Phenotyping of human platelets in response to platelet agonists and inhibitors using multiparameter flow cytometry and unbiased high-dimensional analysis

Background

Despite increasing evidence of functionally distinct platelet subpopulations, the role of receptor remodeling in this process is unclear.

Objectives

To develop a high-dimensional multiparameter flow cytometry panel for platelet phenotyping and subpopulation analysis.

Methods

Using conventional flow cytometry, we assessed the expression of 9 platelet surface receptors in whole blood (WB) samples from healthy adults in response to different platelet activators and inhibitors. Platelet subpopulations were identified by Full Annotation Using Shape-constrained Trees (FAUST).

Results

The 9-parameter assay simultaneously measured the activation of αIIbβ3 (defined by PAC1 antibody binding) and expression of CD62P, CD40L, CD63, CD42b, CD147, CXCR4, CD32a, and CD36. After WB stimulation with adenosine diphosphate, thrombin receptor-activating peptide-6, or convulxin, FAUST identified 8 platelet subpopulations that were defined by differential levels of PAC1, CD62P, and CD63. To determine how inhibitors affect platelet subpopulations, WB was pretreated with prostacyclin, dasatinib, vorapaxar, or ticagrelor prior to stimulation with adenosine diphosphate, thrombin receptor-activating peptide-6, or convulxin. Again, FAUST identified 8 distinct platelet subpopulations, which were defined by differential levels of PAC1, CD40L, and CD62P, showing that under conditions of platelet inhibition, CD40L expression is important for subpopulation annotation.

Conclusion

Platelet subpopulations are dynamic and undergo remodeling in response to different agonists and inhibitors. This high-dimensional single-cell flow cytometry assay is a practicable approach to evaluate the effects of agonists and inhibitors on platelet receptor expression and platelet subpopulations.