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首页> 美国卫生研究院文献>Frontiers in Cardiovascular Medicine >In vitro Incubation of Platelets with oxLDL Does Not Induce Microvesicle Release When Measured by Sensitive Flow Cytometry
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In vitro Incubation of Platelets with oxLDL Does Not Induce Microvesicle Release When Measured by Sensitive Flow Cytometry

机译：用敏感流式细胞术测量时oxLDL在体外孵育血小板不会诱导微囊泡释放

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Microvesicles (MVs) are submicron vesicles with sizes of 0.1–1.0 μm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxidized low-density lipoprotein (oxLDL), a platelet ligand, induces platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study, we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5 μm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5 μm limit. MV size distribution was analyzed in plasma from 11 healthy volunteers (four females and seven males). MVs were identified as <1 μm and positive for lactadherin binding and cell-specific markers. Platelet-rich plasma (PRP) was incubated without and with oxLDL or LDL (as control) to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41+ and CD41+CD36+ MVs increased by six to eightfold in PRP, when left at room temperature, and the presence of cell-specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large interindividual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that procoagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great interindividual variability in size distribution of platelet-derived MVs and thereby stress the importance for generation of standardized protocols for MV quantification by flow cytometry.
机译：微囊泡（MVs）是直径为0.1–1.0μm的亚微米囊泡，在激活或凋亡后会从各种细胞中释放出来。他们广泛参与各种疾病的研究。在血液中，血小板是有效的MV分泌物，而氧化的低密度脂蛋白（oxLDL）（一种血小板配体）则诱导血小板活化，从而潜在地导致MV分泌。这种相互作用是通过oxLDL与位于血小板膜上的CD36结合而发生的。在这项研究中，我们调查了oxLDL体外孵育血小板对MV释放的影响。此外，我们比较了分离大于0.5μm的MV时获得的结果，作为从灵敏度低于0.5μm极限的较不敏感的常规流式细胞仪获得的结果的度量。分析了11名健康志愿者（4名女性和7名男性）血浆中的MV大小分布。 MVs被鉴定为<1μm，对乳黏附素结合和细胞特异性标记物呈阳性。在没有和含oxLDL或LDL的情况下（作为对照）孵育富血小板血浆（PRP），以研究对血小板活化的影响（通过释放MV来证明）。使用尺寸校准的荧光珠来建立MV门，并分离大小囊泡。当放置在室温下并且存在细胞特异性时，CD41 + 和CD41 + CD36 + MV在PRP中增加了六到八倍标记增加。总MV计数不受影响。 oxLDL孵育不会增加MV的释放或影响小尺寸和大型MV的分布。我们发现小型和大型MV的比例之间存在很大的个体差异，为73％。总之，我们建议血小板CD36与oxLDL相互作用诱导的促凝活性和血小板活化可能不涉及MV的释放。此外，我们的研究结果表明，血小板衍生的MV的大小分布存在很大的个体差异，因此强调了通过流式细胞术生成MV量化标准化协议的重要性。

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