VAS2870 and VAS3947 attenuate platelet activation and thrombus formation via a NOX-independent pathway downstream of PKC

AbstractNADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by treatment with ML171, GSK2795039, and GKT136901/GKT137831 did not affect thrombin and U46619-induced platelet aggregation. These findings suggest that VAS compounds may inhibit platelet aggregation via a NOX-independent manner. Thus, we aimed to investigate the detailed antiplatelet mechanisms of VAS compounds. The data revealed that VAS compounds blocked various agonist-induced platelet aggregation, possibly via blocking PKC downstream signaling, including IKKβ and p38 MAPK, eventually reducing platelet granule release, calcium mobilization, and GPIIbIIIa activation. In addition, VAS compounds inhibited mouse platelet aggregation-induced by collagen and thrombin. The in vivo study also showed that VAS compounds delayed thrombus formation without affecting normal hemostasis. This study is the first to demonstrate that, in addition to inhibiting NOX activity, VAS compounds reduced platelet activation and thrombus formation through a NOX-independent pathway downstream of PKC. These findings also indicate that VAS compounds may be safe and potentially therapeutic agents for treating patients with cardiovascular diseases.

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AN IMPORTANT STIMULATORY ROLE FOR THE cGMP-DEPENDENT PROTEIN KINASE II IN PLATELET ACTIVATION, IN VIVO THROMBOSIS AND HAEMOSTASIS

10.22541/au.160802063.39198387/v1 ◽

2020 ◽

Author(s):

Zhenyu Li ◽

Ying Liang ◽

can wang ◽

Guoying Zhang ◽

Jens Schlossmann ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Bleeding Time ◽

Thrombus Formation ◽

Atp Release ◽

Downstream Signaling ◽

Dependent Protein ◽

Deficient Mice

Background and Purpose: The intracellular second messenger cGMP mediates signals by activating two types of cGMP-dependent protein kinases (PKG), PKG I and PKG II, differentially expressed in different cells. In platelets, cGMP mediates biphasic signals that stimulate and inhibit platelet activation, and the downstream signaling of cGMP is mediated by PKG I, the only PKG known to be expressed in platelets. However, functional defects of PKG I knockout platelets did not fully explain the roles of cGMP and the effect of PKG inhibitors on platelet activation. Experimental Approach: To determine if PKG II is present in platelets and plays a role in platelet activation, we performed RT-PCR and isolation of PKG II protein using cGMP-conjugated beads. We further determined platelet aggregation and ATP release in vitro, and FeCl3-injured carotid artery thrombosis as well as tail bleeding time in vivo. Key Results: PKG II is expressed in platelets and plays an important role in selectively stimulating platelet activation but not in the negative regulatory role of cGMP. Collagen-induced platelet aggregation and ATP secretion were reduced in PKG II-deficient mice but not PKG I-deficient mice. In contrast, low-dose thrombin-induced platelet activation depended on PKG I but not PKG II. Tail bleeding time and FeCl3-induced artery thrombus formation were significantly prolonged in PKG II knockout mice. Conclusion and Implication: PKG II-mediated cGMP signals are important in platelet activation, thrombosis and haemostasis in vitro and in vivo.

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FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

Cardiovascular Research ◽

10.1093/cvr/cvy311 ◽

2018 ◽

Vol 115 (11) ◽

pp. 1672-1679 ◽

Cited By ~ 1

Author(s):

Qi Ma ◽

Weilin Zhang ◽

Chongzhuo Zhu ◽

Junling Liu ◽

Quan Chen

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Rich Plasma ◽

Thrombus Formation ◽

Mitochondrial Protein ◽

Dependent Manner ◽

Clot Retraction ◽

Akt Phosphorylation ◽

Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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CalDAG-GEFI Is a Key Signal Integrator in Platelet Activation and Thrombus Formation.

Blood ◽

10.1182/blood.v104.11.326.326 ◽

2004 ◽

Vol 104 (11) ◽

pp. 326-326

Author(s):

Wolfgang Bergmeier ◽

Jill R. Crittenden ◽

Crystal L. Piffath ◽

Denisa D. Wagner ◽

David E. Housman ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Knockout Mice ◽

Thrombus Formation ◽

Es Cells ◽

Embryonic Stem ◽

Premature Stop Codon ◽

Small Gtpase ◽

Integrin Αiibβ3

Abstract Inside-out activation of platelet integrin αIIbβ3 is a key step in agonist-induced platelet aggregation. Recent studies suggested the involvement of the small GTPase Rap1b in this process as it is highly expressed in platelets and becomes activated during platelet activation. In cell lines, overexpression of the Rap activator CalDAG-GEFI increased αIIbβ3-dependent adhesion, while overexpression of RapGAP, which inactivates Rap1, reduced αIIbβ3 activity. Here we provide evidence that CalDAG-GEFI is an essential component of this pathway in vivo. To generate CalDAG-GEFI knockout mice, we engineered mouse embryonic stem (ES) cells with a deletion that results in a frameshift mutation and a premature stop codon at the position encoding the 37th amino acid of CalDAG-GEFI. These ES cells were then used to derive chimeric mice that yielded germline transmission of the CalDAG-GEFI mutation. Deficiency of CalDAG-GEFI in mutant mice was confirmed by immunohistochemistry and western blot analysis. CalDAG-GEFI−/− platelets showed impaired Rap1b activation and aggregation in response to various agonists, with aggregation being completely blocked when platelets were activated with ADP, thromboxaneA2 analog, or calcium ionophore. Under physiological flow conditions in vitro and in vivo, CalDAG-GEFI-deficient platelets showed normal tethering to basement membrane components but failed to form thrombi. Mice deficient in CalDAG-GEFI were further characterized by a greatly increased bleeding time as well as by a strong protection against collagen-induced pulmonary thrombosis. In summary, we identified CalDAG-GEFI as a key signal integrator in the cascade leading through Rap1 and integrin αIIbβ3 to platelet aggregation and thrombus formation. The fact that CalDAG-GEFI knockout mice are resistant to collagen-induced thrombosis, and do not undergo spontaneous hemorrhaging, suggests that CalDAG-GEFI may be a promising new target for antithrombotic therapy.

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Tannic Acid Inhibits Protein Disulfide Isomerase, Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood-2018-99-114418 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2418-2418

Author(s):

Li Zhu

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Tannic Acid ◽

Protein Disulfide Isomerase ◽

Thrombus Formation ◽

Platelet Surface ◽

Disulfide Isomerase ◽

Protein Disulfide

Abstract Tannic acid (TA) was a polyphenol that harbors anti-oxidant capacity. A recent report implied that surface coating with TA might blunt thrombosis via altering the structure of fibrinogen. However, the effect of TA on platelet function and in vivo thrombus formation has not been reported. In this study, we showed that TA inhibits PDI activity and attenuates platelet activation. To explore the effects of TA on platelet aggregation, gel-filtered human platelets from healthy human donors were pretreated with TA (10/30/50 μM) or vehicle (0.9% sodium chloride) before being stimulated by various agonists. Turbidity analyses on a Chronolog aggregometer showed that TA dose-dependently inhibited platelet aggregation induced by thrombin, SFLLRN, GYQGQV, collagen, CRP, U46619, and ristocetin. Next, we employed flow cytometry (FACS) to determine the role of TA in platelet activation, including α-granule secretion and integrin activation. Pretreatment of platelets with TA led to significant reductions in surface P-selectin expression and soluble fibrinogen binding, supporting the inhibition of diverse platelet activation pathways. Supportively, platelet spreading on immobilized fibrinogen was significantly suppressed by TA treatment. In addition, cell viability assay with Almar blue agent showed no detrimental impact of TA on the survival of platelets. To ask whether the antiplatelet role of TA might be translated into an antithrombotic efficacy, we tested the effect of TA in both ex vivo and in vivo thrombosis models. Calcein-labeled human whole blood was perfused through microfluidic channels coated with collagen, and adherent platelets were visualized under a fluorescent microscopy. However, treatment with TA suppressed the number of adherent platelets under flow conditions. Moreover, in laser-induced mouse cremaster muscle arteries, administration of TA (5mg/kg) significantly reduced the size of forming thrombi compared with the vehicle. Verification of bleeding risk using tail truncation assay indicated no prolongation of bleeding time in mice receiving TA. Thus, TA shows an antiplatelet effect and may also attenuate thrombus formation. To gain a mechanistic insight to the role of TA in platelet function, we performed a molecular docking screen of the structure of TA and platelet surface proteins using the Autodock Vina software, which displayed the binding of TA with protein disulfide isomerase at the enzymatic active center. We then measured the impact of TA on PDI reductase activity with the dieosin glutathione disulfide assay in vitro (di-GSSG), showing that TA significantly inhibited PDI activity in a concentration-dependent manner. The results were verified in platelets using the 3-(N-Maleimidylpropionyl) biocytin (MPB) labeling, which showed that TA abrogated thrombin-stimulated free thiol formation on platelet surface. Supportively, FACS demonstrated that TA significantly suppressed the binding of fluorescent-labeled PDI to Mn2+-activated platelet integrin β3. Taken together, our findings demonstrated that TA inhibits PDI activity and may become a novel antithrombotic agent. Disclosures No relevant conflicts of interest to declare.

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Phosphatidylinositol-3,4-Bisphosphate-Akt Signaling Pathway Promotes Platelet Activation

Blood ◽

10.1182/blood-2018-99-115066 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1131-1131

Author(s):

Jasna Marjanovic ◽

Brad Rumancik ◽

Luke Weber ◽

Felix Wangmang ◽

Dane Fickes ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Glycogen Synthase ◽

Thrombus Formation ◽

Type I ◽

Dependent Manner ◽

Wild Type ◽

Phosphorylated Akt

Abstract Phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is a messenger that accumulates in platelets in a phosphoinositide 3-kinase and platelet aggregation-dependent manner. PtdIns(3,4)P2 is broken down by inositol polyphosphate 4-phosphatases, type I (INPP4A) and type II (INPP4B). These enzymes do not catalyze hydrolysis of phosphoinositides other than PtdIns(3,4)P2, and therefore provide unique means for studying the role of this lipid in platelet activation. We have found that the dominant isoform of 4-phosphatases expressed in platelets is INPP4A and we have generated radiation chimera mice with the deficiency in INPP4A restricted to hematopoietic cell lineage. Compared to wild type platelets, agonist-stimulated INPP4A-deficient platelets accumulated higher levels of PtdIns(3,4)P2. An increase in platelet aggregation in INPP4A-deficient platelets was observed with all tested agonists. To study platelet function in vivo, we performed carotid artery injury mouse thrombosis model experiments. Time to occlusion was dramatically reduced in mice with INPP4A deficiency. These data support the hypothesis that by regulating PtdIns(3,4)P2 levels, INPP4A downregulates platelet aggregation and thrombus formation. To investigate mechanisms mediating INPP4A-dependent signals, we compared levels of phosphorylated Akt and phosphorylated glycogen synthase kinase (GSK) in wild type and INPP4A-deficient platelets in response to agonist stimulation. An increase in phospho-Akt levels was observed in INPP4A-deficient platelets, suggesting that in addition to its well-characterized regulator, PtdIns(3,4,5)P3, PtdIns(3,4)P2 can promote Akt activation. Interestingly, this was not accompanied by a significant increase in phospho-GSK levels, suggesting a possible novel mechanism involved in platelet aggregation. Disclosures No relevant conflicts of interest to declare.

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An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

Journal of Clinical Medicine ◽

10.3390/jcm7110440 ◽

2018 ◽

Vol 7 (11) ◽

pp. 440 ◽

Cited By ~ 3

Author(s):

Wan Lu ◽

Chi Chung ◽

Ray Chen ◽

Li Huang ◽

Li Lien ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Phospholipase D ◽

Thrombus Formation ◽

Human Platelets ◽

Platelet Activity ◽

Clot Retraction ◽

First Time

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

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Triggering Receptor Expressed on Myeloid cells-1: a new player in platelet aggregation

Thrombosis and Haemostasis ◽

10.1160/th17-03-0156 ◽

2017 ◽

Vol 117 (09) ◽

pp. 1772-1781 ◽

Cited By ~ 12

Author(s):

Lucie Jolly ◽

Jérémie Lemarié ◽

Kevin Carrasco ◽

Batric Popovic ◽

Marc Derive ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Inflammatory Diseases ◽

Thrombus Formation ◽

Myeloid Cells ◽

Human Platelets ◽

Excessive Bleeding ◽

Supplementary Material ◽

Pharmacologic Inhibition

SummaryTriggering Receptor Expressed on Myeloid cells-1 (TREM-1) is an immunoreceptor initially known to be expressed on neutrophils and monocytes/macrophages. TREM-1 acts as an amplifier of the inflammatory response during both infectious and aseptic inflammatory diseases. Another member of the TREM family, The Triggering receptor expressed on myeloid cells Like Transcript-1 (TLT-1) is exclusively expressed in platelets and promotes platelet aggregation. As the gene that encodes for TLT-1 is located in the TREM-1 gene cluster, this prompted us to investigate the expression of TREM-1 on platelets. Here we show that TREM-1 is constitutively expressed in α-granules and mobilised at the membrane upon platelet activation. Pharmacologic inhibition of TREM-1 reduces platelet activation as well as platelet aggregation induced by collagen, ADP, and thrombin in human platelets. Aggregation is similarly impaired in platelets from Trem-1−/− mice. In vivo, TREM-1 inhibition decreases thrombus formation in a carotid artery model of thrombosis and protects mice during pulmonary embolism without excessive bleeding. These findings suggest that TREM-1 inhibition could be useful adducts in antiplatelet therapies.Supplementary Material to this article is available online at www.thrombosis-online.com.

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Impaired thrombin-induced platelet activation and thrombus formation in mice lacking the Ca2+-dependent tyrosine kinase Pyk2

Blood ◽

10.1182/blood-2012-06-438762 ◽

2013 ◽

Vol 121 (4) ◽

pp. 648-657 ◽

Cited By ~ 31

Author(s):

Ilaria Canobbio ◽

Lina Cipolla ◽

Alessandra Consonni ◽

Stefania Momi ◽

Gianni Guidetti ◽

...

Keyword(s):

Platelet Aggregation ◽

Focal Adhesion Kinase ◽

Platelet Activation ◽

Focal Adhesion ◽

Ex Vivo ◽

Thrombus Formation ◽

Arterial Occlusion ◽

In Vivo Model ◽

Glycoprotein Vi

Abstract In the present study, we used a knockout murine model to analyze the contribution of the Ca2+-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin αIIbβ3 and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A2 production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA2 phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein–coupled receptors in supporting platelet aggregation and thrombus formation.

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Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

BioMed Research International ◽

10.1155/2014/728019 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Ting-Lin Yen ◽

Wan-Jung Lu ◽

Li-Ming Lien ◽

Philip Aloysius Thomas ◽

Tzu-Yin Lee ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Therapeutic Potential ◽

Mapk Pathway ◽

Thrombus Formation ◽

Active Principle ◽

Intravascular Thrombosis ◽

Gentiana Lutea ◽

Mitogen Activated Protein

Amarogentin, an active principle ofGentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibitsin vivothrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

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Abstract 56: Platelet Specific Knockout of Glucose Transporter 3 Leads to Altered Metabolism and Decreased Platelet Activation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.56 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Trevor P Fidler ◽

Elizabeth Middleton ◽

Jesse W Rowley ◽

Luc Boudreau ◽

Robert A Campbell ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Plasma Membrane ◽

Glucose Uptake ◽

Platelet Activation ◽

Glucose Transporter ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Mitochondrial Uncoupling ◽

Granule Release

Patients with diabetes display increased thrombosis and platelet activation. In these disorders, the systemic milieu is characterized by multiple metabolic changes including increased glucose concentrations. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. Therefore we hypothesized that decreasing platelet glucose uptake would limit glycolysis thereby decreasing energy production and platelet reactivity. Platelets import glucose via two glucose transporters GLUT1 and GLUT3. GLUT1 is expressed on the plasma membrane and GLUT3 is expressed predominantly on alpha granule membranes (85%) and to a lesser extent on the plasma membrane (15%). To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout (KO) of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. Platelet glycogen content and glycolytic intermediates were significantly reduced in GLUT3 KO platelets compared to controls, and following mitochondrial uncoupling exhibited reduced glycolysis rates. Interestingly, under these conditions, mitochondrial maximal respiration was increased two-fold, with no change in mitochondrial density, or citric acid cycle intermediates. In vitro , GLUT3 deficient platelets display a 90% reduction of spreading on fibrinogen and collagen matrixes and significant reductions in CD62p surface translocation and GPIIbIIIa activation following stimulation with multiple agonists. Additionally makers of alpha granule release were significantly reduced. In vivo analysis of GLUT3 KO mice using a 10% ferric chloride model of arterial thrombosis and a tail-bleed model indicated no alteration in thrombosis between littermate controls and knockouts. However in a KBx/N model of rheumatoid arthritis GLUT3 KO mice exhibited significantly reduced disease severity. Together, these data indicate that GLUT3-mediated glucose uptake is essential for platelet activation, spreading and alpha granule release. GLUT3 modulates mechanisms that promote rheumatoid arthritis but not those that regulate in vivo thrombus formation.

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