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

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 Atg5f/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

最近研究发现，无核血小板中亦存在基底性自噬/自噬现象。血小板自噬参与血小板活化和血栓形成。然而，无核血小板中自噬的潜在机制尚需进一步阐明。我们的数据揭示，在H2O2激活的人体血小板中，观察到LC3-II的形成和SQSTM1/p62的降解，这一过程可被3-甲基腺嘌呤和巴利福霉素A1阻断，表明血小板激活可能诱发血小板自噬。同时，检测到AMPK磷酸化和MTOR去磷酸化，通过AMPK抑制剂多索莫林阻断AMPK活性，可逆转SQSTM1降解和LC3-II形成。此外，通过透射电子显微镜和解卷积显微镜观察到自噬体的形成。这些发现表明，血小板自噬部分是通过AMPK-MTOR途径诱导的。此外，仅在H2O2处理的Atg5f/f血小板中观察到LC3-II表达增加，而在H2O2处理的atg5−/−血小板中则未观察到，这表明血小板自噬发生在血小板激活过程中。atg5−/−血小板对激动剂的聚集反应也降低，特异性缺失atg5的血小板在回肠微血管中表现出延迟性血栓形成和降低的因肺栓塞导致的死亡率。值得注意的是，代谢分析发现鞘脂代谢参与血小板激活，如观察到的几种代谢物改变，这些改变可通过多索莫林逆转。因此，血小板自噬与血小板激活呈正相关，部分是通过鞘脂代谢相互关联的网络实现的。总之，本研究首次证明AMPK-MTOR信号通路可以调节血小板自噬。在无核血小板自噬中，AMPK-MTOR与鞘脂代谢之间的新型联系也得到了鉴定：血小板自噬与血小板激活呈正相关。缩写：3-MA：3-甲基腺嘌呤；A.C.D.：柠檬酸/柠檬酸三钠/葡萄糖；ADP：腺苷二磷酸；AKT：AKT丝氨酸/苏氨酸激酶；AMPK：AMP激活的蛋白激酶；ANOVA：方差分析；ATG：自噬相关；B4GALT/LacCS：β-1,4-半乳糖基转移酶；Baf-A1：巴利福霉素A1；BECN1：beclin 1；BHT：丁基羟基甲苯；BSA：牛血清白蛋白；DAG：二酰甘油；ECL：增强化学发光；EDTA：乙二胺四乙酸；ELISA：酶联免疫吸附测定；GALC/GCDase：半乳糖脑苷脂酶；GAPDH：甘油醛-3-磷酸脱氢酶；GBA/GluSDase：葡萄糖脑苷脂酶β；GPI：糖基磷脂酰肌醇；H2O2：过氧化氢；HMDB：人类代谢组数据库；HRP：辣根过氧化物酶；IF：免疫荧光；IgG：免疫球蛋白G；KEGG：京都基因与基因组百科全书；LAMP1：溶酶体相关膜蛋白1；LC-MS/MS：液相色谱-串联质谱；mAb：单克隆抗体；MAP1LC3/LC3：微管相关蛋白1轻链3；MPV：平均血小板体积；MTOR：雷帕霉素靶蛋白激酶；ox-LDL：氧化低密度脂蛋白；pAb：多克隆抗体；PC：磷脂酰胆碱；PCR：聚合酶链反应；PI3K：磷脂酰肌醇3激酶；PLS-DA：偏最小二乘判别分析；PRP：富血小板血浆；Q-TOF：四极杆-飞行时间；RBC：红细胞；ROS：活性氧；RPS6KB/p70S6K：核糖体蛋白S6激酶B；SDS：十二烷基硫酸钠；S.E.M.：标准误；SEM：扫描电子显微镜；SGMS：鞘磷脂合成酶；SM：鞘磷脂；SMPD/SMase：鞘磷脂磷酸二酯酶；SQSTM1/p62：隔离素1；TEM：透射电子显微镜；UGT8/CGT：UDP糖基转移酶8；UGCG/GCS：UDP-葡萄糖脑苷脂糖基转移酶；ULK1：unc-51样自噬激活激酶1；UPLC：超高效液相色谱；PIK3C3/VPS34：磷脂酰肌醇3激酶催化亚基型3；PtdIns3P：磷脂酰肌醇-3-磷酸；WBC：白细胞；WT：野生型