Volume 20, Issue 4, Pages (July 2017)

Slides:



Advertisements
Similar presentations
Volume 4, Issue 4, Pages (April 2015)
Advertisements

Wallerian Degeneration Is Executed by an NMN-SARM1-Dependent Late Ca2+ Influx but Only Modestly Influenced by Mitochondria  Andrea Loreto, Michele Di Stefano,
Volume 70, Issue 4, Pages (May 2011)
Volume 13, Issue 5, Pages (November 2015)
TIA-1 Self-Multimerization, Phase Separation, and Recruitment into Stress Granules Are Dynamically Regulated by Zn2+  Joseph B. Rayman, Kevin A. Karl,
by Matt W. Goschnick, Lai-Man Lau, Janet L. Wee, Yong S. Liu, P
PRT , a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model by Michael P. Reilly, Uma Sinha,
Complement-Independent, Peroxide-Induced Antibody Lysis of Platelets in HIV-1- Related Immune Thrombocytopenia  Michael Nardi, Stephen Tomlinson, M.Alba.
Alterations in platelet secretion differentially affect thrombosis and hemostasis by Smita Joshi, Meenakshi Banerjee, Jinchao Zhang, Akhil Kesaraju, Irina.
Modulation of K-Ras-Dependent Lung Tumorigenesis by MicroRNA-21
Β1 integrin−mediated signals are required for platelet granule secretion and hemostasis in mouse by Tobias Petzold, Raphael Ruppert, Dharmendra Pandey,
Impaired activation of platelets lacking protein kinase C-θ isoform
Volume 20, Issue 8, Pages (August 2017)
Volume 20, Issue 10, Pages (September 2017)
Volume 21, Issue 12, Pages (December 2017)
Volume 88, Issue 3, Pages (November 2015)
Volume 18, Issue 1, Pages (January 2017)
Volume 23, Issue 11, Pages (June 2018)
Volume 6, Issue 1, Pages (January 2014)
Volume 26, Issue 3, Pages e5 (September 2017)
The Role of Dendritic Cells in S
Dasatinib enhances megakaryocyte differentiation but inhibits platelet formation by Alexandra Mazharian, Cedric Ghevaert, Lin Zhang, Steffen Massberg,
International Journal of Cardiology
Volume 12, Issue 3, Pages (July 2015)
Volume 20, Issue 3, Pages (September 2016)
Thiazolidinediones Regulate Adipose Lineage Dynamics
Dichloroacetate, an inhibitor of pyruvate dehydrogenase kinases, inhibits platelet aggregation and arterial thrombosis by Manasa K. Nayak, Nirav Dhanesha,
Volume 6, Issue 6, Pages (March 2014)
TRPV3 Channels Mediate Strontium-Induced Mouse-Egg Activation
Volume 18, Issue 13, Pages (March 2017)
Volume 22, Issue 3, Pages (March 2015)
Volume 18, Issue 3, Pages (January 2017)
S.-M. Harwood, D.-A. Allen, M.-J. Raftery, M.M. Yaqoob 
Volume 22, Issue 2, Pages (January 2018)
Volume 25, Issue 1, Pages (January 2017)
Volume 125, Issue 4, Pages (May 2006)
Volume 22, Issue 1, Pages e7 (July 2017)
Volume 6, Issue 1, Pages (January 2014)
Volume 22, Issue 5, Pages (January 2018)
Volume 19, Issue 6, Pages (May 2017)
Volume 18, Issue 11, Pages (March 2017)
Kentaro Abe, Masatoshi Takeichi  Neuron 
Volume 38, Issue 3, Pages (March 2013)
Wallerian Degeneration Is Executed by an NMN-SARM1-Dependent Late Ca2+ Influx but Only Modestly Influenced by Mitochondria  Andrea Loreto, Michele Di Stefano,
Volume 19, Issue 1, Pages (April 2017)
Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance  Valeria R. Fantin, Julie St-Pierre,
Volume 25, Issue 1, Pages (January 2017)
The Mitochondrial Rhomboid Protease PARL Is Regulated by PDK2 to Integrate Mitochondrial Quality Control and Metabolism  Guang Shi, G. Angus McQuibban 
EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy
Volume 18, Issue 12, Pages (March 2017)
Volume 5, Issue 4, Pages (November 2013)
Molecular Therapy - Oncolytics
Volume 12, Issue 1, Pages (July 2015)
Volume 21, Issue 1, Pages (October 2017)
TIA-1 Self-Multimerization, Phase Separation, and Recruitment into Stress Granules Are Dynamically Regulated by Zn2+  Joseph B. Rayman, Kevin A. Karl,
Volume 16, Issue 4, Pages (October 2012)
Volume 19, Issue 1, Pages (April 2017)
by Kelly E. Johnson, Julia R. Ceglowski, Harvey G. Roweth, Jodi A
Volume 10, Issue 4, Pages (April 1999)
Volume 39, Issue 3, Pages (November 2016)
Molecular Therapy - Oncolytics
Arisa Hirano, Daniel Braas, Ying-Hui Fu, Louis J. Ptáček  Cell Reports 
Volume 13, Issue 5, Pages (November 2015)
Molecular Therapy - Nucleic Acids
Volume 39, Issue 1, Pages (July 2003)
Volume 23, Issue 8, Pages (May 2018)
Volume 24, Issue 4, Pages (July 2018)
Volume 28, Issue 1, Pages (January 2008)
Volume 19, Issue 6, Pages (May 2017)
Volume 23, Issue 6, Pages (May 2018)
Presentation transcript:

Volume 20, Issue 4, Pages 881-894 (July 2017) Deletion of GLUT1 and GLUT3 Reveals Multiple Roles for Glucose Metabolism in Platelet and Megakaryocyte Function  Trevor P. Fidler, Robert A. Campbell, Trevor Funari, Nicholas Dunne, Enrique Balderas Angeles, Elizabeth A. Middleton, Dipayan Chaudhuri, Andrew S. Weyrich, E. Dale Abel  Cell Reports  Volume 20, Issue 4, Pages 881-894 (July 2017) DOI: 10.1016/j.celrep.2017.06.083 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 881-894DOI: (10.1016/j.celrep.2017.06.083) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Glucose Metabolism Is Decreased in DKO Platelets (A) Representative western blot of protein lysates from GLUT1-KO, GLUT3-KO, DKO, and respective littermate control platelets. (B) [3H] 2-DOG glucose uptake in platelets. (C and D) 13C-Lactic acid production (C) and 12C-lactic acid (D) in the presence of 13C-Glucose exclusively in the extracellular media (n = 3). (E) Seahorse analysis of DKO platelet extracellular acidification rate (ECAR) under non-stimulated and thrombin (IIa)-stimulated conditions (n = 3). (F) Glycogen analysis of platelets normalized to cell number (n = 5). Error bars are SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 with respect to control genotype; #p < 0.05 with respect to treatment, same genotype; two-way ANOVA followed by Bonferroni multiple comparison post hoc test, B and E; Student’s t test, C, D, and F). Cell Reports 2017 20, 881-894DOI: (10.1016/j.celrep.2017.06.083) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 DKO Platelets Increase Mitochondrial Metabolism in response to Metabolic Stress (A) Estimation of AMP + ADP to ATP ratio in whole platelet lysates (n = 8). (B) Western blot analysis of protein lysates from freshly isolated platelets (n = 3). (C) Mitochondrial membrane potential of platelets incubated in the indicated media for 1 hr (n = 3). (D) Seahorse analysis of platelet O2 consumption in 25 mM glucose + 1 mM glutamate + 1 mM pyruvate media ± thrombin (IIa) (n = 3). (E) Seahorse analysis of DKO and control platelets under basal conditions in the presence of 25 mM glucose ± 1 mM glutamate and 1 mM pyruvate (n = 3). (F) Ratio of intracellular lactic acid to pyruvic acid (n = 6). Error bars are SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001 with respect to control genotype; #p < 0.05 with respect to treatment, same genotype; one-way ANOVA followed by Tukey’s multiple comparison post hoc test, C and E; two-way ANOVA followed by Bonferroni multiple comparison post hoc test, D; Student’s t test, A and F). Cell Reports 2017 20, 881-894DOI: (10.1016/j.celrep.2017.06.083) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Impaired Activation of DKO Platelets (A) Transmission electron microscopy of washed platelets in 5 mM glucose media, treated in the presence or absence of 250 μM Par4 (scale bar, 2 μm). (B–E) Quantification of non-stimulated (B, n = 4) and Par4 peptide-stimulated (C, n = 4) electron micrographs of platelets. Washed platelets pre-incubated for 30 min in the presence of 5 mM glucose ± 1 mM glutamate and 1 mM pyruvate were stimulated with the indicated agonist and analyzed for GPIIbIIIa activation (D, JonA geo. MFI) and CD62p surface translocation (E, CD62p Geo. MFI) (n = 5). Data are mean ± SEM (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001 with respect to control genotype; #p < 0.05 with respect to treatment, same genotype; two-way ANOVA followed by Bonferroni multiple comparison post hoc test, B–E). Cell Reports 2017 20, 881-894DOI: (10.1016/j.celrep.2017.06.083) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 DKO Platelets Demonstrate Decreased Thrombosis (A) Washed platelets in 5 mM glucose media treated with the indicated agonist for 15 min were monitored for annexin V positivity (n = 6). (B) Representative tracing of thrombin-mediated cytoplasmic Ca2+ monitored via fluo-4 MFI, using flow cytometry (n = 3). (C) Fura-2-loaded platelets stimulated with 1 U/mL thrombin + 160 ng/mL convulxin, SEM (dashed lines) (n = 4). (D) Change in Ca2+ following the administration of thrombin + convulxin (n = 4). (E) Fura-2-loaded platelets in 250 μM EGTA, treated with thapsigargin (TG) and then 1 mM Ca2+ SEM (dashed lines) (n = 4). (F) Change in Ca2+ following the administration of thapsigargin (n = 4). (G and H) Relative GPIIbIIIa activation (G, relative JonA Geo. MFI) and CD62p surface translocation (H, relative Geo. MFI) following stimulation with the indicated agonist (n ≥ 3). (I) Tail bleeding was assessed by monitoring time to bleeding cessation (n = 7). (J) Time to occlusion was determined in a 7.5% Ferric chloride-induced arterial thrombosis model (n = 6). (K) Length of survival in a collagen/epinephrine-induced pulmonary embolism model (n = 12). Data are mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; two-way ANOVA followed by Bonferroni multiple comparison post hoc test, A, G, and H; Student’s t test, D, F, I, and J; log rank [Mantel-Cox] test, K). Cell Reports 2017 20, 881-894DOI: (10.1016/j.celrep.2017.06.083) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Platelet Production by DKO Megakaryocytes Is Decreased (A) Platelet counts in whole blood (n = 7). (B) Platelets were depleted through the administration of anti-GP1bα (black arrow), and platelet counts were monitored (n = 6). (C) Representative image of immunohistochemistry for vWF in femurs and spleens from DKO and control mice counterstained with eosin. (D) Quantification of megakaryocyte density in femurs (n = 4) and spleens (n = 3). (E) Glucose uptake in cultured megakaryocytes. (F) Representative image of megakaryocytes derived from control and DKO bone marrow (scale bar, 60 μm). (G) Quantification of pro-platelet formation by bone marrow-derived megakaryocytes (n = 5). Data are mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 with respect to control genotype; one-way ANOVA followed by Tukey’s multiple comparison post hoc test, A; two-way ANOVA followed by Tukey’s multiple comparison post hoc test, B; Student’s t test, D, E, and G). Cell Reports 2017 20, 881-894DOI: (10.1016/j.celrep.2017.06.083) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 DKO Platelets Demonstrate Increased Clearance (A) Platelets were labeled with anti-Gp1bβ-FITC-labeled antibody, then monitored for percentage CD41-positive platelets (n = 6). (B) Whole blood was collected at 72 hr post-anti-Gp1bβ-FITC injection, then monitored for annexin V Geo. MFI. (n = 6). (C) Washed platelets incubated in 5 mM glucose media ± 1 mM glutamate and 1 mM pyruvate at 37°C with 5% CO2 were monitored for annexin V exposure (n = 3). (D) Platelet annexin V binding following 6-hr incubation in specified media at 37°C with 5% CO2 (n = 20). (E) Mitochondrial potential of platelets stratified for annexin V positivity, following 6-hr incubation (n = 3). (F) Western blot analysis of platelet proteins following 6-hr incubation (n = 6). (G) Annexin V binding in platelets incubated for 1 hr in vitro with oligomycin. (H) Mice were injected with 1 mg/kg oligomycin or vehicle and monitored for platelet count following 6 hr (n = 5). Data are mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 with respect to control genotype; #p < 0.05 with respect to treatment, same genotype, $p < 0.05 with respect to annexin V-negative platelets of equivalent genotype and treatment; two-way ANOVA followed by Bonferroni multiple comparison post hoc test, A–E and H). Cell Reports 2017 20, 881-894DOI: (10.1016/j.celrep.2017.06.083) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 Calpain Activation Regulates DKO Platelet Annexin V Binding and Clearance In Vivo (A) Western blot analysis of caspase-3 (black arrow indicates calpain cleavage product). (B and C) Annexin V-negative platelets loaded with Fluo-4 in media with 1 mM EGTA were analyzed for basal cytoplasmic Ca2+ concentration and following stimulation with 1 μM ionomycin at 1 hr (B) and 6 hr post-isolation (C), marked by Fluo-4 Geo. MFI (n = 6). (D) Calpain activity of platelets incubated in glucose media for 6 hr. (E) Western blot analysis of Filamin A cleavage (n = 3). (F) Platelets were incubated in the presence of vehicle (Veh) or calpeptin for 6 hr, then analyzed for annexin V binding (n = 6). (G) Mice were injected with calpeptin (1 mg/kg) daily for 7 days prior to injection with anti-Gp1bβ-FITC-labeled antibody, and calpeptin administration was continued daily until the cessation of half-life experiments. Blood was assayed at 55 hr post-injection (n = 6). Data are mean ± SEM (∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 with respect to control genotype; #p < 0.05 with respect to treatment, same genotype; Student’s t test, D; one-way ANOVA followed by Tukey’s multiple comparison post hoc test, G; two-way ANOVA followed by Bonferroni multiple comparison post hoc test, B, C, and F). Cell Reports 2017 20, 881-894DOI: (10.1016/j.celrep.2017.06.083) Copyright © 2017 The Author(s) Terms and Conditions