Increased small PMPs and thrombin generation with CSPs in vitro. (A) After platelet storage at both RT and 4°C conditions, PPP was prepared by centrifugation with 1 freeze-thaw cycle before testing. (B) Using the CytoFLEX flow cytometer, and nanobeads, we identified MPs with a large MP size gate (1-μm equivalent to 0.5-μm equivalent) and a small MP size gate (between 0.5-μm and 0.1-μm equivalent beads). (C) The gates were applied to MPs in plasma. (D) Within the respective large and small MP size gates, PMPs were defined as all CD41a⁺ events. Both large and small PMPs were further gated for lactadherin (E) or CD62P (gray trace indicates isotype control) (F) positivity within the CD41⁺ gate. (C-F) Example data from stored platelet samples and gating for large-sized events. (G) PMPs of both RSPs and CSPs were identified as events within small and large size gates that were CD41a⁺ and the concentration of each reported (large, ∗P = .0369; small, ∗∗∗∗P < .0001; small vs large, RSPs ∗∗P = .002; CSPs ∗∗∗∗P < .0001). The concentration of large and small PMPs that were also positive for lactadherin (small, ∗P = .0113; small vs large, CSPs ∗∗P = .005, RSPs ∗∗P = .002) (H) or CD62P (small, ∗∗∗P = .0002; small vs large, RSPs ∗P = .04; CSPs ∗∗∗∗P < .0001) (I). The MP-mediated thrombin generation potential of PPP from storage bags was measured and reported as thrombin generation peak (∗P = .0202) (J), start tail time (∗P = .0479), (K), time to thrombin peak (L), and lag time (M). Data are shown as mean ± SEM and individual values. Unpaired data, n = 9, individual P values are shown in the text above. RT, room temperature.