Department of Anaesthesiology and Intensive Care Medicine, University Hospital Tübingen, Germany.
Platelets. 2012;23(5):386-94. doi: 10.3109/09537104.2011.624209. Epub 2011 Oct 31.
Animal models are essential tools for the in vivo evaluation of pharmacological modulation of platelet function and the mechanisms underlying thrombosis. In particular, pigs are being increasingly used in cardiovascular and platelet research. One standard method for the investigation of platelet function under experimental conditions is flow cytometry. However, this approach is limited by a shortage of feasible antibodies and a lack of incubation protocols with regard to porcine platelets. This study aimed to establish a method for the investigation of porcine platelets in flow cytometry. Platelets from pigs and human donors were stained with various commercially available specific antibodies against platelet receptors CD41a, CD42bα, CD62P, activated CD41/CD61, and platelet-bound fibrinogen. Staining procedures were performed in undiluted or diluted whole blood (WB) or platelet-rich plasma (PRP). Samples were treated with PBS buffer as control or with adenosine diphosphate (ADP) to induce platelet activation. Flow cytometry was performed using standard methodology. Furthermore, platelet counts were determined and ADP-induced platelet aggregations of both species were examined to confirm that the agonist ADP reliably activates human as well as porcine platelets. Five of the investigated antibodies bound to human, but not to porcine platelets only. However, two chicken-derived antibodies directed against CD62P (09-143) and fibrinogen (09-038) as well as a monoclonal mouse anti-CD62P (KO2.5) and a polyclonal rabbit anti-fibrinogen antibody (F0111) allowed reliable detection of porcine platelet activation. Moreover, binding intensity of the 09-143 antibody was increased when incubated in porcine PRP compared to WB, whereas antibody binding of both anti-fibrinogen antibodies to porcine platelets was only observed when incubated in a WB-buffer solution. KO2.5 antibody binding was detectable employing PRP as well as the WB-buffer solution. The feasibility of our new incubation protocols was confirmed by successful investigation of platelet activation in a porcine in vivo cardiopulmonary bypass model. In conclusion, we describe a reliable method to detect the activation of porcine platelets and therefore provide a useful tool for platelet flow cytometry in porcine models. Notably, the applied incubation protocol and medium, in which platelets are suspended, have major effects on antibody-binding properties.
动物模型是评估血小板功能药理学调节和血栓形成机制的体内评估的重要工具。特别是，猪越来越多地用于心血管和血小板研究。在实验条件下研究血小板功能的一种标准方法是流式细胞术。然而，这种方法受到可行抗体的短缺和缺乏针对猪血小板的孵育方案的限制。本研究旨在建立一种用于流式细胞术研究猪血小板的方法。使用针对血小板受体 CD41a、CD42bα、CD62P、活化的 CD41/CD61 和血小板结合纤维蛋白原的各种市售特异性抗体，对来自猪和人类供体的血小板进行染色。染色程序在未稀释或稀释的全血（WB）或富含血小板的血浆（PRP）中进行。将样品用 PBS 缓冲液处理作为对照或用二磷酸腺苷（ADP）处理以诱导血小板活化。使用标准方法进行流式细胞术。此外，还测定血小板计数，并检查两种物种的 ADP 诱导的血小板聚集，以确认激动剂 ADP 可靠地激活人类和猪血小板。五种研究的抗体仅与人类血小板结合，而不与猪血小板结合。然而，两种针对 CD62P（09-143）和纤维蛋白原（09-038）的鸡源性抗体、一种单克隆抗 CD62P（KO2.5）抗体和一种多克隆抗纤维蛋白原抗体（F0111）可可靠地检测猪血小板的活化。此外，与在 WB 中孵育相比，在猪 PRP 中孵育时 09-143 抗体的结合强度增加，而只有在 WB-缓冲液溶液中孵育时，两种抗纤维蛋白原抗体与猪血小板的结合才观察到。可以检测到 KO2.5 抗体的结合，既可以使用 PRP，也可以使用 WB-缓冲液溶液。通过成功研究猪心肺旁路模型中的血小板活化，证实了我们新的孵育方案的可行性。总之，我们描述了一种可靠的方法来检测猪血小板的活化，因此为猪模型中的血小板流式细胞术提供了有用的工具。值得注意的是，应用的孵育方案和血小板悬浮的介质对抗体结合特性有重大影响。
