
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Affiliation(s)
- Roger J S Preston
- Department of Haematology, Division of Investigative Science, Hammersmith Campus, Imperial College London, UK
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Ganopolsky JG, Castellino FJ. A protein C deficiency exacerbates inflammatory and hypotensive responses in mice during polymicrobial sepsis in a cecal ligation and puncture model. THE AMERICAN JOURNAL OF PATHOLOGY 2004; 165:1433-46. [PMID: 15466407 PMCID: PMC1618621 DOI: 10.1016/s0002-9440(10)63401-3] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
During the systemic inflammatory state induced by sepsis, the potential for coagulopathy exists because of up-regulation of natural procoagulants and anti-fibrinolytics, and down-regulation of natural anti-coagulants, with protein C (PC) being a critical example of the latter case. PC functions as an anti-coagulant, profibrinolytic, and anti-inflammatory agent, and, thus, its administration or deficiency may affect the course and outcome of sepsis in patients. In this study, a cecal ligation and puncture model of septic peritonitis was applied to wild-type mice and littermates with a targeted heterozygous deficiency of PC (PC(+/-)) to characterize the importance of a PC-deficiency on polymicrobial sepsis. An enhanced mortality rate was found to accompany a PC deficiency. Plasma cytokines, as well as organ-specific expression of cytokine transcripts, were elevated in PC(+/-) mice. No signs of severe disseminated intravascular coagulation (DIC) were observed in wild-type or PC(+/-) mice, as indicated by an increase in fibrinogen levels and the invariability of platelet counts after cecal ligation and puncture. Consumption of coagulation factors was similar in both genotypes and a decrease in the PC mRNA and protein levels was more prominent in PC(+/-) mice. Renal and organ muscle damage was enhanced in PC(+/-) mice, as shown by increases in plasma blood urea nitrogen, creatinine, and creatinine kinase. Hypotension and bradycardia were more enhanced in PC(+/-) mice than in wild-type mice, thus provoking a more severe septic shock response. Thus, the hemodynamic role of PC during sepsis is of critical importance to the outcome of the disease.
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Affiliation(s)
- Jorge G Ganopolsky
- W.M. Keck Center for Transgene Research, Department of Chemistry and Biochemistry, 434 Stepan Hall of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
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Van de Wouwer M, Collen D, Conway EM. Thrombomodulin-protein C-EPCR system: integrated to regulate coagulation and inflammation. Arterioscler Thromb Vasc Biol 2004; 24:1374-83. [PMID: 15178554 DOI: 10.1161/01.atv.0000134298.25489.92] [Citation(s) in RCA: 260] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Late in the 18th century, William Hewson recognized that the formation of a clot is characteristic of many febrile, inflammatory diseases (Owen C. A History of Blood Coagulation. Rochester, Minnesota: Mayo Foundation; 2001). Since that time, there has been steady progress in our understanding of coagulation and inflammation, but it is only in the past few decades that the molecular mechanisms linking these 2 biologic systems have started to be delineated. Most of these can be traced to the vasculature, where the systems most intimately interact. Thrombomodulin (TM), a cell surface-expressed glycoprotein, predominantly synthesized by vascular endothelial cells, is a critical cofactor for thrombin-mediated activation of protein C (PC), an event further amplified by the endothelial cell protein C receptor (EPCR). Activated PC (APC), in turn, is best known for its natural anticoagulant properties. Recent evidence has revealed that TM, APC, and EPCR have activities that impact not only on coagulation but also on inflammation, fibrinolysis, and cell proliferation. This review highlights recent insights into the diverse functions of this complex multimolecular system and how its components are integrated to maintain homeostasis under hypercoagulable and/or proinflammatory stress conditions. Overall, the described advances underscore the usefulness of elucidating the relevant molecular pathways that link both systems for the development of novel therapeutic and diagnostic targets for a wide range of inflammatory diseases.
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Affiliation(s)
- Marlies Van de Wouwer
- The Center for Transgene Technology and Gene Therapy, University of Leuven and the Flanders Interuniversity Institute for Biotechnology (VIB), Belgium
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Abstract
The objective of this study was to review the mechanisms by which thrombomodulin (TM) may modulate inflammation. The data were taken from published research performed by other laboratories and our own experimental results. TM is a transmembrane glycoprotein receptor and cofactor for thrombin in the protein C anticoagulant system. Recent studies have revealed that TM has activities, both dependent and independent of either protein C or thrombin, that affect biological systems beyond the coagulation pathway. This review highlights recent insights, provided by in vitro and in vivo analyses, into how the unique structural domains of TM effectively modify coagulation, fibrinolysis, and inflammation in health and disease. A paradigm is presented to describe how these apparently distinct functions are integrated to maintain homeostasis under stress conditions. Finally, we explore the potential diagnostic and therapeutic utility of dissecting out the structure-function correlates of TM. We conclude that TM plays a central role in regulating not only hemostasis but also inflammation, thus providing a close link between these processes. Elucidation of the molecular mechanisms by which TM functions will likely provide novel targets for therapeutic intervention.
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Affiliation(s)
- Marlies Van de Wouwer
- Flanders Interuniversity Institute for Biotechnology and the Center for Transgene Technology and Gene Therapy, University of Leuven, Leuven, Belgium
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50
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Affiliation(s)
- C T Esmon
- Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
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