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Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets

Spurgeon, B. E. J.; Aburima, A.; Oberprieler, N. G.; Taskén, K.; Naseem, K. M.

Authors

B. E. J. Spurgeon

A. Aburima

N. G. Oberprieler

K. Taskén

K. M. Naseem



Abstract

Background
Dissecting the signaling events that contribute to platelet activation will increase our understanding of platelet function and aid in the development of new antiplatelet agents. However, high‐throughput methodology for the quantitative analysis of platelet signaling events is still lacking.

Objective
To develop a high‐throughput assay for the analysis of platelet signaling events in whole blood.

Methods and Results
We developed a fluorescent barcoding protocol to facilitate multiplexing and enable large‐scale signaling profiling in platelets in whole blood. The methodology allowed simultaneous staining and acquisition of 24–96 samples in a single analysis tube with a standard flow cytometer. This approach significantly reduced experimental numbers, data acquisition time, and antibody consumption, while providing automated statistically rich quantitative data on signaling events. Using vasodilator‐stimulated phosphoprotein (VASP), an established marker of platelet inhibition and antiplatelet drug therapy, we demonstrated that the assay could detect subtle changes in phosphoVASP‐Ser157/239 in response to cAMP‐elevating agents of varying potency and known modulators of the cAMP signaling cascade. The assay could be used with washed platelets or whole blood, analyzed immediately or frozen, without any significant change in assay performance. To demonstrate the usefulness of the assay as a drug discovery platform, we examined a prostaglandin screening library. Our screen of 70 prostaglandin derivatives revealed three previously uncharacterized lipids that stimulated phosphorylation of VASP‐Ser157. Follow‐up analyses demonstrated that these agents elevated intraplatelet cAMP and inhibited collagen‐induced platelet aggregation.

Conclusions
This novel method enables rapid, large‐scale quantitative signaling profiling and compound screening in human platelets present in whole blood.

Citation

Spurgeon, B. E. J., Aburima, A., Oberprieler, N. G., Taskén, K., & Naseem, K. M. (2014). Multiplexed phosphospecific flow cytometry enables large-scale signaling profiling and drug screening in blood platelets. Journal of thrombosis and haemostasis : JTH, 12(10), 1733-1743. doi:10.1111/jth.12670

Journal Article Type Article
Acceptance Date Jul 23, 2014
Online Publication Date Sep 18, 2014
Publication Date 2014-10
Deposit Date Feb 7, 2019
Journal Journal of Thrombosis and Haemostasis
Print ISSN 1538-7933
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 12
Issue 10
Pages 1733-1743
DOI https://doi.org/10.1111/jth.12670
Keywords Hematology
Public URL https://hull-repository.worktribe.com/output/497129
Publisher URL https://onlinelibrary.wiley.com/doi/full/10.1111/jth.12670