Engagement of β3 integrin and Fg is required for maintenance of platelet cell surface and intracellular P-selectin expression. (A) Gel-filtered platelets were incubated with either thrombin (left histogram) or TRAP (right histogram), and P-selectin expression on the platelet surface was analyzed via flow cytometry. P-selectin expression was significantly decreased on β3^−/− platelets (black) after activation compared with wild type (shaded); bar graph indicates MFI plus or minus SEM (n = 3; **P < .01). A decrease in total platelet P-selectin levels in β3^−/− platelets was also observed by immunoblot (right panel). The immunoblots in each panel were developed from the same nitrocellulose membrane and the experiments were repeated at least 3 times. (B) P-selectin expression was also significantly decreased on FgγΔ5 platelets after TRAP activation compared with wild type (left panel, arrow indicated); bar graph indicates MFI plus or minus SEM (middle panel) (n = 4; **P < .001). A decrease in total platelet P-selectin levels in FgγΔ5 platelets was also observed by immunoblot (right panel). The immunoblots were developed from the same PVDF membrane and the experiments were repeated at least 3 times. (C) No significant differences in the average number of platelet α-granules (arrow indicated) in Fg^−/− and β3^−/− mice compared with those of wild-type (WT) mice were observed (P > .05). The quantitative morphometric comparisons were performed via electron microscopy in 50 platelets per mouse and 2 mice in each group were quantitated. (D) Total platelet mRNA was isolated and reverse transcribed. PCR amplification of cDNA was performed using primers for mouse P-selectin and β-actin (internal control). The levels of P-selectin mRNA in Fg^−/− and β3^−/− platelets appear similar to wild-type controls after individual bands are normalized to their β-actin controls (left panel). These results were confirmed by quantitative real-time PCR (Fg^−/− versus WT: P = .44; β3^−/− versus WT: P = .96; right panel). (E) Gel-filtered platelets from Fg^−/− mice (β3 integrin positive) were transfused to β3^−/− mice (Fg positive) and Fg^−/− mice (negative control). Two days after transfusion, platelets were isolated and analyzed via flow cytometry. β3 integrin–positive Fg^−/− platelets were gated (left panel), and a significant increase in Fg and P-selectin expression was observed on those β3 integrin–positive platelets isolated from β3^−/− mice (arrow indicated), but not from Fg^−/− mice, after treatment with TRAP (right panel). The experiments were repeated at least 3 times. (F) Washed platelets from Fg^−/− mice were incubated in culture media supplemented with Fg (+ Fg) or media alone (no Fg). Platelet P-selectin levels in cultured platelets and uncultured platelets (pre-: platelets were lysed prior to incubation) were examined via Western blot. Platelets cultured with Fg had significantly greater levels of P-selectin. The immunoblots were developed from the same PVDF membrane and the experiments were repeated at least 3 times.