Unit of Heart-Brain Axis: Cellular and Molecular Mechanisms, Centro Cardiologico Monzino IRCCS, 20138 Milano, Italy.
Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy.
Front Biosci (Landmark Ed). 2022 May 18;27(5):161. doi: 10.31083/j.fbl2705161.
Platelet-derived extracellular vesicles (PEVs) are small vesicles released by activated platelets that are gaining growing interest in the field of vascular biology. The mode of platelet activation is a critical determinant of PEVs release, phenotype and function. However, only very limited information is available concerning the impact of the platelet purification procedure on PEVs release.
Washed or isolated platelets were separated by differential centrifugations. For washed platelets, the platelet pellet was washed by resuspension in PIPES buffer and finally resuspended in HEPES buffer. Isolated platelets were obtained by directly resuspending the platelet pellet in HEPES, skipping the washing steps in PIPES buffer. PEVs release was induced in washed or isolated platelets by stimulation with different agonist and analysed by Nanoparticle Tracking Analysis.
Isolated platelets showed a higher release of PEVs upon adenosine diphosphate (ADP) stimulation compared to washed platelets, whereas PEVs released upon stimulation with strong agonists (thrombin, collagen, A23187, U46619) were similar in the two groups. This different responsiveness to ADP was also observed as a higher α-granules release and protein kinase C activation in isolated platelets compared to washed ones. Residual plasma contamination appeared to be essential for the ability of platelets to release PEVs in response to ADP.
In conclusion, our study strongly suggests that procedure adopted for platelets preparation is a critical determinant of PEVs release upon ADP stimulation.
血小板衍生的细胞外囊泡（PEVs）是由活化的血小板释放的小囊泡，在血管生物学领域受到越来越多的关注。血小板的激活方式是决定 PEVs 释放、表型和功能的关键因素。然而，关于血小板纯化过程对 PEVs 释放的影响，仅有非常有限的信息。
通过差速离心分离洗涤或分离的血小板。对于洗涤的血小板，将血小板沉淀通过在 PIPES 缓冲液中重悬并最终在 HEPES 缓冲液中重悬来洗涤。分离的血小板通过直接在 HEPES 中重悬血小板沉淀来获得，跳过 PIPES 缓冲液中的洗涤步骤。通过用不同的激动剂刺激洗涤或分离的血小板来诱导 PEVs 释放，并通过纳米颗粒跟踪分析进行分析。
与洗涤的血小板相比，ADP 刺激时分离的血小板释放的 PEVs 更高，而在两组中，用强激动剂（凝血酶、胶原、A23187、U46619）刺激时释放的 PEVs 相似。与洗涤的血小板相比，分离的血小板对 ADP 的反应性更高，α-颗粒释放和蛋白激酶 C 激活也更高。残留的血浆污染似乎是血小板在 ADP 刺激下释放 PEVs 的能力的关键决定因素。
总之，我们的研究强烈表明，血小板制备所采用的程序是 ADP 刺激时 PEVs 释放的关键决定因素。
