Fig. 4: Activation and functional characterization of platelets in nMG patients.

a Gating strategy for activated platelets and representative flow cytometry scatter plots of CD62P+ platelet populations in nMG, HC, and MMS participants. The left panel shows the initial gating of CD61+ events, followed by further analysis of CD62P expression (right panels). b The percentage of CD61+CD62P+ platelets is higher in the nMG (n = 50) group compared to HC (n = 20, p = 0.0307) and MMS (n = 35, p = 0.0037) groups. c A significant increase of sCD40L levels was observed in the nMG (n = 24) group compared to the HC group (n = 18, p = 0.0264). No significant difference was found between nMG and MMS (n = 18). d Positive association were observed between plasma sCD40L levels and QMG score (n = 24, R = 0.57, p = 0.0034). e Influence of APT on the frequency of CD62P+ platelet in nMG patients, with no significant difference observed (n = 9 for APT and n = 41 for N-APT; p = 0.216). f Representative flow cytometry scatter plot illustrating the gating strategy for free platelets. The CD45⁻CD61⁺ population was identified as free platelets, and the FSC/SSC parameters for this specific population were automatically calculated using FlowJo software. g, h Comparative analysis of FSC and SSC profiles of platelets from nMG (n = 50), HC (n = 20), and MMS (n = 35) participants, showing significant differences in platelet size and internal complexity (FSC: nMG vs HC, p = 0.0124; SSC: nMG vs HC, p = 0.0032, nMG vs MMS, p = 0.0109). i Representative transmission electron microscopy images showing platelet morphology in nMG, HC, and MMS groups. Red arrows indicate platelet microparticles. Scale bars: 2 μm in the left column and 1 μm in the middle and right columns. j For each patient, we randomly selected five microscopic fields and counted the number of non-spheroid platelets (platelets exhibiting signs of activation, such as filopodia or lamellipodia, deviating from the typical discoid shape) and total platelets within each field. The percentage of non-spheroid platelets was determined by calculating the ratio of non-spheroid platelets to total platelets for each field, followed by averaging the ratios across the five fields. The percentage of non-spheroid platelets was increased in nMG patients (n = 4) compared to both HC (n = 4, p = 0.0266) and MMS (n = 4, p = 0.0295) groups. k The PMP count per platelet was obtained by calculating the PMPs/total platelet ratio for each field and averaging these values across the five fields. The PMPs per platelet count were also increased in the nMG group (n = 4) compared with HC (n = 4, p = 0.0466). For (b, c, e, g, h, j, k), data are presented as mean ± s.d., with individual biological replicates shown. Statistical significance was assessed using ordinary one-way ANOVA (two-sided), followed by Tukey’s multiple comparisons test, except for (e), where an unpaired two-tailed Student’s t-test was used. For correlation analysis in (d), two-sided Spearman’s rank correlation was applied, and the regression line is shown with a shaded area indicating the 95% confidence interval. Significance levels are denoted by asterisks: *p < 0.05; **p < 0.01. Non-significant results are not depicted. nMG immunotherapy-naïve MG, HC healthy controls, MMS minimal clinical status, ADL activity of daily living, QMG score quantitative myasthenia gravis scores, FSC forward scatter, SSC side scatter, APT antiplatelet therapy, Conc. concentration, PMPs platelet-derived microparticles. Source data are provided as a Source data file.