Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin

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.

Platelet Phenotyping by Full Spectrum Flow Cytometry

“…Platelet modulators that can be used alone or in combination include the agonists adenosine diphosphate (ADP), collagenrelated peptide (CRP-XL), and thrombin receptor activator peptide (TRAP-6), and the inhibitors prostacyclin (PGI 2 ) and prostaglandin E 1 (PGE 1 ). Dual-agonist stimulation with CRP-XL and TRAP-6 is often used to induce annexin V binding for the evaluation of procoagulant potential (Hindle, Spurgeon, Cheah, Webb, and Naseem, 2021). 2.…”

Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry

“…, suggest that procoagulant platelets are actually undergoing cell death 34 ; referring to them as necrotic cells 34 , 50 . However, it is clear that there are knowledge gaps in the current understanding of platelet phenotypes, and there is a growing body of evidence suggesting additional distinct platelet subpopulations may exist 59 – 62 .…”

Comprehensive phenotyping of human platelets by single‐cell cytometry

“…Alternatives to PAC-1 include anti-fibrinogen, which allows the detection of fibrinogen that is bound to the surface of activated platelets [23], and fluorescent fibrinogen, which competes with native fibrinogen for binding to the platelet surface [29]. Recent work has identified subsets, with distinct capacities for CD62P expression and fibrinogen binding, that have disparate sensitivity to prostacyclin [21]. Furthermore, these subsets show distinct staining for annexin V, suggesting different procoagulant properties.…”