Analyzing Platelet Subpopulations by Multi-color Flow Cytometry

Platelets, or thrombocytes, are small anucleated blood cells that play a crucial role in hemostasis and thrombosis. Defects in platelet functions can cause bleeding or thrombotic events in patients with cardiovascular diseases. Therefore, it is important to characterize platelets phenotypically to be able to assign platelet subpopulations to platelet function. Stimulation of human platelets with platelet agonists and activators induces morphological and physiological changes in platelets, accompanied by changes in the surface receptor population. This leads to functionally diverse platelet subpopulations. Classically defined platelet subpopulations are resting, aggregatory, procoagulant, and apoptotic platelets. To characterize the effect of agonists on platelet subtypes, we established an assay using multi-color flow cytometry consisting of 10 different antibodies and dyes (anti-CD62P, anti-CD63, anti-CD61, anti-CD41, anti-CD42b, anti-IntegrinαIIbβ3 (clone: PAC-1), anti-CXCR4, anti-ACKR3, Annexin V, Zombie NIR). Isolated human platelets were incubated with platelet agonists and stained with specific fluorophore-conjugated antibodies and dyes. Afterwards, they were measured by flow cytometry. This allows us to define agonist-specific subtypes within the classic four subpopulations. In conclusion, the combination of activation markers (anti-CD62P, anti-CD63, anti-IntegrinαIIbβ3 (clone: PAC-1)), inflammatory markers (anti-CXCR4, anti-ACKR3) and apoptotic markers (Annexin V, Zombie NIR) that compose the 10-color flow cytometry panel described in this manuscript opens up the possibility to define further platelet subtypes that could be linked to specific platelet function. This method can be applied in basic research on platelet function and physiology, as well as in defining new platelet subtypes in disease models and patient studies.