Introduction

Precision mapping of the functional structure of platelet populations holds great promise for the identification of hyper-reactive subtypes that are likely to be disease drivers, having value in prognostics and as therapeutic targets. However, the ability to measure the intrinsic functional capacity of individual platelets is confounded by potent paracrine cross-talk, resulting in phenotypic remodeling of the entire platelet population, and in doing so obscuring the identity of hyper-reactive platelets.

Methods

To address this we have developed a droplet microfluidics strategy for single platelet confinement to exclude paracrine signaling. Consideration of the Poisson distribution was used for high throughput single platelet encapsulation and the preparation of minimal platelet collectives serving as digital models for understanding the role of hyper-reactive platelets coordinating system-level behavior by paracrine signaling. Platelets are retrieved from the droplets for phenotyping using standard flow cytometry. In addition, we have incorporated a staggered herringbone micromixing element for accurate agonist and antibody dispensing in droplets.

Results

The methodology was used for characterizing sensitivity distributions from healthy blood donors in response to convulxin (agonist of the GPVI receptor, the major platelet receptor for collagen). P-selectin exposure and α_IIbβ₃ integrin activation were used as analytical end-points to demonstrate the existence of hyper-reactive platelets that direct 20-fold gains in system level sensitivity.

Conclusions

The analytical workflow represents an enabling tool for the accurate classification of platelet subtypes and description of their underlying biology.

中文翻译：

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用于研究血小板内在功能的具有试剂微混合的液滴微流体

介绍

血小板群功能结构的精确映射对于识别可能是疾病驱动因素的高反应性亚型具有很大的前景，在预后和治疗靶点方面具有价值。然而，测量单个血小板的内在功能能力的能力被强大的旁分泌串扰所混淆，导致整个血小板群的表型重塑，并且这样做掩盖了高反应性血小板的身份。

方法

为了解决这个问题，我们开发了一种液滴微流体策略，用于单血小板限制以排除旁分泌信号。泊松分布的考虑用于高通量单血小板封装和最小血小板集合体的制备，作为数字模型，以了解高反应性血小板通过旁分泌信号协调系统级行为的作用。使用标准流式细胞仪从液滴中提取血小板用于表型分析。此外，我们还采用了交错的人字形微混合元件，用于在液滴中准确分配激动剂和抗体。

结果

该方法用于表征健康献血者对惊厥蛋白（GPVI 受体激动剂，胶原蛋白的主要血小板受体）的敏感性分布。P-选择素暴露和α _IIb β ₃整合素激活被用作分析终点，以证明存在导致系统水平灵敏度提高 20 倍的高反应性血小板的存在。

结论

分析工作流程代表了一种准确分类血小板亚型和描述其潜在生物学特性的工具。