Study of platelets from patients with mutated DOCK11. (A) DOCK11 expression was evaluated in platelets of HDs, and patients (A, C, D1, D2, F and G) by western blotting. Dotted lines indicate that the samples were derived from the same gel but were noncontiguous. The graph shows the mean of the relative expression of DOCK11 vs HD (set to 1) ± SEM after normalization against β-actin expression from several independent experiments (HDs, n = 69; A, n = 22; C, n = 14; G, n = 16; D1, D2, n = 8; F, n = 15). Statistical difference was evaluated by one-way analysis of variance (ANOVA) with Dunnett correction test for multiple comparisons (∗P < .05; ∗∗∗P < .001). (B) Platelet count for each patient (A, C, D1, D2, F, and G) was determined by automated blood cell counter. Shaded area represents the normal range between 150 × 10⁹ and 400 × 10⁹ platelets per L. Patients A and C were investigated for their platelet count several times (2 and 3 times, respectively). (C) Platelet size was evaluated by flow cytometry. Each dot represents the mean size measured by the mean forward scatter height (FSC-H; a.u., arbitrary units) of washed platelets from HDs and patients./ The box-and-whisker plot shows the normal range, determined from HDs. Whiskers represent the 5th to 95th percentiles, the box correspond to the interquartile range, the center line is the median, and the cross indicates the mean of 65 platelets. (D) Platelet ultrastructure, which was analyzed once for each patient using transmission electron microscopy (TEM). Scale bar represents 1 μm. (E-F) TEM analysis of platelet ultrastructure in each patient. Platelet surface (E) and platelet shape (F), which is defined as the ratio between the largest diameter and the smallest diameter, are derived from the TEM images. Graphs represent the mean ± SEM of at least 1100 HD platelets and 100 patient platelets. Statistical significance was determined in a one-way ANOVA with the Dunnett posttest for multiple comparisons. (G) Graph represents the mean percentage ± SEM of discoid platelets (dark gray), platelets with filopodia (light gray) and platelets with lamellipodia or full spreading (white) of at least 150 analyzed platelets from several independent experiments (HDs, n = 17; patient A, n = 4; patient C, n = 3). Statistical significance was determined only for patients A and C compared with HDs in a one-way ANOVA with the Dunnett posttest for multiple comparisons (∗P < .05; ∗∗P < .01). Patients D1, D2, F, and G were not statistically analyzed because only 1 experiment was performed. Scale bar represents 10 μm. (H) Spreading of HD and patient platelets onto fibrinogen matrix for 30 minutes was analyzed by epifluorescence microscopy in the presence of apyrase (5 U/mL) and indomethacin (4.5 μM). Platelet morphology was visualized using F-actin detection by fluorescently labeled phalloidin. (I) CDC42 activity evaluated in HD platelets and patient platelets by G-LISA after stimulation by 0.5 U/mL thrombin for 1 minute in unstirred conditions. Graph represents the relative CDC42 activity of each patient compared with that of HDs (set to 100%). G-LISA, G-protein–linked immunosorbent assay.