Multidimensional Flow Cytometry Reveals Novel Platelet Subpopulations In Response To Prostacyclin

Background Robust platelet activation leads to the generation of subpopulations characterized by differential expression of phosphatidylserine (PS). Prostacyclin (PGI(2)) 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 PGI(2) 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/fb(hi) (68%), PShi/fb(lo) (23%), and PShi/fb(hi) (8%), all expressed CD62P and partially shed CD42b. PGI(2) 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)/PSlo/fb(lo) (64%), CD62P(hi)/PSlo/fb(hi) (22%), CD62P(hi)/PShi/fb(lo) (3%), and CD62P(lo)/PSlo/fb(lo) (12%). Mechanistically this was linked to PGI(2)-mediated inhibition of mitochondrial depolarization upstream of PS exposure. Combining phosphoflow with surface staining, we showed that PGI(2)-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 PGI(2) 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 PGI(2). Development of this approach with increased surface and intracellular markers will allow the identification of rare platelet subtypes and novel biomarkers.