Sundaramoorthi Hemalatha, Panapakam Rekha, Jagadeeswaran Pudur
a Department of Biological Sciences , University of North Texas , Denton , TX , USA.
Platelets. 2015;26(7):613-9. doi: 10.3109/09537104.2015.1018879. Epub 2015 Apr 22.
Zebrafish has become an excellent model system to study mammalian hemostasis. Despite our extensive efforts to develop technologies to measure zebrafish hemostasis and even with previously established thrombocyte qualitative and quantitative functional assays, quantifying thrombocyte function for high throughput applications has been a challenge. In this paper, we have developed two quantitative methods to estimate thrombocyte aggregation: one by whole blood aggregometry and the other by flow cytometry. We found that it is possible to conduct whole blood aggregometry using only 2 µl of blood and the currently available aggregometer. Each of three agonists, arachidonic acid, ADP, and collagen yielded impedance curves similar to those obtained with human blood. We were also able to use flow cytometry to indirectly quantify the extent of thrombocyte aggregation by labeling whole blood with mepacrine, aggregating in the presence of each of the above agonists, separating the aggregates from the white blood cells by centrifugation, and then sorting the resulting white cell fraction for thrombocyte numbers. These methods have high throughput capabilities and have the potential to be used in large scale screens to detect and characterize mutants with thrombocyte functional defects or to identify genes involved in thrombocyte function by large scale knockdowns.
斑马鱼已成为研究哺乳动物止血的优秀模型系统。尽管我们付出了巨大努力来开发测量斑马鱼止血的技术，甚至已有先前建立的血小板定性和定量功能检测方法，但对高通量应用中的血小板功能进行量化仍是一项挑战。在本文中，我们开发了两种定量方法来评估血小板聚集：一种是通过全血凝集测定法，另一种是通过流式细胞术。我们发现，仅使用2微升血液和现有的凝集仪就可以进行全血凝集测定。三种激动剂花生四烯酸、ADP和胶原蛋白各自产生的阻抗曲线与用人血获得的曲线相似。我们还能够通过用美帕林标记全血、在上述每种激动剂存在的情况下进行聚集、通过离心将聚集体与白细胞分离，然后对所得白细胞部分进行分选以确定血小板数量，利用流式细胞术间接量化血小板聚集程度。这些方法具有高通量能力，有潜力用于大规模筛选，以检测和表征具有血小板功能缺陷的突变体，或通过大规模基因敲低来鉴定参与血小板功能的基因。
