Droplet microfluidics reports digital and tuneable single-platelet ADP secretion describing primed, active and hyper-active states

New platelet function assays that provide an accurate assessment of platelet reactivity as a surrogate for thrombosis risk could change the paradigm for acute myocardial infarction and stroke prevention. Considering ADP secretion as a hallmark of platelet activation, we developed a method for encapsulating single platelets with a fluorescent reporter for extracellular ADP in picolitre droplets. The method, termed the Droplet ADP Secretion Assay (DASA), robustly detected dose-dependent ADP secretion responses to agonists. Digital ADP secretion heterogeneity was observed with the major fraction of platelets responding and the remaining fraction being non-responders. Primed platelets had elevated responses to agonists, with increased ADP secretion and an increased fraction of secreting platelets, demonstrating that priming can convert otherwise inactive platelets to a reactive state. Uniquely, the method detected ADP secretion from primed platelets in the absence of agonists. In contrast, primed platelets were not detected by flow cytometry involving CD63 and CD62P endpoints. In a preliminary clinical study, some patients had platelets with a priming-like ADP secretion signature, suggesting treatment with P2Y12 inhibitors may be warranted. In vitro aspirin and ticagrelor treatments reduced ADP secretion responses to agonists. In summary, DASA offers a novel approach to assess platelet reactivity and holds promise for assessing thrombotic tendency and personalised therapeutics.