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Lorenzoni S, Rodríguez-Nogales C, Blanco-Prieto MJ. Targeting tumor microenvironment with RGD-functionalized nanoparticles for precision cancer therapy. Cancer Lett 2025; 614:217536. [PMID: 39924081 DOI: 10.1016/j.canlet.2025.217536] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2024] [Revised: 01/21/2025] [Accepted: 02/06/2025] [Indexed: 02/11/2025]
Abstract
The need for precision therapies arises from the complexities associated with high-risk types of cancer, due to their aggressiveness and resistance to treatment. These diseases represent a global issue that requires transversal strategies involving cooperation among oncology specialists and experts from related fields, including nanomedicine. Nanoparticle-mediated active targeting of tumors has proven to be a revolutionary approach to address the most challenging neoplasms by overcoming the poor permeation at tumor site of untargeted, and nowadays questioned, strategies that rely solely on Enhanced Permeability and Retention (EPR) effects. The decoration of nanoparticles with Arg-Gly-Asp (RGD) peptides, which selectively target integrins on the cell membrane, marks a turning point in tumor microenvironment (TME) targeted strategies, enabling precision and efficiency in the delivery of chemotherapeutics. This review delves into the intricacies of the TME's features and targetable components (i.e. integrins), and the development of RGDs for nanoparticles' functionalization for active TME targeting. It provides a translational perspective on the integration of RGD-functionalized nanoparticles in oncology, highlighting their potential to overcome current therapeutic challenges, particularly in precision medicine. The current landscape of targeted nanomedicines in the clinic, and the development of RGD-nanomedicine for pediatric cancers are also discussed.
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Affiliation(s)
- Sara Lorenzoni
- Department of Pharmaceutical Sciences, School of Pharmacy and Nutrition, Universidad de Navarra, C/Irunlarrea 1, 31008, Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, Pamplona, 31008, Pamplona, Spain; Cancer Center Clínica Universidad de Navarra (CCUN), Avenida Pio XII 36, 31008, Pamplona, Spain
| | - Carlos Rodríguez-Nogales
- Department of Pharmaceutical Sciences, School of Pharmacy and Nutrition, Universidad de Navarra, C/Irunlarrea 1, 31008, Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, Pamplona, 31008, Pamplona, Spain; Cancer Center Clínica Universidad de Navarra (CCUN), Avenida Pio XII 36, 31008, Pamplona, Spain.
| | - María J Blanco-Prieto
- Department of Pharmaceutical Sciences, School of Pharmacy and Nutrition, Universidad de Navarra, C/Irunlarrea 1, 31008, Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, Pamplona, 31008, Pamplona, Spain; Cancer Center Clínica Universidad de Navarra (CCUN), Avenida Pio XII 36, 31008, Pamplona, Spain.
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Coffman RE, Bidone TC. Application of Funnel Metadynamics to the Platelet Integrin αIIbβ3 in Complex with an RGD Peptide. Int J Mol Sci 2024; 25:6580. [PMID: 38928286 PMCID: PMC11203998 DOI: 10.3390/ijms25126580] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Revised: 06/11/2024] [Accepted: 06/11/2024] [Indexed: 06/28/2024] Open
Abstract
Integrin αIIbβ3 mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the αIIb and β3 subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to αIIbβ3 integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the αIIb and β3 subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the β3 subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin.
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Affiliation(s)
- Robert E. Coffman
- Scientific Computing and Imaging Institute, University of Utah, Salt Lake City, UT 84112, USA;
| | - Tamara C. Bidone
- Scientific Computing and Imaging Institute, University of Utah, Salt Lake City, UT 84112, USA;
- Department of Biomedical Engineering, University of Utah, Salt Lake City, UT 84112, USA
- Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA
- Department of Molecular Pharmaceutics, University of Utah, Salt Lake City, UT 84112, USA
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Hu HJ, Xiao XR, Li T, Liu DM, Geng X, Han M, Cui W. Integrin beta 3-overexpressing mesenchymal stromal cells display enhanced homing and can reduce atherosclerotic plaque. World J Stem Cells 2023; 15:931-946. [PMID: 37900938 PMCID: PMC10600744 DOI: 10.4252/wjsc.v15.i9.931] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Revised: 06/22/2023] [Accepted: 08/23/2023] [Indexed: 09/25/2023] Open
Abstract
BACKGROUND Umbilical cord (UC) mesenchymal stem cell (MSC) transplantation is a potential therapeutic intervention for atherosclerotic vascular disease. Integrin beta 3 (ITGB3) promotes cell migration in several cell types. However, whether ITGB-modified MSCs can migrate to plaque sites in vivo and play an anti-atherosclerotic role remains unclear. AIM To investigate whether ITGB3-overexpressing MSCs (MSCsITGB3) would exhibit improved homing efficacy in atherosclerosis. METHODS UC MSCs were isolated and expanded. Lentiviral vectors encoding ITGB3 or green fluorescent protein (GFP) as control were transfected into MSCs. Sixty male apolipoprotein E-/- mice were acquired from Beijing Vital River Lab Animal Technology Co., Ltd and fed with a high-fat diet (HFD) for 12 wk to induce the formation of atherosclerotic lesions. These HFD-fed mice were randomly separated into three clusters. GFP-labeled MSCs (MSCsGFP) or MSCsITGB3 were transplanted into the mice intravenously via the tail vein. Immunofluorescence staining, Oil red O staining, histological analyses, western blotting, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction were used for the analyses. RESULTS ITGB3 modified MSCs successfully differentiated into the "osteocyte" and "adipocyte" phenotypes and were characterized by positive expression (> 91.3%) of CD29, CD73, and CD105 and negative expression (< 1.35%) of CD34 and Human Leukocyte Antigen-DR. In a transwell assay, MSCsITGB3 showed significantly faster migration than MSCsGFP. ITGB3 overexpression had no effects on MSC viability, differentiation, and secretion. Immunofluorescence staining revealed that ITGB3 overexpression substantially enhanced the homing of MSCs to plaque sites. Oil red O staining and histological analyses further confirmed the therapeutic effects of MSCsITGB3, significantly reducing the plaque area. Enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction revealed that MSCITGB3 transplantation considerably decreased the inflammatory response in pathological tissues by improving the dynamic equilibrium of pro- and anti-inflammatory cytokines. CONCLUSION These results showed that ITGB3 overexpression enhanced the MSC homing ability, providing a potential approach for MSC delivery to plaque sites, thereby optimizing their therapeutic effects.
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Affiliation(s)
- Hai-Juan Hu
- First Division, Department of Cardiology, The Second Hospital of Hebei Medical University and Institute of Cardiocerebrovascular Disease of Hebei Province, Shijiazhuang 050000, Hebei Province, China
| | - Xue-Ru Xiao
- Department of Obstetrics, Shijiazhuang People's Hospital, Shijiazhuang 050030, Hebei Province, China
| | - Tong Li
- First Division, Department of Cardiology, The Second Hospital of Hebei Medical University and Institute of Cardiocerebrovascular Disease of Hebei Province, Shijiazhuang 050000, Hebei Province, China
| | - De-Min Liu
- First Division, Department of Cardiology, The Second Hospital of Hebei Medical University and Institute of Cardiocerebrovascular Disease of Hebei Province, Shijiazhuang 050000, Hebei Province, China
| | - Xue Geng
- First Division, Department of Cardiology, The Second Hospital of Hebei Medical University and Institute of Cardiocerebrovascular Disease of Hebei Province, Shijiazhuang 050000, Hebei Province, China
| | - Mei Han
- Key Laboratory of Medical Biotechnology of Hebei Province, Department of Biochemistry and Molecular Biology, College of Basic Medicine, Cardiovascular Medical Science Center, Hebei Medical University, Shijiazhuang 050017, Hebei Province, China
| | - Wei Cui
- First Division, Department of Cardiology, The Second Hospital of Hebei Medical University and Institute of Cardiocerebrovascular Disease of Hebei Province, Shijiazhuang 050000, Hebei Province, China.
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Khamessi O, Ben Mabrouk H, Hkimi C, Rtima R, Kamoun S, Kharrat R, Ghedira K. DisintegrinDB: The first integrated database resource of disintegrins from snake venoms. Biochem Biophys Res Commun 2022; 597:77-82. [PMID: 35124463 DOI: 10.1016/j.bbrc.2022.01.117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Revised: 01/25/2022] [Accepted: 01/29/2022] [Indexed: 11/20/2022]
Abstract
Nowadays, a large number of databases have been developed gathering different types of therapeutic peptides including antimicrobial, antiviral and scorpion toxins peptides facilitating the searching for these molecules and their structural characteristics and pharmacology. Disintegrins, a family of small non-enzymatic and cysteine-rich proteins found in the snake venom may have a potential role in terms of novel therapeutic leads for cancer treatment. Despite their therapeutic effect, no database dedicated to disintegrins is available yet. Indeed, accessible information related to disintegrins are either scattered or fragmented in different databases from which it becomes extremely difficult to collect all the properties related to a particular disintegrin without exploring numerous databases available through distinct websites. Here, we propose DisintegrinDB as a first unique resource centralizing data related to disintegrins from snake venom. DisintegrinDB aims to facilitate the search on a given disintegrin and centralizes all the information on these peptides, helping researchers to retrieve all relevant related information.
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Affiliation(s)
- Oussema Khamessi
- Laboratoire des Venins et Molécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13 Place Pasteur BP74, Tunis Belvédère, University of Tunis El Manar, Tunisia.
| | - Hazem Ben Mabrouk
- Laboratoire des Venins et Molécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13 Place Pasteur BP74, Tunis Belvédère, University of Tunis El Manar, Tunisia
| | - Chaima Hkimi
- Laboratory of Bioinformatics, Biomathematics and Biostatistics LR20IPT09, Pasteur Institute of Tunis, 1002, University of Tunis El Manar, Tunis, Tunisia
| | - Rawa Rtima
- Laboratoire des Venins et Molécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13 Place Pasteur BP74, Tunis Belvédère, University of Tunis El Manar, Tunisia
| | - Selim Kamoun
- Laboratory of Bioinformatics, Biomathematics and Biostatistics LR20IPT09, Pasteur Institute of Tunis, 1002, University of Tunis El Manar, Tunis, Tunisia
| | - Riadh Kharrat
- Laboratoire des Venins et Molécules Thérapeutiques LR11IPT08, Institut Pasteur de Tunis, 13 Place Pasteur BP74, Tunis Belvédère, University of Tunis El Manar, Tunisia
| | - Kais Ghedira
- Laboratory of Bioinformatics, Biomathematics and Biostatistics LR20IPT09, Pasteur Institute of Tunis, 1002, University of Tunis El Manar, Tunis, Tunisia
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Wang J, Peng H, Timur AA, Pasupuleti V, Yao Y, Zhang T, You SA, Fan C, Yu Y, Jia X, Chen J, Xu C, Chen Q, Wang Q. Receptor and Molecular Mechanism of AGGF1 Signaling in Endothelial Cell Functions and Angiogenesis. Arterioscler Thromb Vasc Biol 2021; 41:2756-2769. [PMID: 34551592 PMCID: PMC8580577 DOI: 10.1161/atvbaha.121.316867] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Objective Angiogenic factor AGGF1 (angiogenic factor with G-patch and FHA [Forkhead-associated] domain 1) promotes angiogenesis as potently as VEGFA (vascular endothelial growth factor A) and regulates endothelial cell (EC) proliferation, migration, specification of multipotent hemangioblasts and venous ECs, hematopoiesis, and vascular development and causes vascular disease Klippel-Trenaunay syndrome when mutated. However, the receptor for AGGF1 and the underlying molecular mechanisms remain to be defined. Approach and Results Using functional blocking studies with neutralizing antibodies, we identified [alpha]5[beta]1 as the receptor for AGGF1 on ECs. AGGF1 interacts with [alpha]5[beta]1 and activates FAK (focal adhesion kinase), Src (proto-oncogene tyrosine-protein kinase), and AKT (protein kinase B). Functional analysis of 12 serial N-terminal deletions and 13 C-terminal deletions by every 50 amino acids mapped the angiogenic domain of AGGF1 to a domain between amino acids 604-613 (FQRDDAPAS). The angiogenic domain is required for EC adhesion and migration, capillary tube formation, and AKT activation. The deletion of the angiogenic domain eliminated the effects of AGGF1 on therapeutic angiogenesis and increased blood flow in a mouse model for peripheral artery disease. A 40-mer or 15-mer peptide containing the angiogenic domain blocks AGGF1 function, however, a 15-mer peptide containing a single amino acid mutation from -RDD- to -RGD- (a classical RGD integrin-binding motif) failed to block AGGF1 function. Conclusions We have identified integrin [alpha]5[beta]1 as an EC receptor for AGGF1 and a novel AGGF1-mediated signaling pathway of [alpha]5[beta]1-FAK-Src-AKT for angiogenesis. Our results identify an FQRDDAPAS angiogenic domain of AGGF1 crucial for its interaction with [alpha]5[beta]1 and signaling.
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Affiliation(s)
- Jingjing Wang
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
- Institute of Genetics and Development, Chinese Academy of Sciences, Beijing, China
| | - Huixin Peng
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
| | - Ayse Anil Timur
- Robert J. Tomsich Pathology & Laboratory Medicine Institute Cleveland Clinic, Cleveland, OH 44195, USA
- Department of Molecular Cardiology, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Vinay Pasupuleti
- Department of Molecular Cardiology, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
- Department of Biological Sciences, Kent State University, Kent, OH 44242, USA
- Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA
| | - Yufeng Yao
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
| | - Teng Zhang
- Yueyang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Sun-Ah You
- Department of Molecular Cardiology, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
- Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA
| | - Chun Fan
- Department of Molecular Cardiology, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
- Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA
| | - Yubing Yu
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
| | - Xinzhen Jia
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
| | - Jing Chen
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
| | - Chengqi Xu
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
| | - Qiuyun Chen
- Department of Molecular Cardiology, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
- Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA
- Present Address, Department of Pathology, Case Western Reserve University School of Medicine, Cleveland OH 44106, USA
| | - Qing Wang
- Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
- Department of Molecular Cardiology, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
- Department of Biological Sciences, Kent State University, Kent, OH 44242, USA
- Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA
- Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland OH 44106, USA
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Alvandi Z, Al-Mansoori LJR, Opas M. Calreticulin regulates Src kinase in osteogenic differentiation from embryonic stem cells. Stem Cell Res 2020; 48:101972. [PMID: 32916637 DOI: 10.1016/j.scr.2020.101972] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2019] [Revised: 06/16/2020] [Accepted: 08/25/2020] [Indexed: 11/26/2022] Open
Abstract
Calreticulin, the major Ca2+ buffer of the endoplasmic reticulum plays an important role in the choice of fate by embryonic stem cells. Using the embryoid body method of organogenesis, we showed impaired osteogenesis in crt-/- cells vis-à-vis calreticulin-containing osteogenic WT cells. In the non-osteogenic crt-/- cells, c-Src- a non-receptor tyrosine kinase- was activated and its inhibition rescued osteogenesis. Most importantly, we demonstrated that calreticulin-containing cells had lower c-Src kinase activity, and this was accomplished via the Ca2+-homeostatic function of calreticulin. Specifically, lowering cytosolic [Ca2+] in calreticulin-containing osteogenic WT cells with BAPTA-AM, activated c-Src and impaired osteogenic differentiation. Conversely, increasing cytosolic [Ca2+] in crt-/- cells with ionomycin deactivated c-Src kinase and restored osteogenesis. The immediate effector of calreticulin, the Ser/Thr phosphatase calcineurin, was less active in crt-/- cells, however, its activity was rescued upon inhibition of c-Src activity by small molecule inhibitors. Finally, we showed that higher activity of calcineurin correlated with increased level of nuclear Runx2, a transcription factor that is the master regulator of osteogenesis. Collectively, our work has identified a novel pathway involving calreticulin regulated Ca2+ signalling via c-Src in osteogenic differentiation of embryonic stem cells.
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Affiliation(s)
- Zahra Alvandi
- Department of Lab Medicine & Pathobiology, University of Toronto, Toronto, ON M5S1A8, Canada.
| | - Layla J R Al-Mansoori
- Department of Lab Medicine & Pathobiology, University of Toronto, Toronto, ON M5S1A8, Canada
| | - Michal Opas
- Department of Lab Medicine & Pathobiology, University of Toronto, Toronto, ON M5S1A8, Canada
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Liu L, Boyd SD, Bulla LA, Winkler DD. "The Defined Toxin-binding Region of the Cadherin G-protein Coupled Receptor, BT-R 1, for the Active Cry1Ab Toxin of Bacillus thuringiensis". ACTA ACUST UNITED AC 2018; 11:201-210. [PMID: 30740004 PMCID: PMC6366636 DOI: 10.4172/0974-276x.1000487] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The bacterium Bacillus thuringiensis (Bt) produces protoxin proteins in parasporal crystals. Proteolysis of the protoxin generates an active toxin which is a potent microbial insecticide. Additionally, Bt toxin genes have been introduced into genetically modified crops to produce insecticidal toxins which protect crops from insect invasion. The insecticidal activity of Cry toxins is mediated by specific interaction between toxins and their respective cellular receptors. One such toxin (Cry1Ab) exerts toxicity by first targeting the 12th ectodomain region (EC12) of the moth cadherin receptor BT-R1. Binding promotes a highly regulated signaling cascade event that concludes in oncotic-like cell death. We previously determined that conserved sequence motifs near the N- and C-termini of EC12 are critical for toxin binding in insect cells. Here, we have established that Cry1Ab specifically binds to EC12 as a soluble heterodimeric complex with extremely high affinity (Kd = 19.5 ± 1.6 nM). Binding assays using Cry1Ab toxin and a fluorescently labeled EC12 revealed that the heterodimeric complex is highly specific in that no such formation occurs between EC12 and other Cry toxins active against beetle and mosquito. Disruption of one or both terminal sequence motifs in EC12 eliminates complex formation. Until now, comprehensive biophysical characterization of Cry1Ab recognition and binding by the BT-R1 receptor was unresolved. The findings presented here provide insight on the molecular determinants in the Cry family of toxins and should facilitate the assessment and advancement of their use as pesticidal agents.
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Affiliation(s)
- Li Liu
- Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75083, USA
| | - Stefanie D Boyd
- Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75083, USA
| | - Lee A Bulla
- Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75083, USA.,CustomGene, LLC, Tioga, TX 76271, USA
| | - Duane D Winkler
- Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75083, USA
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Characterization of fibrinogen binding on platelet-derived extracellular vesicles. Thromb Res 2018; 172:135-138. [DOI: 10.1016/j.thromres.2018.10.021] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2018] [Revised: 10/11/2018] [Accepted: 10/24/2018] [Indexed: 11/30/2022]
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Li Y, Shang W, Liang X, Zeng C, Liu M, Wang S, Li H, Tian J. The diagnosis of hepatic fibrosis by magnetic resonance and near-infrared imaging using dual-modality nanoparticles. RSC Adv 2018; 8:6699-6708. [PMID: 35540380 PMCID: PMC9078292 DOI: 10.1039/c7ra10847h] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2017] [Accepted: 01/24/2018] [Indexed: 12/13/2022] Open
Abstract
Hepatic fibrosis (HF), as the only reversible process of chronic liver disease, remains a big diagnostic challenge. Development of noninvasive and effective methods to assess quantitatively early-stage HF is of great clinical importance. Compared with conventional diagnostic methods, near-infrared fluorescence imaging (NIR) and magnetic resonance imaging (MRI) could offer highly sensitive and spatial resolution signals for HF detection. However, precise detection using contrast agents is not possible. Superparamagnetic iron oxide (SPIO) nanoparticles have low toxicity, high sensitivity and excellent biocompatibility. Integration of Fe3O4 nanoparticles and indocyanine green (ICG), coupled with targeting ligand of integrin αvβ3, arginine–glycine–aspartic acid (RGD) expressed on hepatic stellate cells (HSCs), were used to detect HF. Both in vivo and in vitro results showed that the SPIO@SiO2–ICG–RGD had high stability and low cytotoxicity. The biodistribution of SPIO@SiO2–ICG–RGD was significantly different between mice with HF and healthy controls. SPIO@SiO2–ICG–RGD was characterized and the results of imaging in vitro and in vivo demonstrated the expression of integrin αvβ3 on activated HSCs. These data suggest that our SPIO@SiO2–ICG–RGD probe could be used for the diagnosis of early-stage HF. This new nanoprobe with a dual-modality imaging approach holds great potential for the diagnosis and classification of HF. Schematic diagram for the synthesis of SPIO@SiO2–ICG–RGD.![]()
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Affiliation(s)
- Yunfang Li
- Department of Radiology
- Beijing YouAn Hospital
- Capital Medical University
- Beijing
- China
| | - Wenting Shang
- Key Laboratory of Molecular Imaging
- Institute of Automation
- Chinese Academy of Sciences
- Beijing 100190
- P. R. China
| | - Xiaoyuan Liang
- Key Laboratory of Molecular Imaging
- Institute of Automation
- Chinese Academy of Sciences
- Beijing 100190
- P. R. China
| | - Chaoting Zeng
- Key Laboratory of Molecular Imaging
- Institute of Automation
- Chinese Academy of Sciences
- Beijing 100190
- P. R. China
| | - Mingming Liu
- Department of Radiology
- Beijing YouAn Hospital
- Capital Medical University
- Beijing
- China
| | - Sudan Wang
- Department of Radiology
- Beijing YouAn Hospital
- Capital Medical University
- Beijing
- China
| | - Hongjun Li
- Department of Radiology
- Beijing YouAn Hospital
- Capital Medical University
- Beijing
- China
| | - Jie Tian
- Key Laboratory of Molecular Imaging
- Institute of Automation
- Chinese Academy of Sciences
- Beijing 100190
- P. R. China
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10
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Grassmé H, Henry B, Ziobro R, Becker KA, Riethmüller J, Gardner A, Seitz AP, Steinmann J, Lang S, Ward C, Schuchman EH, Caldwell CC, Kamler M, Edwards MJ, Brodlie M, Gulbins E. β1-Integrin Accumulates in Cystic Fibrosis Luminal Airway Epithelial Membranes and Decreases Sphingosine, Promoting Bacterial Infections. Cell Host Microbe 2017; 21:707-718.e8. [PMID: 28552668 PMCID: PMC5475347 DOI: 10.1016/j.chom.2017.05.001] [Citation(s) in RCA: 78] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2016] [Revised: 03/10/2017] [Accepted: 05/02/2017] [Indexed: 11/18/2022]
Abstract
Chronic pulmonary colonization with bacterial pathogens, particularly Pseudomonas aeruginosa, is the primary cause of morbidity and mortality in patients with cystic fibrosis (CF). We observed that β1-integrins accumulate on the luminal membrane of upper-airway epithelial cells from mice and humans with CF. β1-integrin accumulation is due to increased ceramide and the formation of ceramide platforms that trap β1-integrins on the luminal pole of bronchial epithelial cells. β1-integrins downregulate acid ceramidase expression, resulting in further accumulation of ceramide and consequent reduction of surface sphingosine, a lipid that kills bacteria. Interrupting this vicious cycle by triggering surface β1-integrin internalization via anti-β1-integrin antibodies or the RGD peptide ligand-or by genetic or pharmacological correction of ceramide levels-normalizes β1-integrin distribution and sphingosine levels in CF epithelial cells and prevents P. aeruginosa infection in CF mice. These findings suggest a therapeutic avenue to ameliorate CF-associated bacterial infections.
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Affiliation(s)
- Heike Grassmé
- Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
| | - Brian Henry
- Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany; Department of Surgery, University of Cincinnati, 231 Albert Sabin Way, ML 0558, Cincinnati, Ohio 45229, USA
| | - Regan Ziobro
- Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
| | - Katrin Anne Becker
- Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
| | - Joachim Riethmüller
- Center for Pediatric Clinical Studies, Children's Clinic, University of Tuebingen, Hoppe-Seyler-Strasse 1, 72076 Tübingen, Germany
| | - Aaron Gardner
- Institute of Cellular Medicine, Newcastle University, c/o Level 3, Clinical Resource Building, Great North Children's Hospital, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP, UK
| | - Aaron P Seitz
- Department of Surgery, University of Cincinnati, 231 Albert Sabin Way, ML 0558, Cincinnati, Ohio 45229, USA
| | - Joerg Steinmann
- Department of Medical Microbiology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
| | - Stephan Lang
- Department of Otorhinolaryngology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
| | - Christopher Ward
- Institute of Cellular Medicine, Newcastle University, c/o Level 3, Clinical Resource Building, Great North Children's Hospital, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP, UK
| | - Edward H Schuchman
- Department of Genetics & Genomic Sciences, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA
| | - Charles C Caldwell
- Department of Surgery, University of Cincinnati, 231 Albert Sabin Way, ML 0558, Cincinnati, Ohio 45229, USA
| | - Markus Kamler
- West German Heart and Vascular Center Essen, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany
| | - Michael J Edwards
- Department of Surgery, University of Cincinnati, 231 Albert Sabin Way, ML 0558, Cincinnati, Ohio 45229, USA
| | - Malcolm Brodlie
- Institute of Cellular Medicine, Newcastle University, c/o Level 3, Clinical Resource Building, Great North Children's Hospital, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP, UK
| | - Erich Gulbins
- Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany; Department of Surgery, University of Cincinnati, 231 Albert Sabin Way, ML 0558, Cincinnati, Ohio 45229, USA.
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11
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Boudreaux MK, Lipscomb DL. Clinical, Biochemical, and Molecular Aspects of Glanzmann's Thrombasthenia in Humans and Dogs. Vet Pathol 2016; 38:249-60. [PMID: 11355654 DOI: 10.1354/vp.38-3-249] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Glanzmann's thrombasthenia (GT) is an inherited, intrinsic platelet function defect that involves the platelet glycoprotein complex IIb–IIIa, also known as the fibrinogen receptor and the integrin αIIbβ3. The defect was originally described by Dr. Glanzmann in humans in 1918 as a bleeding disorder that differed clinically from other known coagulopathies. Over the decades that followed, researchers determined the biochemical and molecular basis for the disease in humans. Otterhounds with thrombasthenic thrombopathia, described in the 1960s, were the only animal model that closely resembled the disease described in humans until 1996. At that time, a Great Pyrenees dog was identified with unequivocal clinical and biochemical features of Type I GT. The cDNA encoding for glycoproteins IIb and IIIa were sequenced in normal dogs in 1999, allowing for identification of specific mutations causing Type I GT in both Otterhounds and Great Pyrenees dogs. Knowing the molecular basis for Type I GT in dogs as well as the cDNA sequences in normal dogs should enhance the understanding of structure/function relationships of the αIIbβ3 integrin and provide an excellent animal model for studies aimed at correction of GT in humans. The following review focuses on the structure and function of this platelet receptor and reviews the molecular, biochemical, and clinical aspects of Glanzmann's thrombasthenia in humans and dogs.
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Affiliation(s)
- M K Boudreaux
- Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519, USA.
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12
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Srokowski EM, Woodhouse KA. Evaluation of the bulk platelet response and fibrinogen interaction to elastin-like polypeptide coatings. J Biomed Mater Res A 2013; 102:540-51. [DOI: 10.1002/jbm.a.34699] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2012] [Revised: 02/18/2013] [Accepted: 02/21/2013] [Indexed: 11/10/2022]
Affiliation(s)
- Elizabeth M. Srokowski
- Department of Chemical Engineering and Applied Chemistry; University of Toronto; Ontario Canada
- Institute of Biomaterials and Biomedical Engineering; University of Toronto; Ontario Canada
| | - Kimberly A. Woodhouse
- Department of Chemical Engineering and Applied Chemistry; University of Toronto; Ontario Canada
- Institute of Biomaterials and Biomedical Engineering; University of Toronto; Ontario Canada
- Department of Chemical Engineering; Queen's University; Ontario Canada
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13
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Cai H, Conti PS. RGD-based PET tracers for imaging receptor integrin αv β3 expression. J Labelled Comp Radiopharm 2013; 56:264-79. [PMID: 24285371 DOI: 10.1002/jlcr.2999] [Citation(s) in RCA: 74] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2012] [Revised: 11/02/2012] [Accepted: 11/06/2012] [Indexed: 12/20/2022]
Abstract
Positron emission tomography (PET) imaging of receptor integrin αv β3 expression may play a key role in the early detection of cancer and cardiovascular diseases, monitoring disease progression, evaluating therapeutic response, and aiding anti-angiogenic drugs discovery and development. The last decade has seen the development of new PET tracers for in vivo imaging of integrin αv β3 expression along with advances in PET chemistry. In this review, we will focus on the radiochemistry development of PET tracers based on arginine-glycine-aspartic acid (RGD) peptide, present an overview of general strategies for preparing RGD-based PET tracers, and review the recent advances in preparations of (18) F-labeled, (64) Cu-labeled, and (68) Ga-labeled RGD tracers, RGD-based PET multivalent probes, and RGD-based PET multimodality probes for imaging receptor integrin αv β3 expression.
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Affiliation(s)
- Hancheng Cai
- PET Center, Children's Hospital of Michigan, Detroit Medical Center, Detroit, MI, 48201, USA; Wayne State University School of Medicine, Detroit, MI, 48201, USA
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14
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Identification of Ata, a multifunctional trimeric autotransporter of Acinetobacter baumannii. J Bacteriol 2012; 194:3950-60. [PMID: 22609912 DOI: 10.1128/jb.06769-11] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Acinetobacter baumannii has recently emerged as a highly troublesome nosocomial pathogen, especially in patients in intensive care units and in those undergoing mechanical ventilation. We have identified a surface protein adhesin of A. baumannii, designated the Acinetobacter trimeric autotransporter (Ata), that contains all of the typical features of trimeric autotransporters (TA), including a long signal peptide followed by an N-terminal, surface-exposed passenger domain and a C-terminal domain encoding 4 β-strands. To demonstrate that Ata encoded a TA, we created a fusion protein in which we replaced the entire passenger domain of Ata with the epitope tag V5, which can be tracked with specific monoclonal antibodies, and demonstrated that the C-terminal 101 amino acids of Ata were capable of exporting the heterologous V5 tag to the surface of A. baumannii in a trimeric form. We found that Ata played a role in biofilm formation and bound to various extracellular matrix/basal membrane (ECM/BM) components, including collagen types I, III, IV, and V and laminin. Moreover, Ata mediated the adhesion of whole A. baumannii cells to immobilized collagen type IV and played a role in the survival of A. baumannii in a lethal model of systemic infection in immunocompetent mice. Taken together, these results reveal that Ata is a TA of A. baumannii involved in virulence, including biofilm formation, binding to ECM/BM proteins, mediating the adhesion of A. baumannii cells to collagen type IV, and contributing to the survival of A. baumannii in a mouse model of lethal infection.
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15
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Chen CC, Sirsi SR, Borden MA. Effect of surface architecture on in vivo ultrasound contrast persistence of targeted size-selected microbubbles. ULTRASOUND IN MEDICINE & BIOLOGY 2012; 38:492-503. [PMID: 22305060 PMCID: PMC3273728 DOI: 10.1016/j.ultrasmedbio.2011.12.007] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/28/2011] [Revised: 11/29/2011] [Accepted: 12/01/2011] [Indexed: 05/04/2023]
Abstract
Ultrasound molecular imaging is a powerful diagnostic modality using microbubbles coated with targeting ligands specific for endothelial biomarkers. The circulation persistence of ligand-bearing contrast agents is a key determinant in their contrast enhancement and targeting capability. Prior studies have shown that targeted microbubbles with ligands attached to the shell using the conventional exposed-ligand architecture (ELA) could trigger undesired ligand-induced complement activation and decreased circulation time. Microbubbles with the buried-ligand architecture (BLA), however, were found to inhibit complement activation and prolong circulation time. In the present study, we extended the stealth BLA microbubble design to size-selected (4 to 5-μm diameter) microbubbles targeted with cyclic RGD peptide using the postlabeling technique. Microbubble circulation persistence was measured in the healthy mouse kidney using a Visualsonics Vevo 770 scanner operating at 40 MHz in fundamental mode. The circulation persistence for targeted BLA microbubbles was significantly longer compared with their ELA counterparts and similar to no-ligand controls. Use of the BLA instead of the ELA increased the circulation half-life approximately two-fold. Analysis of the time-intensity and time-fluctuation curves with a two-compartment pharmacokinetic model showed a minimal degree of nonspecific vascular adhesion for any group. These results demonstrate the importance of surface architecture in the design of targeted microbubbles for ultrasound molecular imaging.
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Affiliation(s)
- Cherry C. Chen
- Department of Chemical Engineering, Columbia University, New York, NY 10027
| | - Shashank R. Sirsi
- Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309
| | - Mark A. Borden
- Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309
- Corresponding Author Address: Mark A. Borden, PhD, Department of Mechanical Engineering, University of Colorado, 1111 Engineering Drive, Boulder, CO 80309-0427, Phone: 303-492-7750, Fax: 303-492-3498,
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16
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Borovjagin AV, Dong J, Passineau MJ, Ren C, Lamani E, Mamaeva OA, Wu H, Keyser E, Murakami M, Chen S, MacDougall M. Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells. PLoS One 2011; 6:e24281. [PMID: 22003382 PMCID: PMC3189176 DOI: 10.1371/journal.pone.0024281] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2010] [Accepted: 08/09/2011] [Indexed: 12/31/2022] Open
Abstract
To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including “pK7” and/or “RGD” motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber “knob” domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both αvβ3/αvβ5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.
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Affiliation(s)
- Anton V. Borovjagin
- Department of Periodontics, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
- Institute of Oral Health Research, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
| | - Juan Dong
- Department of Orthodontics, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
- Institute of Oral Health Research, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
| | - Michael J. Passineau
- Division of Cardiovascular Medicine and Allegheny-Singer Research Institute, West-Penn Allegheny Health System, Pittsburgh, Pennsylvania, United States of America
| | - Changchun Ren
- Department of Oral and Maxillofacial Surgery, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
- Institute of Oral Health Research, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
| | - Ejvis Lamani
- Department of Oral and Maxillofacial Surgery, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
- Institute of Oral Health Research, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
| | - Olga A. Mamaeva
- Institute of Oral Health Research, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
| | - Hongju Wu
- Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
- Division of Human Gene Therapy, Department of Medicine, The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
| | - Enid Keyser
- Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
| | - Miho Murakami
- Division of Human Gene Therapy, Department of Medicine, The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
| | - Shuo Chen
- Department of Pediatric Dentistry, Dental School University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America
| | - Mary MacDougall
- Department of Oral and Maxillofacial Surgery, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
- Institute of Oral Health Research, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America
- * E-mail:
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Liu J, Jin TC, Chang S, Czajka-Jakubowska A, Clarkson BH. Adhesion and growth of dental pulp stem cells on enamel-like fluorapatite surfaces. J Biomed Mater Res A 2011; 96:528-34. [PMID: 21254384 DOI: 10.1002/jbm.a.33002] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2010] [Revised: 10/06/2010] [Accepted: 10/25/2010] [Indexed: 01/08/2023]
Abstract
UNLABELLED To study how apatite crystal alignment of an enamel-like substrate affects DPSC cellular adhesion and growth as a precursor to produce an in vitro enamel/dentin superstructure for future studies. The cells were subcultured in 10% FBS DMEM up to seven weeks on the two surfaces. Specimens were observed under SEM, counted, and analyzed using the human pathway-focused matrix and adhesion PCR array. After three days, the cell number on ordered FA surface was significantly higher than on the disordered surface. Of the 84 focused pathway genes, a total of 20 genes were either up or down regulated in the cells on ordered FA surface compared to the disordered surface. More interestingly, of the cell-matrix adhesion molecules, integrin alpha 7 and 8 (ITGA 7 and 8), integrin beta 3 and 4 (ITGB3 and 4), and the vitronectin receptor-integrin alpha V (ITGAV) and the key adhesion protein-fibronectin1 (FN1) were up-regulated. In SEM, both surfaces showed good biocompatibility and supported long term growth of DPSC cells but with functional cell-matrix interaction on the ordered FA surfaces. SIGNIFICANCE The enhanced cellular response of DPSC cell to the ordered FA crystal surface involves a set of delicately regulated matrix and adhesion molecules which could be manipulated by treating the cells with a dentin extract, to produce a dentin/enamel superstructure.
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Affiliation(s)
- J Liu
- Department of Cardiology, Restorative Sciences and Endodontics, Dental School, University of Michigan, Ann Arbor, Michigan, USA
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18
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Pillitteri D, Pilgrimm AK, Kirchmaier CM. Novel Mutations in the GPIIb and GPIIIa Genes in Glanzmann Thrombasthenia. Transfus Med Hemother 2010; 37:268-277. [PMID: 21113249 PMCID: PMC2980511 DOI: 10.1159/000320258] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2010] [Accepted: 08/17/2010] [Indexed: 02/03/2023] Open
Abstract
BACKGROUND: Glanzmann thrombasthenia (GT) is an inherited autosomal recessive platelet disorder characterized by a complete or partial lack, or mutation, of the GPIIb/IIIa complex (integrin α(IIb)β(3)) on the thrombocytes' surface, leading to a severe bleeding syndrome. MATERIAL AND METHODS: Molecular genetic analysis was performed in patients with suspected GT. The aim of the present study was the identification of new natural variants, their impact on platelet function, and their relation to the risk of bleeding. RESULTS: Expression of the platelet integrin α(IIb)β(3) was determined by flow cytometry. Mutations were identified through sequencing of cDNA and genomic DNA. In addition, platelet function studies (PAC-binding, aggregations) were implemented. The study included 25 patients revealing 13 mutations (GPIIb: n = 9; GPIIIa: n = 4). Two of the 13 mutations were previously described (T207I; L214P). The remaining mutations have not been published yet, whereas 1 mutation in 2 unrelated families was identical (3062 T→C). CONCLUSION: All patients with less than 25% of present α(IIb)β(3) have a medical history of bleeding.
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Affiliation(s)
- Daniele Pillitteri
- Deutsche Klinik für Diagnostik, Sektion Innere Medizin I, Arbeitsgruppe: «Thrombose, Hämostase und vaskuläre Medizin», Wiesbaden, Germany
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19
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Park HY. The glycoprotein IIIa gene polymorphism and the risk of myocardial infarction. Future Cardiol 2010; 1:207-14. [PMID: 19804165 DOI: 10.1517/14796678.1.2.207] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Abstract
The hemostatic platelet response of an individual may be influenced by the genetic profile of the platelet membrane glycoprotein (GP) receptors. The GP IIIa, as a part of von Willebrand factor and fibrinogen receptor, plays a central role in platelet aggregation. Polymorphism in GP IIIa has been extensively studied for its association with myocardial infarction or coronary artery diseases. To date, the role of GP IIIa polymorphism in genetic susceptibility to thrombotic disease still remains controversial. The results of case-control association studies vary, even with the same ethnic background, and the association was hardly found in studies with larger sample size, suggesting publication-bias toward positive findings. In meta-analysis, the GP IIIa Pl(A2) allele carriers did not show increased risk for myocardial infarction compared with Pl(A1/A1) homozygotes. The functional studies also showed conflicting results. In conclusion, GP IIIa Pl(A1/A2) polymorphism does not seem to have a major role either in determining the individual variance of platelet function or the risk of myocardial infarction according to the currently available data. Therefore, the genotype determination of GP IIIa Pl(A1/A2 )polymorphism may not be useful for risk assessment of myocardial infarction at this time. Nevertheless, the author can not completely exclude its possible role in coronary thrombosis after angioplasty or sudden cardiac death. Thus, further evaluation in larger prospective or multicenter studies is required to elucidate the role of GPs in cardiovascular system.
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Affiliation(s)
- Hyun-Young Park
- Yonsei Cardiovascular Research Institute, Division of Cardiology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, South Korea.
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Sánchez-Cortés J, Mrksich M. The platelet integrin alphaIIbbeta3 binds to the RGD and AGD motifs in fibrinogen. ACTA ACUST UNITED AC 2010; 16:990-1000. [PMID: 19778727 DOI: 10.1016/j.chembiol.2009.08.012] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2009] [Revised: 08/27/2009] [Accepted: 08/27/2009] [Indexed: 10/20/2022]
Abstract
Fibrinogen (Fbg) mediates platelet aggregation by binding the alphaIIbbeta3 integrin receptor, but the interaction of the receptor with peptide motifs of Fbg remains unresolved. This paper describes the use of self-assembled monolayers (SAMs) to study the adhesion of alphaIIbbeta3-transfected CHO cells to the GRGDS and HHLGGAKQAGDV motifs within Fbg. Cells adhered to and spread on monolayers presenting either peptide. Cell adhesion could be inhibited by either soluble peptide, demonstrating that the peptides bind competitively to the integrin. A peptide array was used to show that AGD was the minimal binding sequence in HHLGGAKQAGDV and that the receptor recognizes ligands of the form GXGDSC, where X is a hydrophobic or basic residue. This work revises our understanding of the alphaIIbbeta3 specificity and also suggests a new class of antithrombotic agents.
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Affiliation(s)
- Juan Sánchez-Cortés
- Department of Chemistry and Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA
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21
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Mazoyer E, Caen JP, Tenza D, Cramer EM. The Anti-aggregating Peptide KRDS Impairs a-granule Release, Whereas RGDS Does Not. Platelets 2009; 6:91-8. [DOI: 10.3109/09537109509078449] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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22
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Meyer M, Thieme A, Jablonka B, Just M, Ströhl C, Schellenberg I, Kirchmaier CM. A new variant of Glanzmann's thrombasthenia with defective activation-dependent fibrinogen binding and altered expression of epitopes for several monoclonal antibodies against GP IIb-IIIa. Platelets 2009; 7:215-24. [DOI: 10.3109/09537109609023581] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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23
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Agnihotri A, Soman P, Siedlecki CA. AFM measurements of interactions between the platelet integrin receptor GPIIbIIIa and fibrinogen. Colloids Surf B Biointerfaces 2009; 71:138-47. [DOI: 10.1016/j.colsurfb.2009.01.019] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2008] [Revised: 01/24/2009] [Accepted: 01/25/2009] [Indexed: 11/16/2022]
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24
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Papamichael ND, Stathopoulou EM, Roussa VD, Tsironis LD, Kotsia AP, Stanica RM, Moussis V, Tsikaris V, Katsouras CS, Tselepis AD, Michalis LK. Effect of a synthetic peptide corresponding to residues 313 to 320 of the alphaIIb subunit of the human platelet integrin alphaIIbbeta3 on carotid artery thrombosis in rabbits. J Pharmacol Exp Ther 2009; 329:634-40. [PMID: 19244095 DOI: 10.1124/jpet.108.150086] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/07/2025] Open
Abstract
The platelet integrin receptor alpha(IIb)beta(3) plays a critical role in thrombosis. We have shown previously that the octapeptide YMESRADR, corresponding to sequences 313 to 320 of the human alpha(IIb) subunit, inhibits human platelet activation and fibrinogen binding to alpha(IIb)beta(3), possibly interacting with the ligand. We investigated the effect of YMESRADR on electrically induced carotid artery thrombosis in New Zealand white rabbits. Peptide was administered via the femoral vein, starting 60 min before and continuing for 90 min after the electrical stimulation. Carotid blood flow was monitored for 90 min after the electrical stimulation. The peptide effects on platelet aggregation, in vitro and ex vivo, and on various coagulation, bleeding, and hemostatic parameters were evaluated. YMESRADR significantly inhibited rabbit platelet aggregation in vitro in a dose-dependent manner. It is important that peptide administration in vivo, at doses ranging from 3 to 15 mg/kg, prolonged the duration of the patency of the carotid artery, and no artery occlusion was observed until the end of the study (90 min after electrical stimulation). Furthermore, YMESRADR administration reduced platelet aggregation ex vivo and thrombus weight; however, these reductions reached statistical significance, compared with the control group, at the peptide doses of 12 and 15 mg/kg. YMESRADR did not affect any coagulation parameter studied and the hemostatic response observed in control animals. Thus, YMESRADR represents a novel antiplatelet agent that can inhibit thrombus formation effectively and carotid artery occlusion without causing hemorrhagic complications in a rabbit model of arterial thrombosis.
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26
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Sun CX, Chen P, Lu W, Liu JN. Tyr178 of beta3 is critical for alphaIIb maturation and macromolecular ligand binding to alphaIIbbeta3. Thromb Res 2008; 122:203-10. [PMID: 18201749 DOI: 10.1016/j.thromres.2007.11.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2007] [Revised: 10/14/2007] [Accepted: 11/12/2007] [Indexed: 11/17/2022]
Abstract
To explore the structural basis of ligand binding to alphaIIbbeta3, we conducted a site-directed mutagenesis of Y178, which is located in the ligand-specificity region (C177-C184) of the beta3 subunit. Two mutant beta3 constructs, Y178A and Y178I, were transfected into CHO cells and co-expressed with human alphaIIb subunit on the cell surface. Our results showed that the Y178A mutation affected processing and cell surface exposure of recombinant alphaIIbbeta3 receptor, abrogated the binding of PAC-1, a ligand-mimetic antibody, to alphaIIbbeta3 pre-treated with the known activator DTT. The Y178A mutation also resulted in reduced adhesion of alphaIIbbeta3 on immobilized fibrinogen. In contrast, the interaction of alphaIIbbeta3 with the small molecular ligand RGDS was unaffected by Y178A mutation, as evidenced by the elevated LIBS-1 epitope expression following RGDS addition. Interestingly however, Y178I mutation did not affect the receptor synthesis and function at all. As for post-receptor occupancy, neither Y178A nor Y178I prevented alphaIIbbeta3 translocation to focal adhesion contacts. These results suggest that Y178 is involved in alphaIIb maturation and alphaIIbbeta3 complex expression. This residue is also critical for alphaIIbbeta3 interaction with its macromolecular ligand or ligand-mimetic mAb, possibly due to its involvement in other ligand-binding sites distinct from the RGD-binding pocket. We also propose that a residue with appropriate side-chain size and hydrophobicity at position 178 is spatially required for formation of the correct tertiary structure of the site.
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Affiliation(s)
- Chong-Xiu Sun
- Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing, 210093, China
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27
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Wang PC, Vilaire G, DeGrado WF, Bennett JS. Interactions of ADP-stimulated human platelets with PEGylated polystyrene substrates prepared by surface amidation. Colloids Surf B Biointerfaces 2007; 58:225-30. [PMID: 17499487 DOI: 10.1016/j.colsurfb.2007.03.012] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2006] [Revised: 02/05/2007] [Accepted: 03/14/2007] [Indexed: 11/17/2022]
Abstract
A study primarily focused on the interactions between ADP-stimulated human platelets and PEGylated polystyrene substrates is described in this paper. The platelet-surface interactions were investigated using colorimetric acid phosphatase assay. Two types of amine-containing polymeric hydrogel materials based on poly(ethylene glycol) (PEG), H(2)N-PEG-OCH(3) and H(2)N-PEG-NH(2), were used to PEGylate polystyrene surfaces derivatized with maleic anhydride by amidation at alkaline pH. In addition, comparative studies using surfaces non-covalently adsorbed by bovine serum albumin (BSA) or fibrinogen (Fg) were also conducted. The assay results showed that no significant platelet adhesion was observed when PEGylated surfaces or BSA-coated surfaces were exposed to unstimulated gel-filtered platelets (GFP). However, upon ADP-stimulation, platelet adhesion to the surfaces under investigation in this study all increased to varying degrees. Most importantly, the results showed that polystyrene surfaces PEGylated using H(2)N-PEG-NH(2) were most effective in resisting platelet adhesion when assays were performed using ADP-stimulated GFP. By PEGylating the surfaces of polystyrene microtiter wells via the amidation reaction described in this paper, it is demonstrated that (i) higher degree of surface PEGylation is favored at more alkaline pH and (ii) polystyrene substrates capable of more effectively resisting the adhesion of ADP-stimulated GFP can be obtained by the PEGylation reaction carried out at pH 9.1 using H(2)N-PEG-NH(2).
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Affiliation(s)
- Pen-Cheng Wang
- Hematology-Oncology Division, Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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Maile LA, Busby WH, Sitko K, Capps BE, Sergent T, Badley-Clarke J, Clemmons DR. Insulin-like growth factor-I signaling in smooth muscle cells is regulated by ligand binding to the 177CYDMKTTC184 sequence of the beta3-subunit of alphaVbeta3. Mol Endocrinol 2005; 20:405-13. [PMID: 16195248 DOI: 10.1210/me.2005-0241] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
The response of smooth muscle cells to IGF-I requires ligand occupancy of the alphaVbeta3 integrin. We have shown that vitronectin (Vn) is required for IGF-I-stimulated migration or proliferation, whereas the anti-alphaVbeta3 monoclonal antibody, LM609, which inhibits ligand binding, blocks responsiveness of these cells to IGF-I. The amino acids 177-184 ((177)CYDMKTTC(184)) within the extracellular domain of beta3 have been proposed to confer the ligand specificity of alphaVbeta3; therefore, we hypothesized that ligand binding to the 177-184 cysteine loop of beta3 may be an important regulator of the cross talk between alphaVbeta3 and IGF-I in SMCs. Here we demonstrate that blocking ligand binding to a specific amino acid sequence within the beta3 subunit of alphaVbeta3 (i.e. amino acids 177-184) blocked Vn binding to the beta3 subunit of alphaVbeta3 and correspondingly beta3 phosphorylation was decreased. In the presence of this antibody, IGF-I-stimulated Shc phosphorylation and ERK 1/2 activation were impaired, and this was associated with an inhibition in the ability of IGF-I to stimulate an increase in migration or proliferation. Furthermore, in cells expressing a mutated form of beta3 in which three critical residues within the 177-184 sequence were altered beta3 phosphorylation was decreased. This was associated with a loss of IGF-I-stimulated Shc phosphorylation and impaired smooth muscle cell proliferation in response to IGF-I. In conclusion, we have demonstrated that the 177-184 sequence of beta3 is necessary for Vn binding to alphaVbeta3 and that ligand occupancy of this site is necessary for an optimal response of smooth muscle cells to IGF-I.
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Affiliation(s)
- Laura A Maile
- Division of Endocrinology, University of North Carolina, Chapel Hill, North Carolina 27599-7170, USA.
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Bella J, Humphries MJ. Calpha-H...O = C hydrogen bonds contribute to the specificity of RGD cell-adhesion interactions. BMC STRUCTURAL BIOLOGY 2005; 5:4. [PMID: 15710040 PMCID: PMC551611 DOI: 10.1186/1472-6807-5-4] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/18/2004] [Accepted: 02/14/2005] [Indexed: 11/11/2022]
Abstract
BACKGROUND The Arg-Gly-Asp (RGD) cell adhesion sequence occurs in several extracellular matrix molecules known to interact with integrin cell-surface receptors. Recently published crystal structures of the extracellular regions of two integrins in complex with peptides containing or mimicking the RGD sequence have identified the Arg and Asp residues as key specificity determinants for integrin recognition, through hydrogen bonding and metal coordination interactions. The central Gly residue also appears to be in close contact with the integrin surface in these structures. RESULTS When hydrogen atoms are modelled on the central Gly residue with standard stereochemistry, the interaction between this residue and a carbonyl group in the integrin surface shows all the hallmarks of Calpha-H...O = C hydrogen bonding, as seen in the collagen triple helix and in many crystal structures of small organic molecules. Moreover, molecular dynamic simulations of the docking of RGD-containing fragments on integrin surfaces support the occurrence of these interactions. There appears to be an array of four weak and conventional hydrogen bonds lining up the RGD residues with main chain carbonyl groups in the integrin surface. CONCLUSIONS The occurrence of weak Calpha-H...O = C hydrogen bonds in the RGD-integrin interaction highlights the importance of the conserved Gly residue in the RGD motif and its contribution to integrin-ligand binding specificity. Our analysis shows how weak hydrogen bonds may also play important biological roles by contributing to the specificity of macromolecular recognition.
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Affiliation(s)
- Jordi Bella
- Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester
| | - Martin J Humphries
- Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester
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31
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El-Amin SF, Kofron MD, Attawia MA, Lu HH, Tuan RS, Laurencin CT. Molecular regulation of osteoblasts for tissue engineered bone repair. Clin Orthop Relat Res 2004:220-5. [PMID: 15552161 DOI: 10.1097/01.blo.0000137556.51604.0c] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The use of biodegradable polymers in medicine and biomedical research is increasing. A key growth area has been the use of these materials in tissue engineering, especially for guided regeneration of bone and cartilage. Our interest has been in determining the mechanisms by which cellular attachment and growth occurs on these materials. In the current study, we examined human osteoblast cell adhesion, growth, and morphologic changes on polymeric scaffolds composed of polylactic-co-glycolic acid and polylactic acid materials. We examined these characteristics in association with measurements of levels of key adhesion integrin receptors in the presence and absence of antibodies against alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits, and the adhesion ligand peptides RGD (Arg-Gly-Asp) and RGE (Arg-Gly-Ser). At 2 hours, results showed initial cell adhesion was considerably decreased on polylactic-co-glycolic acid and polylactic acid in the presence of the alpha2 and beta1, antibodies with a 70% adhesion rate difference observed among the groups evaluated. Higher levels of inhibition were observed on polylactic-co-glycolic acid relative to polylactic acid, which may be correlated to a higher number of cells being able to interact with the surface initially. The presence of known competitive peptide (RGD) at 2 hours, revealed its ability to block cellular adhesion to these matrices relative to the control and noncompetitive peptide RGE on polylactic-co-glycolic acid matrices. Overall adhesion rate was affected by the presence of the integrin antibodies to the alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits with highest differences among polylactic-co-glycolic acid relative to its control, therefore suggesting that initial osteoblastic cell adhesion to commonly used biomaterials is regulated through integrin binding.
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Affiliation(s)
- Saadiq F El-Amin
- Department of Orthopaedic Surgery, University of Virginia, Charlottesville, VA 22903, USA
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32
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Tsai WB, Grunkemeier JM, Horbett TA. Variations in the ability of adsorbed fibrinogen to mediate platelet adhesion to polystyrene-based materials: a multivariate statistical analysis of antibody binding to the platelet binding sites of fibrinogen. J Biomed Mater Res A 2004; 67:1255-68. [PMID: 14624512 DOI: 10.1002/jbm.a.20024] [Citation(s) in RCA: 64] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Platelet adhesion to the surfaces of biomaterials preadsorbed with plasma previously has been shown to be mediated exclusively by surface-bound fibrinogen and does not seem to involve the other adhesion proteins in plasma (Tsai et al., J Biomed Mater Res 2002;60:348-359). In this study, the influence of surface-bound fibrinogen on platelet adhesion to five different types of polystyrene-based microtiter plates preadsorbed with plasma was analyzed relative to the amount of adsorbed fibrinogen and monoclonal antibody binding to the adsorbed fibrinogen. There was no significant correlation between platelet adhesion and the absolute amount of adsorbed fibrinogen. However, platelet adhesion was positively correlated to the ability of the adsorbed fibrinogen to bind three types of monoclonal antibodies. The antibodies used bound to the sites on fibrinogen thought to be involved in platelet binding (the two gamma chain C-terminal dodecapeptides and the RGDF and RGDS sequences in each of the Aalpha chains). A partial least-squares calibration model was used to analyze the relative importance of these binding sites in fibrinogen to platelet adhesion. The gamma chain C-terminal dodecapeptide was shown to be the most important site in adsorbed fibrinogen in mediating platelet adhesion.
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Affiliation(s)
- Wei-Bor Tsai
- Department of Bioengineering, Box 351750, University of Washington, Seattle, Washington 98195, USA
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33
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Lee I, Marchant RE. Molecular interaction studies of hemostasis: fibrinogen ligand-human platelet receptor interactions. Ultramicroscopy 2003; 97:341-52. [PMID: 12801687 DOI: 10.1016/s0304-3991(03)00059-7] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The interactions between fibrinogen ligands and platelet receptor alpha(IIb)beta(3) were studied under physiological conditions by atomic force microscopy (AFM). Two linear peptide sequences in fibrinogen, RGD and HHLGGAKQAGDV, play central roles in the regulation of hemostasis and thrombosis by facilitating adhesion and aggregation of platelets. In order to measure the interactions (i.e., debonding force), oligopeptides, GSSSGaaa, where aaa is -RGDSPA or -HHLGGAKQAGDV, were synthesized and grafted on to the surface of AFM probe tips. The interaction forces between a peptide-modified AFM probe tip and platelet surface were determined from pN to nN levels using AFM force measurements. Our results show that the zero kinetic off-rate, K(off)(0), for RGDSPA is significantly smaller than that for HHLGGAKQAGDV, under the consideration of flexible receptor surfaces. From our analysis, the K(off)(0), the single molecular binding energy E(b), and the transition state x(b), were extracted from the data, and estimated to be 1.53s(-1), -2.64x10(-20)J and 1.03A for the RGD-alpha(IIb)beta(3) system, and 47.58s(-1), 2.67x10(-20), 1.09A for the HHLGGAKQAGDV-alpha(IIb)beta(3) system, respectively.
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Affiliation(s)
- Imshik Lee
- Department of Biomedical Engineering, Case Western Reserve University, 10900 Eculid Avenue, Wickenden Building, Cleveland, OH 44106-7207, USA
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Biris N, Abatzis M, Mitsios JV, Sakarellos-Daitsiotis M, Sakarellos C, Tsoukatos D, Tselepis AD, Michalis L, Sideris D, Konidou G, Soteriadou K, Tsikaris V. Mapping the binding domains of the alpha(IIb) subunit. A study performed on the activated form of the platelet integrin alpha(IIb)beta(3). EUROPEAN JOURNAL OF BIOCHEMISTRY 2003; 270:3760-7. [PMID: 12950259 DOI: 10.1046/j.1432-1033.2003.03762.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.
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Affiliation(s)
- Nikolaos Biris
- Department of Chemistry, University of Ioannina, Ioannina, Greece
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35
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Abstract
AlphaIIbbeta3, the major membrane protein on the surface of platelets, is a member of the integrin family of heterodimeric adhesion receptors. The alphaIIb and beta3 subunits are each composed of a short cytoplasmic tail, a single transmembrane domain, and a large, extracellular region that consists of a series of linked domains. Recent structural analyses have provided insights into the organization of this and other integrins and how a signal is initiated at its cytoplasmic tail to transform the extracellular domain of alphaIIbbeta3 into a functional receptor for fibrinogen or von Willebrand factor to support platelet aggregation and thrombus formation. These functions of alphaIIbbeta3 have been targeted for antithrombotic therapy, and intravenous alphaIIbbeta3 antagonists have been remarkably effective in the setting of percutaneous coronary interventions, showing both short-term and long-term mortality benefits. However, the development of oral antagonists has been abandoned on the basis of excess of mortality in clinical trials, and the extension of therapy with existing alphaIIbbeta3 antagonists to broadly treat acute coronary syndromes has not fully met expectations. An in-depth understanding of how antagonists engage and influence the function of alphaIIbbeta3 and platelets in the context of the new structural insights may explain its salutary and potential deleterious effects.
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Affiliation(s)
- Martin J Quinn
- Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology/NB50, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, Ohio 44195, USA
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36
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37
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Candas M, Francis BR, Griko NB, Midboe EG, Bulla LA. Proteolytic cleavage of the developmentally important cadherin BT-R1 in the midgut epithelium of Manduca sexta. Biochemistry 2002; 41:13717-24. [PMID: 12427034 DOI: 10.1021/bi026323k] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
BT-R1 (M(r) = 210 kDa) represents a new type of insect cadherin that is expressed specifically in the midgut epithelium during growth and development of Manduca sexta larvae. It also is a target receptor for the Cry1A toxins of the entomopathogenic bacterium Bacillus thuringiensis. Expression of BT-R1, which varies during larval development, correlates with the abundance of the protein and with the differential cleavage of the molecule at each developmental stage. The cleavage of BT-R1 is calcium dependent, and consequently, Ca2+ directly influences the structural integrity of BT-R1. Indeed, removal of calcium ions by chelating agents promotes cleavage of the BT-R1 ectodomain, resulting in formation of fragments that are similar to those observed during larval development. Partial purification of proteins from brush border membrane vesicles (BBMVs) by gel filtration chromatography hinders the cleavage of BT-R1 in the presence of EDTA and EGTA, indicating that there is specific proteolytic activity associated with the BBMV. This specific proteolytic cleavage of BT-R1 not only alters the integrity of BT-R1 but it most likely is implicated in cell adhesion events during differentiation and development of M. sexta midgut epithelium. We propose a model for calcium-dependent protection of BT-R1 as well as a cleavage pattern that may modulate the molecular interactions and adhesive properties of its ectodomain. Molecular characterization of such a protection mechanism should lead to a better understanding of how the function of specific cadherins is modulated during tissue differentiation and insect development.
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Affiliation(s)
- Mehmet Candas
- Center for Biotechnology and Bioinformatics and Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083, USA
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38
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Abstract
In their roles as major adhesion receptors, integrins signal across the plasma membrane in both directions. Recent structural and cell biological data suggest models for how integrins transmit signals between their extracellular ligand binding adhesion sites and their cytoplasmic domains, which link to the cytoskeleton and to signal transduction pathways. Long-range conformational changes couple these functions via allosteric equilibria.
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Affiliation(s)
- Richard O Hynes
- Howard Hughes Medical Institute, Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
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39
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Abstract
Integrins are alphabeta heterodimeric cell-surface receptors that are vital to the survival and function of nucleated cells. They recognize aspartic-acid- or a glutamic-acid-based sequence motifs in structurally diverse ligands. Integrin recognition of most ligands is divalent cation dependent and conformationally sensitive. In addition to this common property, there is an underlying binding specificity between integrins and ligands for which there has been no structural basis. The recently reported crystal structures of the extracellular segment of an integrin in its unliganded state and in complex with a prototypical Arg-Gly-Asp (RGD) ligand have provided an atomic basis for cation-mediated binding of aspartic-acid-based ligands to integrins. They also serve as a basis for modelling other integrins in complex with larger physiologic ligands. These models provide new insights into the molecular basis for ligand binding specificity in integrins and its regulation by activation-driven tertiary and quaternary changes.
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Affiliation(s)
- M Amin Arnaout
- Renal Unit, Leukocyte Biology and Inflammation Program, Massachusetts General Hospital, and Harvard Medical School, Charlestown, MA 02129, USA.
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40
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Acute thrombocytopenia after treatment with tirofiban or eptifibatide is associated with antibodies specific for ligand-occupied GPIIb/IIIa. Blood 2002. [DOI: 10.1182/blood.v100.6.2071] [Citation(s) in RCA: 140] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractAcute thrombocytopenia is a recognized complication of treatment with GPIIb/IIIa inhibitors whose cause is not yet known. We studied 9 patients who developed severe thrombocytopenia (platelets less than 25 × 109/L) within several hours of treatment with the GPIIb/IIIa inhibitors tirofiban (4 patients) and eptifibatide (5 patients). In each patient, acute-phase serum contained a high titer (range, 1:80-1:20 000) IgG antibody that reacted with the glycoprotein IIb/IIIa complex only in the presence of the drug used in treatment. Four patients had been previously treated with the same drug, but 5 had no known prior exposure. Pretreatment serum samples from 2 of the latter patients contained drug-dependent antibodies similar to those identified after treatment. No tirofiban- or eptifibatide-dependent antibodies were found in any of 100 randomly selected healthy blood donors, and only 2 of 23 patients receiving tirofiban or eptifibatide who did not experience significant thrombocytopenia had extremely weak (titer, 1:2) tirofiban-dependent antibodies. In preliminary studies, evidence was obtained that the 9 antibodies recognize multiple target epitopes on GPIIb/IIIa complexed with the inhibitor to which the patient was sensitive, indicating that they cannot all be specific for the drug-binding site. The findings indicate that acute thrombocytopenia after the administration of tirofiban or eptifibatide can be caused by drug-dependent antibodies that are “naturally occurring” or are induced by prior exposure to drug. These antibodies may be human analogs of mouse monoclonal antibodies that recognize ligand-induced binding sites (LIBS) induced in the GPIIb/IIIa heterodimer when it reacts with a ligand-mimetic drug.
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41
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Acute thrombocytopenia after treatment with tirofiban or eptifibatide is associated with antibodies specific for ligand-occupied GPIIb/IIIa. Blood 2002. [DOI: 10.1182/blood.v100.6.2071.h81802002071_2071_2076] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Acute thrombocytopenia is a recognized complication of treatment with GPIIb/IIIa inhibitors whose cause is not yet known. We studied 9 patients who developed severe thrombocytopenia (platelets less than 25 × 109/L) within several hours of treatment with the GPIIb/IIIa inhibitors tirofiban (4 patients) and eptifibatide (5 patients). In each patient, acute-phase serum contained a high titer (range, 1:80-1:20 000) IgG antibody that reacted with the glycoprotein IIb/IIIa complex only in the presence of the drug used in treatment. Four patients had been previously treated with the same drug, but 5 had no known prior exposure. Pretreatment serum samples from 2 of the latter patients contained drug-dependent antibodies similar to those identified after treatment. No tirofiban- or eptifibatide-dependent antibodies were found in any of 100 randomly selected healthy blood donors, and only 2 of 23 patients receiving tirofiban or eptifibatide who did not experience significant thrombocytopenia had extremely weak (titer, 1:2) tirofiban-dependent antibodies. In preliminary studies, evidence was obtained that the 9 antibodies recognize multiple target epitopes on GPIIb/IIIa complexed with the inhibitor to which the patient was sensitive, indicating that they cannot all be specific for the drug-binding site. The findings indicate that acute thrombocytopenia after the administration of tirofiban or eptifibatide can be caused by drug-dependent antibodies that are “naturally occurring” or are induced by prior exposure to drug. These antibodies may be human analogs of mouse monoclonal antibodies that recognize ligand-induced binding sites (LIBS) induced in the GPIIb/IIIa heterodimer when it reacts with a ligand-mimetic drug.
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42
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Dorsch JA, Candas M, Griko NB, Maaty WSA, Midboe EG, Vadlamudi RK, Bulla LA. Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R(1) in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2002; 32:1025-1036. [PMID: 12213239 DOI: 10.1016/s0965-1748(02)00040-1] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R(1) (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K(d) approximately 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R(1) revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R(1) cDNA, subsequently killing the cells. Recruitment of BT-R(1) by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.
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Affiliation(s)
- J A Dorsch
- Center for Biotechnology and Bioinformatics, The University of Texas at Dallas, P.O. Box 830688, FO31, Richardson, TX 75083-0688, USA
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Abstract
The divalent-cation-dependent binding of alphabeta heterodimeric integrins to their ligands regulates most cellular processes. Integrin-ligand interactions are tightly controlled by inside-out activation signals. Ligand-bound integrins in turn transduce outside-in signals typical of other receptors. Precise information of how ligands bind to integrins is restricted to that of a small vWF A-type domain present in some alpha-subunits (alphaA). Both inside-out and outside-in signals elicit tertiary and quaternary changes in integrins, but the precise nature and scope and of these changes are unknown. The recently solved structures of the extracellular segment of integrin alphaVbeta3 in its unliganded and liganded states are generating exciting new insights into the design, wiring, function and regulation of this protein family. The structures reveal a surprising degree of flexibility at defined regions in the structure that is potentially controlled by cations. The quaternary structure of the ligand-binding region bears a striking resemblance to the nucleotide-binding pocket of G-proteins, implying analogous activation and signaling mechanisms. Structural links exist through which ligand-induced tertiary changes may be translated into quaternary changes and vice versa. The structures also raise the tantalizing hypothesis that alphaA is a regulated endogenous integrin ligand, so that no special regulatory features are needed in this integrin. These findings provide the framework for new investigations of structure-activity relationships in integrins, with important implications for targeting these receptors therapeutically [corrected].
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Affiliation(s)
- M Amin Arnaout
- Renal Unit, Leukocyte Biology & Inflammation Program, Structural Biology Program, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
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O'Shea JC, Tcheng JE. Eptifibatide: a potent inhibitor of the platelet receptor integrin glycoprotein IIb/IIIa. Expert Opin Pharmacother 2002; 3:1199-210. [PMID: 12150697 DOI: 10.1517/14656566.3.8.1199] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The platelet glycoprotein (GP) IIb/IIIa integrin plays a key role in mediating platelet aggregation. Blockade of the platelet GP IIb/IIIa receptor prevents arterial thrombosis in animal models much better than does aspirin. Among the most specific inhibitors in this class of drugs is eptifibatide (Integrilin(TM), Millennium Pharmaceuticals, Inc.), a cyclic heptapeptide based on a peptide recognition sequence found in snake venom. Peptide inhibitors, such as eptifibatide, bind competitively to GP IIb/IIIa and have a short half-life, allowing the effect to be rapidly reversible and providing a favourable overall safety profile. Eptifibatide has been studied in a broad range of ischaemic coronary conditions including percutaneous coronary intervention (PCI), ST-segment and non-ST-segment acute myocardial infarction (MI) and unstable angina. In PCI and non-ST-segment MI, therapy with eptifibatide has been shown to reduce acute ischaemic complications without any increased risk of life-threatening adverse events. In the recently reported Enhanced Suppression of the Platelet IIb/IIIa Receptor with Integrilin Therapy (ESPRIT) trial, two 180 microg/kg boluses of eptifibatide, 10 min apart, followed by an 18 - 24 h infusion at 2 microg/kg/min given as adjunctive therapy in non-urgent PCI reduced the 30-day composite of death, MI and need for urgent target vessel revascularisation from 10.4 to 6.8% compared with placebo. These results were achieved under conditions of typical contemporary PCI, namely the implantation of second- and third-generation stents deployed at high balloon pressures along with modern adjunctive pharmacological treatment, particularly the universal use of thienopyridines and lower-dose heparin. Few significant pharmacological effects other than inhibition of platelet aggregation and the effect on bleeding time have been reported. Future research will focus on alternative clinical applications and combinations with other therapies to further improve cardiovascular outcomes.
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Affiliation(s)
- J Conor O'Shea
- Duke Clinical Research Institute, Durham, NC 27705, USA.
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Jallu V, Meunier M, Brément M, Kaplan C. A new platelet polymorphism Duv(a+), localized within the RGD binding domain of glycoprotein IIIa, is associated with neonatal thrombocytopenia. Blood 2002; 99:4449-56. [PMID: 12036875 DOI: 10.1182/blood.v99.12.4449] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We report here the identification and characterization of a new platelet alloantigen, Duv(a+), implicated in a case of neonatal thrombocytopenia. Immunochemical studies demonstrated that the epitope was localized on glycoprotein (GP) IIIa. Sequencing of the exons 2 to 15 of GP IIIa gene polymerase chain reaction products from both parents revealed a single base substitution 517C>T (complementary DNA) present in a heterozygous state in DNA from the father leading to amino acid substitution Thr140Ile (ACC>ATC) within the Arg-Gly-Asp binding domain of GP IIIa. Flow cytometry and immunoprecipitation studies of IIb-C517 or T517 IIIa transfected Cos cells allowed us to demonstrate this mutation was responsible for expression of the Duv(a+) epitope. By polymerase chain reaction-single-strand conformational-polymorphism analysis, the mutated allele could not be detected in a population of 100 healthy unrelated donors, indicating a low frequency of occurrence. The Thr140/Ile dimorphism, localized 3 amino acids upstream from the Arg143 involved in the expression of HPA-4a, did not interfere with the binding of an anti-HPA-4a antibody in flow cytometry. Results of functional analysis of wild-type or mutated transfected CHO cells-(1) aggregation in the presence of Ca(++) and soluble fibrinogen after complex activation by dithiothreitol, (2) adhesion on coated fibrinogen, (3) binding of monoclonal antibody PAC-1 or LIBS antibody D3, and (4) outside-in signaling-all suggest that the Thr140Ile polymorphism localized in the Arg-Gly-Asp binding domain of GP IIIa does not affect significantly, if at all, the integrin function. We have shown that the anti-Duv(a+) antibody may inhibit platelet GP IIb-IIIa function.
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46
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Park HS, Kim C, Kang YK. Preferred conformations of RGDX tetrapeptides to inhibit the binding of fibrinogen to platelets. Biopolymers 2002; 63:298-313. [PMID: 11877740 DOI: 10.1002/bip.10067] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The conformational study on Arg-Gly-Asp (RGD)-containing tetrapeptides in the unhydrated and hydrated states has been carried out using the force field ECEPP/3 and the hydration shell model. The tetrapeptides studied here are H-RGDX-OH (X = Trp, Tyr, Phe, Leu, Val, Cys, Gln, and Ser), which show the inhibitory activity for binding of fibrinogen to platelets in the order of RGDW approximately equal to RGDY approximately equal to RGDF approximately equal to RGDL > RGDV > or = RGDC > or = RGDQ > or = RGDS. The backbone conformations with two C(7) backbone-to-backbone hydrogen bonds between Asp and Arg residues and between Xaa and Gly residues are in common most probable for the RGD sequence of RGDX tetrapeptides in the hydrated state. The dominant beta-turns for RGDX are found to be the types V' and IV at Gly-Asp and Asp-Xaa sequences, respectively, which are quite similar to the types II' and I (or II), respectively. However, it cannot be ruled out that the extended conformations are also remarkably feasible for RGDX tetrapeptides in water by peering the distributions of backbone conformations. These calculated results are consistent with the experimental results on RGD-containing proteins and conformationally constrained RGD-containing peptides. The reason why the RGDX becomes more potent as the side chain of the X residue is more hydrophobic may be ascribed to that the more hydrophobic is the residue X, the more populated are beta-turn structures for the Gly-Asp sequence. The hydrophobic side chain of X residue exposed to water is likely to interact with the hydrophobic region of receptor easily.
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Affiliation(s)
- Hae Sook Park
- Department of Radiotechnology, Cheju-halla College, Cheju 690-708, Korea
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47
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Wu Y, Chen L, Zheng PS, Yang BB. beta 1-Integrin-mediated glioma cell adhesion and free radical-induced apoptosis are regulated by binding to a C-terminal domain of PG-M/versican. J Biol Chem 2002; 277:12294-301. [PMID: 11805102 DOI: 10.1074/jbc.m110748200] [Citation(s) in RCA: 104] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Integrins are cell-surface glycoproteins that mediate cell activities, including tissue morphogenesis, development, immune response, and cancer, through interaction with extracellular proteins. Here we report a novel means by which integrin signaling and functions are regulated. In pull-down assays and immunoprecipitation, beta(1)-integrin bound to the C-terminal domain of PG-M/versican, an extracellular chondroitin sulfate proteoglycan. This was confirmed by cell-surface binding assays. Binding was calcium- and manganese-dependent. Upon native gel electrophoresis, beta(1)-integrin comigrated with the C-terminal domain of PG-M/versican. The interaction of beta(1)-integrin with the C-terminal domain of PG-M/versican activated focal adhesion kinase, enhanced integrin expression, and promoted cell adhesion. As a result, cells expressing the C-terminal domain of PG-M/versican were resistant to free radical-induced apoptosis. As the PG-M/versican peptide used in this study does not contain the RGD consensus-binding motif for integrins, the mechanism of the observed binding represents an entirely new function.
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Affiliation(s)
- Yaojiong Wu
- Sunnybrook and Women's College Health Sciences Centre and the Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M4N 3M5, Canada
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48
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Eriksson M, Christensen K, Lindahl TL, Larsson A. Pharmaceutical thrombosis prevention in cardiovascular disease. Expert Opin Investig Drugs 2002; 11:553-63. [PMID: 11922863 DOI: 10.1517/13543784.11.4.553] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Cardiovascular diseases are a leading cause of morbidity and mortality in modern society. As a result of this, great efforts have been made to establish regimens for prophylaxis and treatment of such disorders. Pharmacological intervention is also a prerequisite for the success of other therapeutic approaches, e.g. coronary angioplasty. Prevention of platelet aggregation is a goal that can be achieved by counteracting various receptors on the platelet surface. The main attentions for such interventions are focused on inhibiting the glycoprotein IIb/IIIa receptor. So far, they are limited to intravenous usage. Adenosine diphosphate receptor inhibitors are available for intravenous and oral usage. Their effect is, at least partly, also exerted via the counteraction of adenosine diphosphate-mediated activation of the glycoprotein IIb/IIIa complex. An oral direct thrombin inhibitor is under clinical evaluation. This review focuses on atherothrombotic disorders, but recent advances within new fields of anticoagulation (i.e., treatment of severe septic shock and a novel approach to prevent thromboembolic disorder during surgery) should not be overlooked.
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Affiliation(s)
- Mats Eriksson
- Department of Surgical Sciences, Institute of Anesthesiology and Intensive Care, Uppsala University Hospital, SE-751 85 Uppsala, Sweden.
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49
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Cierniewska-Cieslak A, Cierniewski CS, Bledzka K, Blecka K, Papierak M, Michalec L, Zhang L, Haas TA, Plow EF. Identification and characterization of two cation binding sites in the integrin beta 3 subunit. J Biol Chem 2002; 277:11126-34. [PMID: 11796735 DOI: 10.1074/jbc.m112388200] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
The midsegment of the beta(3) subunit has been implicated in the ligand and cation binding functions of the beta(3) integrins. This region may contain a metal ion-dependent adhesion site (MIDAS) and fold into an I domain-like structure. Two recombinant fragments, beta(3)-(95-373) and beta(3)-(95-301), were expressed and found to bind fibrinogen. Whereas 0.1 mm Ca(2+) supported ligand binding to both recombinant fragments, 1.0 mm Ca(2+) suppressed binding to the longer but not the shorter fragment. These properties suggest that beta(3)-(95-373) contains both the ligand-competent (LC) and inhibitory (I) cation binding sites, and beta(3)-(95-301) lacks the I site. In equilibrium dialysis experiments, beta(3)-(95-373) contained two divalent cation binding sites, one reactive with either Mg(2+) or Ca(2+) and one Ca(2+)-specific, whereas beta(3)-(95-301) lacked the Ca(2+)-specific site. Mutant forms of beta(3)-(95-373) suggested that the LC site is a MIDAS motif involving Asp(119), Ser(121), Ser(123), Asp(217), and/or Glu(220) as coordination sites, and the I site was dependent upon residues within beta(3)-(301-323). In a molecular model of beta(3)-(95-373), a second Ca(2+) could be docked onto a flexible loop in close proximity to the MIDAS. These results indicate that the ligand competent and Ca(2+)-specific inhibitory cation binding sites are distinct and reside in beta(3)-(95-373).
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50
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Novel synthetic RGD analogs incorporating salicylic acid derivatives show antiplatelet activityin vitro. ACTA ACUST UNITED AC 2002. [DOI: 10.1007/bf02576871] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
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