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      Antiplatelet Activity, P2Y 1 and P2Y 12 Inhibition, and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P 1,P 4-dithio-P 2,P 3-chloromethylenetetraphosphate

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          Abstract

          Background

          Diadenosine tetraphosphate (Ap 4A), a constituent of platelet dense granules, and its P 1,P 4-dithio and/or P 2,P 3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y 1 and P2Y 12. The most active of those analogs, diadenosine 5′,5″″-P 1,P 4-dithio-P 2,P 3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

          Objective

          To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y 1 and P2Y 12 receptor antagonism, and their metabolism in human plasma.

          Methods

          We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y 1-mediated changes in cytosolic Ca 2+, P2Y 12-mediated changes in VASP phosphorylation, and metabolism in human plasma.

          Results

          The inhibition of ADP-induced human platelet aggregation and human platelet P2Y 12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P 1- and P 4-phosphorothioate groups, the S PS P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R PR P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S PR P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P 2,P 3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S PS P and the R PR P diastereomers.

          Conclusions

          The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y 1 and P2Y 12 receptors.

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          Most cited references28

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          NPP-type ectophosphodiesterases: unity in diversity.

          Nucleotide pyrophosphatase/phosphodiesterase (NPP)-type ectophosphodiesterases are found at the cell surface as type-I or type-II transmembrane proteins, but are also found extracellularly as secreted or shedded enzymes. They hydrolyze pyrophosphate or phosphodiester bonds in a variety of extracellular compounds including nucleotides, (lyso)phospholipids and choline phosphate esters. Despite their structurally related catalytic domain, each enzyme has well-defined substrate specificity. Catalysis by NPPs affects processes as diverse as cell proliferation and motility, angiogenesis, bone mineralization and digestion. In addition, there is emerging evidence for non-catalytic functions of NPPs in cell signaling. NPP-type ectophosphodiesterases are also implicated in the pathophysiology of cancer, insulin resistance and calcification diseases, and they hold great promise as easily accessible therapeutic targets.
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            Antiplatelet therapies for the treatment of cardiovascular disease.

            Antiplatelet therapy has been successful in reducing mortality and morbidity in acute myocardial infarction. Recent advances in understanding the molecular basis of the role of platelets in cardiovascular thrombosis have enabled the development of new agents with the potential to further reduce mortality and morbidity. This article reviews the role of platelets in haemostasis and cardiovascular thrombosis, and discusses the benefits and limitations of current and investigational antiplatelet agents in the treatment of cardiovascular disease.
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              Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family.

              The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells. NPP4-5 detected by database searches have a predicted type I membrane orientation but have not yet been functionally characterized. E-NPPs have been detected in almost all tissues often confined to specific substructures or cell types. In some cell types, NPP1 expression is constitutive or can be induced by TGF-beta and glucocorticoids, but the signal transduction pathways that control expression are poorly documented. NPP1-3 have a broad substrate specificity which may reflect their role in a host of physiological and biochemical processes including bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor signalling, nucleotide recycling, and cell motility. Abnormal NPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type 2 diabetes. In this review we summarize the present knowledge on the structure and the physiological and biochemical functions of E-NPP and their contribution to the pathogenesis of diseases.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                10 April 2014
                : 9
                : 4
                : e94780
                Affiliations
                [1 ]Center for Platelet Function Studies, Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America
                [2 ]Hematology Division, Chang Gung Memorial Hospital, Chang Gung University, Taipei, Taiwan
                [3 ]GLSynthesis Inc., Worcester, Massachusetts, United States of America
                [4 ]Center for Platelet Research Studies, Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America
                Royal College of Surgeons, Ireland
                Author notes

                Competing Interests: A.L. Frelinger and A.D. Michelson have been investigators on research grants to Boston Children's Hospital from GLSynthesis and Eli Lilly. A.D. Michelson has been a member of the Data Monitoring Committee of clinical trials sponsored by Lilly. I.B. Yanachkov, M. Yanachkova and G.E. Wright are employees of GLSynthesis, Inc. E.J. Dix has been a consultant to GLSynthesis, Inc. The other authors state that they have no competing interests. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: HC IBY EJD MY YFL MRB GEW ADM ALF. Performed the experiments: HC IBY EJD MY YFL MRB. Analyzed the data: HC ALF IBY. Wrote the paper: HC IBY ALF GEW ADM.

                Article
                PONE-D-13-46712
                10.1371/journal.pone.0094780
                3983250
                24722456
                2c61818e-75c1-448f-9a0c-54921cfc06c2
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 6 November 2013
                : 19 March 2014
                Page count
                Pages: 8
                Funding
                This work was supported by SBIR grants HL081992 and HL088828 (to I.B. Yanachkov) from the National Heart, Lung and Blood Institute. Support for H. Chang was provided by grant CMRP G380211, provided by Chang Gung Memorial Hospital. There were no other funding sources for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Platelets
                Biochemistry
                Enzymology
                Enzymes
                Nucleic Acids
                Nucleotides
                Cell Biology
                Signal Transduction
                Cell Signaling
                Membrane Receptor Signaling
                Nucleotide Receptor Signaling
                Molecular Cell Biology
                Medicine and Health Sciences
                Cardiology
                Cardiovascular Pharmacology
                Hematology
                Physical Sciences
                Chemistry
                Medicinal Chemistry
                Stereochemistry

                Uncategorized
                Uncategorized

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