Impaired inside-out αIIbβ3 activation in patient platelets. (A-B) Aggregation response of control (black) and patient (gray) platelets activated with ADP (A) and collagen (B). (C-E) Flow cytometry analysis of integrin αIIbβ3 activation by activated αIIbβ3-specific antibody PAC-1 (n = 3: mean ± standard deviation [SD]; Student t test, *P < .05, **P < .01). Mean fluorescence intensity (MFI) is indicated after subtracting the MFI of basal PAC-1 binding. (C) Washed platelets were stimulated with ADP, CRP, PAR1-AP, and U46619. (D) Washed platelets were treated with αIIbβ3-activating antibody PT-25-2, or 100 ng/mL PMA. (E) Washed platelets were simultaneously stimulated with the cocktail of 50 μM ADP, 200 μM PAR1-AP, and 20 μM U46619. (F) Electron microscopy image of platelets from patient. The arrow and arrowhead indicate α-granules and dense granule. Image of control platelet is shown in supplemental Figure 4. (G) Western blot analysis of talin, FAK, Kindlin-3, and CalDAG-GEFI in platelet lysate of control and patient. β-Actin was probed as a loading control. (H) Pull-down assay for activated Rap1 in platelets. Washed control and patient platelets were stimulated with 100 μM PAR1-AP for indicated time. Platelet lysates were incubated GST-RalGDS, and the binding of active GTP-Rap1 was analyzed by western blotting (representative image from 3 independent experiments). The density of each band was measured, and the relative changes of Rap1 activation to unstimulated platelets were indicated as a fold change (n = 3: mean ± SD; Student t test, *P < .05). (I) Time course of intraplatelet calcium mobilization. Washed control and patient fluo-4–loaded platelets were stimulated with 100 μM PAR1-AP, 5.0 μg/mL CRP, or A23187, and fluorescence was analyzed by flow cytometry (MFI).