Effects of Platelet Agonists and Priming on the Formation of Platelet Populations

Platelets from healthy donors display heterogeneity in responsiveness to agonists. The response thresholds of platelets are controlled by multiple bioactive molecules, acting as negatively or positively priming substances. Higher circulating levels of priming substances adenosine and succinate, as well as the occurrence of hypercoagulability, have been described for patients with ischaemic heart disease. Here, we present an improved methodology of flow cytometric analyses of platelet activation and the characterisation of platelet populations following activation and priming by automated clustering analysis.Platelets were treated with adenosine, succinate, or coagulated plasma before stimulation with CRP-XL, 2-MeSADP, or TRAP6 and labelled for activated integrin α_IIbβ₃ (PAC1), CD62P, TLT1, CD63, and GPIX. The Super-Enhanced Dmax subtraction algorithm and 2% marker (quadrant) setting were applied to identify populations, which were further defined by state-of-the-art clustering techniques (tSNE, FlowSOM).Following activation, five platelet populations were identified: resting, aggregating (PAC1 + ), secreting (α- and dense-granules; CD62P + , TLT1 + , CD63 + ), aggregating plus α-granule secreting (PAC1 + , CD62P + , TLT1 + ), and fully active platelet populations. The type of agonist determined the distribution of platelet populations. Adenosine in a dose-dependent way suppressed the fraction of fully activated platelets (TRAP6 > 2-MeSADP > CRP-XL), whereas succinate and coagulated plasma increased this fraction (CRP-XL > TRAP6 > 2-MeSADP). Interestingly, a subset of platelets showed a constant response (aggregating, secreting, or aggregating plus α-granule secreting), which was hardly affected by the stimulus strength or priming substances.