Department of Vascular Biology and Thrombosis Research, Center of Physiology and Pharmacology, Medical University Vienna, Vienna, Austria.
Department of Surgery, General Hospital, Medical University Vienna, Vienna, Austria.
PLoS One. 2017 Dec 8;12(12):e0188921. doi: 10.1371/journal.pone.0188921. eCollection 2017.
Platelets store a plethora of different molecules within their granules, modulating numerous pathways, not only in coagulation, but also in angiogenesis, wound healing, and inflammatory diseases. These molecules get rapidly released upon activation and therefore represent an easily accessible indirect marker for platelet activation. Accurate analysis of platelet-derived molecules in the plasma requires appropriate anticoagulation to avoid in vitro activation and subsequent degranulation of platelets, potentially causing artificially high levels and masking biologically relevant differences within translational research studies. However, there is still enormous heterogeneity among anticoagulants used to prevent unwanted platelet activation, so that plasma levels reported for platelet granule contents range over several orders of magnitude. To address this problem and to define the most robust method of plasma preparation to avoid in vitro platelet activation during processing, we compared plasma concentrations of the three platelet-stored factors thrombospondin (TSP-1), platelet factor 4 (PF4), and soluble P-selectin (sCD62P) between human blood samples anticoagulated with either citrate-theophylline-adenosine-dipyridamole (CTAD), acid-citrate-dextrose (ACD), citrate, ethylenediaminetetraacetic acid (EDTA) or heparin. Additionally, we assessed the effect of storage temperature and time between blood drawing and sample processing within the differentially anticoagulated samples. Our data strongly support the use of CTAD as anticoagulant for determining plasma concentrations of platelet-stored molecules, as anticoagulation with heparin or EDTA led to a 12.4- or 8.3-fold increase in plasma levels of PF4, respectively. Whereas ACD was similar effective as CTAD, citrate only showed comparable PF4 plasma levels when plasma was kept at 4°C. Moreover, blood sampling with CTAD as anticoagulant resulted in the most reproducible values, even when samples were processed at ambient temperature or after storage over 6 hours. In the latter case, anticoagulation with heparin or EDTA led to artificially high plasma levels indicative of in vitro platelet activation. Therefore, we want to raise scientific awareness for choosing CTAD as optimal anticoagulant for the detection of platelet-stored molecules in plasma.
血小板在其颗粒中储存着大量不同的分子，调节着众多途径，不仅在凝血中，而且在血管生成、伤口愈合和炎症性疾病中也是如此。这些分子在激活后迅速释放，因此代表了血小板激活的一种易于获得的间接标志物。要准确分析血浆中的血小板衍生分子，需要适当的抗凝剂来避免体外血小板激活和随后的脱颗粒，这可能导致人为的高水平，并掩盖转化研究中生物学相关的差异。然而，用于预防不必要的血小板激活的抗凝剂之间仍然存在巨大的异质性，因此报告的血小板颗粒内容物的血浆水平跨越了几个数量级。为了解决这个问题，并定义最稳健的血浆制备方法，以避免在处理过程中的体外血小板激活，我们比较了用柠檬酸-茶碱-腺嘌呤-二吡啶（CTAD）、柠檬酸-柠檬酸钠-葡萄糖（ACD）、柠檬酸、乙二胺四乙酸（EDTA）或肝素抗凝的人类血液样本中三种血小板储存因子（血小板反应蛋白 1[TSP-1]、血小板因子 4[PF4]和可溶性 P-选择素[sCD62P]）的血浆浓度。此外，我们还评估了在不同抗凝的样本中，从采血到样本处理之间的储存温度和时间对血小板储存分子的影响。我们的数据强烈支持使用 CTAD 作为抗凝剂来确定血小板储存分子的血浆浓度，因为肝素或 EDTA 的抗凝作用分别导致 PF4 的血浆水平增加了 12.4 倍或 8.3 倍。而 ACD 与 CTAD 同样有效，只有当血浆在 4°C 下时，枸橼酸盐才能显示出可比的 PF4 血浆水平。此外，用 CTAD 作为抗凝剂进行采血可获得最具重现性的结果，即使在室温下处理样本或在储存 6 小时后也是如此。在后一种情况下，肝素或 EDTA 的抗凝作用导致人为的高血浆水平，表明存在体外血小板激活。因此，我们希望提高科学界对选择 CTAD 作为检测血浆中血小板储存分子的最佳抗凝剂的认识。
