Depletion of syntaxin-11 affects aggregation, ATP release, and P-selectin exposure, but not integrin activation or platelet ultrastructure. (A) Aggregation (i-iii) and ATP release (iv-vi) were monitored concurrently in a lumi-aggregometer. Washed platelets from a control donor (black traces) and FHL4 patient (gray traces) were stimulated with thrombin (0.1 U/mL; i, iv), collagen (10 μg/mL; ii, v), and A23187 (100nM; iii, vi) for 2-3 minutes; B-C) Washed platelets from a control donor (Control) and FHL4 patient (Patient) were stimulated with 0.1 U/mL of thrombin for 1 minute and then incubated with FITC-conjugated anti–P-selectin (B) or FITC-conjugated PAC-1 (C) Abs. The reactions were stopped with hirudin and the fluorescent intensities were measured by flow cytometry. The data were plotted as a histogram (left panels) and as the geometric mean fluorescence intensity (GMFI; right panels). Because of limited samples, the experiments in panels B and C were done only once. (D) Washed platelets from a control donor (Control) and FHL4 patient (Patient) were kept resting with 1 ng/mL of PGI2 (i-ii) or stimulated with 0.1 U/mL of thrombin (iii-iv) for 5 minutes. The platelets were fixed and processed for electron microscopic analysis as described in “Methods.” The samples were analyzed with a transmission electron microscope and images were obtained using Gatan software. The scale bars are indicated.
