血小板的线粒体呼吸:分离方法的比较

Mitochondrial Respiration of Platelets: Comparison of Isolation Methods

 

 

 

Keywords:density gradient centrifugation; flow cytometry; high-resolution respirometry; human platelets; mitochondria; oxidative phosphorylation; platelet apheresis; thrombocytes
关键词:密度梯度离心; 流式细胞仪; 高分辨率呼吸测量; 人血小板; 线粒体; 氧化磷酸化; 血小板单采术; 血小板

细胞类型:血小板
作者:Vernerova A, Garcia-Souza LF, Soucek O, Kostal M, Rehacek V, Krcmova LK, Gnaiger E, Sobotka O
出版期刊:《Biomedicines》 2021/12/8

Abstract:

Multiple non-aggregatory functions of human platelets (PLT) are widely acknowledged, yet their functional examination is limited mainly due to a lack of standardized isolation and analytic methods. Platelet apheresis (PA) is an established clinical method for PLT isolation aiming at the treatment of bleeding diathesis in severe thrombocytopenia. On the other hand, density gradient centrifugation (DC) is an isolation method applied in research for the analysis of the mitochondrial metabolic profile of oxidative phosphorylation (OXPHOS) in PLT obtained from small samples of human blood. We studied PLT obtained from 29 healthy donors by high-resolution respirometry for comparison of PA and DC isolates. ROUTINE respiration and electron transfer capacity of living PLT isolated by PA were significantly higher than in the DC group, whereas plasma membrane permeabilization resulted in a 57% decrease of succinate oxidation in PA compared to DC. These differences were eliminated after washing the PA platelets with phosphate buffer containing 10 mmol·L-1 ethylene glycol-bis (2-aminoethyl ether)-N,N,N',N'-tetra-acetic acid, suggesting that several components, particularly Ca2+ and fuel substrates, were carried over into the respiratory assay from the serum in PA. A simple washing step was sufficient to enable functional mitochondrial analysis in subsamples obtained from PA. The combination of the standard clinical PA isolation procedure with PLT quality control and routine mitochondrial OXPHOS diagnostics meets an acute clinical demand in biomedical research of patients suffering from thrombocytopenia and metabolic diseases.

 

文章摘要:

人类血小板 (PLT) 的多种非聚集功能已被广泛认可,但其功能检查主要由于缺乏标准化的分离和分析方法而受到限制。血小板单采(PA)是一种成熟的PLT分离临床方法,旨在治疗严重血小板减少症的出血素质。另一方面,密度梯度离心 (DC) 是一种用于研究的分离方法,用于分析从少量人体血液样本中获得的 PLT 中氧化磷酸化 (OXPHOS) 的线粒体代谢特征。我们通过高分辨率呼​​吸测量法研究了从 29 名健康供体获得的 PLT,以比较 PA 和 DC 分离株。 PA 分离的活 PLT 的常规呼吸和电子传递能力显着高于 DC 组,而质膜透化导致 PA 中的琥珀酸氧化与 DC 相比降低了 57%。用含有 10 mmol·L-1 乙二醇-双(2-氨基乙基醚)-N,N,N',N'-四乙酸的磷酸盐缓冲液洗涤 PA 血小板后,这些差异消失了,这表明有几种成分,特别是 Ca2+ 和燃料底物,从 PA 中的血清转移到呼吸测定中。一个简单的洗涤步骤就足以对从 PA 获得的子样品进行功能性线粒体分析。标准临床 PA 隔离程序与 PLT 质量控制和常规线粒体 OXPHOS 诊断相结合,满足了血小板减少症和代谢性疾病患者生物医学研究的迫切临床需求。

 


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