Hindle, MS, Spurgeon, BEJ, Cheah, LT et al. (2 more authors) (2021) Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin. Journal of Thrombosis and Haemostasis, 19 (7). pp. 1800-1812. ISSN 1538-7933
Abstract
Background
Robust platelet activation leads to the generation of subpopulations characterized by differential expression of phosphatidylserine (PS). Prostacyclin (PGI2) modulates many aspects of platelet function, but its influence on platelet subpopulations is unknown.
Objectives and Methods
We used fluorescent flow cytometry coupled to multidimensional fast Fourier transform‐accelerated interpolation‐based t‐stochastic neighborhood embedding analysis to examine the influence of PGI2 on platelet subpopulations.
Results
Platelet activation (SFLLRN/CRP‐XL) in whole blood revealed three platelet subpopulations with unique combinations of fibrinogen (fb) binding and PS exposure. These subsets, PSlo/fbhi (68%), PShi/fblo (23%), and PShi/fbhi (8%), all expressed CD62P and partially shed CD42b. PGI2 significantly reduced fibrinogen binding and prevented the majority of PS exposure, but did not significantly reduce CD62P, CD154, or CD63 leading to the generation of four novel subpopulations, CD62Phi/PSlo/fblo (64%), CD62Phi/PSlo/fbhi (22%), CD62Phi/PShi/fblo (3%), and CD62Plo/PSlo/fblo (12%). Mechanistically this was linked to PGI2‐mediated inhibition of mitochondrial depolarization upstream of PS exposure. Combining phosphoflow with surface staining, we showed that PGI2‐treated platelets were characterized by both elevated vasodilator‐stimulated phosphoprotein phosphorylation and CD62P. The resistance to cyclic AMP signaling was also observed for CD154 and CD63 expression. Consistent with the functional role of CD62P, exposure of blood to PGI2 failed to prevent SFLLRN/CRP‐XL‐induced platelet‐monocyte aggregation despite reducing markers of hemostatic function.
Conclusion
The combination of multicolor flow cytometry assays with unbiased computational tools has identified novel platelet subpopulations that suggest differential regulation of platelet functions by PGI2. Development of this approach with increased surface and intracellular markers will allow the identification of rare platelet subtypes and novel biomarkers.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Keywords: | cAMP; flow cytometry; mitochondria; platelets; procoagulant; subpopulations |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM) > Discovery & Translational Science Dept (Leeds) |
Funding Information: | Funder Grant number British Heart Foundation RG/16/5/32250 |
Depositing User: | Symplectic Publications |
Date Deposited: | 21 Jun 2023 15:47 |
Last Modified: | 21 Jun 2023 15:47 |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1111/jth.15330 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:200649 |