Key Laboratory of Shenzhen Respiratory Diseases, Department of Pulmonary and Critical Care Medicine, Shenzhen Institute of Respiratory Disease, The First Affiliated Hospital of Southern University of Science and Technology, The Second Clinical Medical College of Jinan University, Shenzhen People's Hospital, Shenzhen, Guangdong, China.
Department of Pediatrics, The First Affiliated Hospital of Southern University of Science and Technology, The Second Clinical Medical College of Jinan University, Shenzhen People's Hospital, Shenzhen, Guangdong, China.
J Cell Biochem. 2019 Aug;120(8):12300-12310. doi: 10.1002/jcb.28494. Epub 2019 Feb 27.
The disorders of hemostasis and coagulation were believed to be the main contributors to the pathogenesis of pulmonary thromboembolism (PTE), and platelets are the basic factors regulating hemostasis and coagulation and play important roles in the process of thrombosis. This study investigated the proteome of human umbilical vein endothelial cells (HUVECs) with platelet endothelial aggregation receptor-1 (PEAR1) knockdown using the isobaric tags for relative and absolute quantitation (iTRAQ) method and analyzed the role of differential abundance proteins (DAPs) in the regulation of platelets aggregation. Our results showed that the conditioned media-culturing HUVECs with PEAR1 knockdown partially suppressed the adenosine diphosphate (ADP)-induced platelet aggregation. The proteomics analysis was performed by using the iTRAQ technique, and a total of 215 DAPs (124 protein was upregulated and 91 protein were downregulated) were identified. The Gene Ontology (GO) enrichment analysis showed that proteins related to platelet α granule, adenosine triphosphate metabolic process, and endocytosis were significantly enriched. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also identified the significant enrichment of endocytosis-related pathways. The real-time polymerase chain reaction assay confirmed that the expression of P2Y , mitochondrial carrier 2, NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, and ubiquinol-cytochrome c reductase hinge protein are significantly downregulated in the HUVECs with PEAR1 knockdown. In conclusion, our in vitro results implicated that DAPs induced by PEAR1 knockdown might contribute to the platelet aggregation. Proteomic studies by employing GO enrichment and KEGG pathway analysis suggested that the potential effects of DAPs on platelet aggregation may be linked to the balance of ADP synthesis or degradation in mitochondria.
止血和凝血障碍被认为是肺血栓栓塞症（PTE）发病机制的主要原因，血小板是调节止血和凝血的基本因素，在血栓形成过程中发挥重要作用。本研究采用同位素相对和绝对定量（iTRAQ）方法研究了血小板内皮聚集受体-1（PEAR1）敲低的人脐静脉内皮细胞（HUVEC）的蛋白质组，并分析了差异丰度蛋白（DAP）在调节血小板聚集中的作用。我们的研究结果表明，用 PEAR1 敲低的条件培养基培养 HUVEC 部分抑制了二磷酸腺苷（ADP）诱导的血小板聚集。采用 iTRAQ 技术进行蛋白质组学分析，共鉴定出 215 个 DAP（124 个蛋白上调，91 个蛋白下调）。基因本体论（GO）富集分析显示，与血小板α颗粒、三磷酸腺苷代谢过程和内吞作用相关的蛋白显著富集。进一步的京都基因与基因组百科全书（KEGG）通路分析也鉴定出内吞作用相关通路的显著富集。实时聚合酶链反应（PCR）检测证实，PEAR1 敲低的 HUVEC 中 P2Y 、线粒体载体 2、NADH 脱氢酶（泛醌）铁硫蛋白 3 和泛醌-细胞色素 c 还原酶铰链蛋白的表达显著下调。综上所述，我们的体外研究结果表明，PEAR1 敲低诱导的 DAP 可能导致血小板聚集。GO 富集和 KEGG 通路分析的蛋白质组学研究表明，DAP 对血小板聚集的潜在影响可能与线粒体中 ADP 合成或降解的平衡有关。
