ICMR, School of Biological Sciences, University of Reading.
ICMR, School of Biological Sciences, University of Reading; Institute of Cardiovascular Sciences, University of Birmingham.
J Vis Exp. 2021 Oct 7(176). doi: 10.3791/62947.
Platelets react rapidly to vascular injury and undergo activation in response to a range of stimuli to limit blood loss. Many platelet function tests measure endpoint responses after a defined time period and not the rate of platelet activation. However, the rate at which platelets convert extracellular stimuli into a functional response is an essential factor in determining how efficiently they can respond to injury, bind to a forming thrombus, and signal to recruit other platelets. This paper describes a flow cytometry-based platelet function assay that enables simultaneous data acquisition and sample stimulation and utilizes newly developed bespoke open-source software (Kinetx) to enable quantitative kinetic measurements of platelet granule release, fibrinogen binding, and intracellular calcium flux. Kinetix was developed in R so that users can alter parameters such as degree of smoothing, identification of outlying data points, or time scales. To aid users unfamiliar with the R environment, Kinetix analysis of data can be performed by a single command. Together, this allows real-time platelet activation metrics, such as rate, acceleration, time to peak-rate, time to peak-calcium, and qualitative shape changes, to be accurately and reproducibly measured and categorized. Kinetic measurements of platelet activation give a unique insight into platelets' behavior during the first stages of activation and may provide a method of predicting the recruitment of platelets into a forming thrombus.
血小板对血管损伤迅速作出反应，并对一系列刺激作出反应，以激活血小板，从而限制血液流失。许多血小板功能测试测量特定时间段后的终点反应，而不是血小板激活的速度。然而，血小板将细胞外刺激转化为功能反应的速度是决定它们对损伤、与正在形成的血栓结合以及向其他血小板发出信号以招募血小板的效率的一个重要因素。本文描述了一种基于流式细胞术的血小板功能检测方法，该方法能够实现数据采集和样本刺激的同步，并利用新开发的专用开源软件（Kinetx）实现血小板颗粒释放、纤维蛋白原结合和细胞内钙流的定量动力学测量。Kinetx 是在 R 语言中开发的，因此用户可以更改平滑程度、异常数据点的识别或时间尺度等参数。为了帮助不熟悉 R 环境的用户，Kinetx 可以通过单个命令对数据进行分析。总之，这使得实时血小板激活指标，如速率、加速度、达到峰值速率的时间、达到峰值钙的时间和定性形状变化，能够被准确、可重复地测量和分类。血小板激活的动力学测量为血小板在激活的最初阶段的行为提供了独特的见解，并可能为预测血小板招募到正在形成的血栓中提供一种方法。
