Procoagulant platelets have supramaximal [Ca²⁺]cyt signaling that is detected by low-affinity Fluo-5N but not high-affinity Fluo-4. (A) Human platelets were loaded with Fluo-4 then treated with CsA (2 µM) or dimethyl sulfoxide (DMSO) (as control) before stimulation with Thr/CRP-XL (arrowhead) in the presence of AnV-APC. Samples were analyzed by using real-time flow cytometry. Platelets were gated on an FSC/SSC profile to exclude microparticles. Fluo-4 fluorescence is shown. Color represents event density from low (blue) to high (red). (B) A comparison of Fluo-4 fluorescence and AnV-APC fluorescence after 10 minutes of stimulation. The horizontal line defines platelets AnV⁺ (procoagulant) and AnV^– (noncoagulant) subpopulations. The vertical line indicates the Fluo-4 fluorescence of unstimulated platelets. All activated platelets exhibit high Fluo-4 fluorescence. (C) The Fluo-4 MFI in all platelets, AnV⁺ platelets, and AnV^– platelets. Data are mean ± standard error of the mean (n = 5; 2-way analysis of variance with the Šidák multiple comparison test). (D) Mean Fluo-4 fluorescence (MFI normalized to initial MFI; F/F₀) of all platelets from real-time flow cytometry (n = 5). The dotted line is the standard error of the mean. (E) Mean Fluo-4 fluorescence (F/F₀) of platelets stimulated with Thr/CRP-XL measured in a microplate reader. (F-J) show the equivalent experiments in Fluo-5N–loaded platelets. ***P < .001 for indicated comparison; ^†††P < .001 for difference in MFI between AnV⁺ vs AnV^– for DMSO or CSA, as appropriate. n.s., not significantly different for the indicated comparison.