Platelet autophagic machinery involved in thrombosis through a novel linkage of AMPK-MTOR to sphingolipid metabolism

Tzu-Yin Lee; Wan-Jung Lu; Chun A Changou; Yuan-Chin Hsiung; Nguyen T T Trang; Cheng-Yang Lee; Tzu-Hao Chang; Thanasekaran Jayakumar; Cheng-Ying Hsieh; Chih-Hao Yang; Chao-Chien Chang; Ray-Jade Chen; Joen-Rong Sheu; Kuan-Hung Lin

doi:10.1080/15548627.2021.1904495

Platelet autophagic machinery involved in thrombosis through a novel linkage of AMPK-MTOR to sphingolipid metabolism

Autophagy. 2021 Dec;17(12):4141-4158. doi: 10.1080/15548627.2021.1904495. Epub 2021 Apr 5.

Authors

Tzu-Yin Lee¹, Wan-Jung Lu^{2

3

4}, Chun A Changou^{5

6

7}, Yuan-Chin Hsiung⁷, Nguyen T T Trang⁸, Cheng-Yang Lee⁹, Tzu-Hao Chang¹⁰, Thanasekaran Jayakumar², Cheng-Ying Hsieh², Chih-Hao Yang², Chao-Chien Chang^{2

11

12}, Ray-Jade Chen^{13

14}, Joen-Rong Sheu^{1

2

3}, Kuan-Hung Lin^{2

15}

Affiliations

¹ Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
² Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
³ Graduate Institute of Metabolism and Obesity Sciences, College of Nutrition, Taipei Medical University, Taipei, Taiwan.
⁴ Department of Medical Research, Taipei Medical University Hospital, Taipei, Taiwan.
⁵ Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
⁶ Integrated Laboratory, Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan.
⁷ Core Facility, Taipei Medical University, Taipei, Taiwan.
⁸ International Ph.D. Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
⁹ Research Information Session, Office of Information Technology, Taipei Medical University, Taipei, Taiwan.
¹⁰ Graduate Institute of Biomedical Informatics, Taipei Medical University, Taipei, Taiwan.
¹¹ Department of Cardiovascular Center, Cathay General Hospital, Taipei, Taiwan.
¹² Division of Cardiology, Department of Internal Medicine, School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan.
¹³ Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
¹⁴ Division of General Surgery, Department of Surgery, Taipei Medical University Hospital, Taipei, Taiwan.
¹⁵ Institute of Biomedical Sciences, MacKay Medical College, New Taipei City, Taiwan.

Abstract

Basal macroautophagy/autophagy has recently been found in anucleate platelets. Platelet autophagy is involved in platelet activation and thrombus formation. However, the mechanism underlying autophagy in anucleate platelets require further clarification. Our data revealed that LC3-II formation and SQSTM1/p62 degradation were noted in H₂O₂-activated human platelets, which could be blocked by 3-methyladenine and bafilomycin A₁, indicating that platelet activation may cause platelet autophagy. AMPK phosphorylation and MTOR dephosphorylation were also detected, and block of AMPK activity by the AMPK inhibitor dorsomorphin reversed SQSTM1 degradation and LC3-II formation. Moreover, autophagosome formation was observed through transmission electron microscopy and deconvolution microscopy. These findings suggest that platelet autophagy was induced partly through the AMPK-MTOR pathway. In addition, increased LC3-II expression occurred only in H₂O₂-treated Atg5^f/f platelets, but not in H₂O₂-treated atg5^-/- platelets, suggesting that platelet autophagy occurs during platelet activation. atg5^-/- platelets also exhibited a lower aggregation in response to agonists, and platelet-specific atg5^-/- mice exhibited delayed thrombus formation in mesenteric microvessles and decreased mortality rate due to pulmonary thrombosis. Notably, metabolic analysis revealed that sphingolipid metabolism is involved in platelet activation, as evidenced by observed several altered metabolites, which could be reversed by dorsomorphin. Therefore, platelet autophagy and platelet activation are positively correlated, partly through the interconnected network of sphingolipid metabolism. In conclusion, this study for the first time demonstrated that AMPK-MTOR signaling could regulate platelet autophagy. A novel linkage between AMPK-MTOR and sphingolipid metabolism in anucleate platelet autophagy was also identified: platelet autophagy and platelet activation are positively correlated.Abbreviations: 3-MA: 3-methyladenine; A.C.D.: citric acid/sod. citrate/glucose; ADP: adenosine diphosphate; AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy-related; B4GALT/LacCS: beta-1,4-galactosyltransferase; Baf-A1: bafilomycin A₁; BECN1: beclin 1; BHT: butylate hydrooxytoluene; BSA: bovine serum albumin; DAG: diacylglycerol; ECL: enhanced chemiluminescence; EDTA: ethylenediamine tetraacetic acid; ELISA: enzyme-linked immunosorbent assay; GALC/GCDase: galactosylceramidase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/GluSDase: glucosylceramidase beta; GPI: glycosylphosphatidylinositol; H₂O₂: hydrogen peroxide; HMDB: human metabolome database; HRP: horseradish peroxidase; IF: immunofluorescence; IgG: immunoglobulin G; KEGG: Kyoto Encyclopedia of Genes and Genomes; LAMP1: lysosomal associated membrane protein 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MPV: mean platelet volume; MTOR: mechanistic target of rapamycin kinase; ox-LDL: oxidized low-density lipoprotein; pAb: polyclonal antibody; PC: phosphatidylcholine; PCR: polymerase chain reaction; PI3K: phosphoinositide 3-kinase; PLS-DA: partial least-squares discriminant analysis; PRP: platelet-rich plasma; Q-TOF: quadrupole-time of flight; RBC: red blood cell; ROS: reactive oxygen species; RPS6KB/p70S6K: ribosomal protein S6 kinase B; SDS: sodium dodecyl sulfate; S.E.M.: standard error of the mean; SEM: scanning electron microscopy; SGMS: sphingomyelin synthase; SM: sphingomyelin; SMPD/SMase: sphingomyelin phosphodiesterase; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; UGT8/CGT: UDP glycosyltransferase 8; UGCG/GCS: UDP-glucose ceramide glucosyltransferase; ULK1: unc-51 like autophagy activating kinase 1; UPLC: ultra-performance liquid chromatography; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; WBC: white blood cell; WT: wild type.

Keywords: AMPK; autophagy; hydrogen peroxide; platelets; sphingolipid metabolism.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

AMP-Activated Protein Kinases / metabolism
Animals
Autophagy* / physiology
Blood Platelets / metabolism
Chromatography, Liquid
Hydrogen Peroxide
Mice
Phosphatidylinositol 3-Kinases / metabolism
Sphingolipids
TOR Serine-Threonine Kinases / metabolism
Tandem Mass Spectrometry
Thrombosis*

Substances

Sphingolipids
Hydrogen Peroxide
TOR Serine-Threonine Kinases
AMP-Activated Protein Kinases

Grants and funding

This work was supported by grants provided by the Ministry of Science and Technology of Taiwan (MOST 105-2320-B-341-001, MOST105-2311-B-038-005-MY3, MOST106-2320-B-715-006-MY3, MOST107-2320-B-038-035-MY2, MOST108-23“20-B-038-031-MY3, MOST108-2320-B-038-063, MOST108-2320-B-038-029, MOST 109-2320-B-715-003-MY3, MOST 109-2320-B-038-044-MY3, and MOST 109-2320-B-038-054), Taipei Medical University Hospital (107TMU-TMUH-14 and 108TMU-TMUH-11), and MacKay Medical College (1071B13 and 1081A02);