Novel aspects of blood platelet function and biology measured by fluorescent flow cytometry.

Fluorescent flow cytometry (FFC) is an accessible, high-throughput and sensitive technique that is well suited to analysis of cells suspended in complex heterogeneous media. Using a wide variety of live, fixed, extracellular, intracellular, dye/antibody/peptide based FFC assays, multiple and novel aspects of platelet biology including; activation, inflammation and mitochondrial function were examined. Through the examination of platelet activation with multiparameter and multidimensional analysis three platelet subsets in PAR1/GPVI activated whole blood samples were described. This same assay in the presence of PGI2 suggested a dichotomy in inhibition when comparing fibrinogen binding, phosphatidylserine (PS) exposure and CD62P. Using novel phosphoflow protocols the inhibition resistant expression of CD62P was shown to be independent of robust cAMP signalling. This CD62P expression was then shown to mediate platelet monocyte interactions – in the presence of inhibition. Caspase-1 cleavage as a marker of NLRP3 inflammasome activity was measured using fluorescent peptide dyes. Activation was induced by canonical activators of the complex, but it was observed that this was significantly potentiated by oxidised LDL (oxLDL). ROS and calcium were also shown to have a vital role in activation of platelet NLRP3 and scavenging or chelation of either induced a loss of signal. Furthermore, active caspase-1 signal also correlated with PS exposure. Finally, mitochondrial function in response to oxLDL in vitro and in the context of murine hyperlipidaemia ex vivo was measured using live cell dyes. In vitro stimulation with oxLDL drove an increase in both mitochondrial superoxide production and mitochondrial membrane potential. However, this was demonstrated to be important in vivo, as transgenic models of hyperlipidaemia showed a significant increase in basal mitochondrial superoxide compared to wild type control. This was recapitulated diet-induced obesity murine models and a trend towards increased mitochondrial superoxide and membrane potential was described.