Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin

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.

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.

Platelet activation (SFLLRN/CRP-XL) in whole blood revealed three platelet subpopulations with unique combinations of fibrinogen (fb) binding and PS exposure. These subsets, PS^lo /fb^hi (68%), PS^hi /fb^lo (23%), and PS^hi /fb^hi (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, CD62P^hi /PS^lo /fb^lo (64%), CD62P^hi /PS^lo /fb^hi (22%), CD62P^hi /PS^hi /fb^lo (3%), and CD62P^lo /PS^lo /fb^lo (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.

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.