Figure 1
Figure 1. Ectopic expression of FcγRIIa in murine platelet enhances outside-in signaling initiated by platelet adhesion to immobilized fibrinogen. (A) Washed platelets from WT (FcγRIIaneg) and FcγRIIa transgenic (FcγRIIapos) mice were plated on BSA or fibrinogen-coated coverslips in the presence of apyrase (0.25 U/mL) and indomethacin (10μM) and allowed to spread for 45 minutes in the presence or absence of 10 μg/mL IV.3 Fab. After spreading, platelets were fixed, permeabilized, and stained with rhodamine-phalloidin to visualize F-actin. Images are representative of 3 independent experiments. (B) Platelet spreading was quantified using Metamorph software, with each bar representing the mean micrometers-squared ± SEM of at least 300 platelets. Note that FcγRIIapos platelets exhibit enhanced αIIbβ3-dependent spreading on fibrinogen, and preincubation of platelets with IV.3 Fab significantly inhibits FcγRIIapos platelet spreading on immobilized fibrinogen. (C-D) Washed platelets from WT and FcγRIIa transgenic mice were plated on BSA or fibrinogen-coated dishes and allowed to spread for 15, 30, or 45 minutes in the presence of apyrase (0.25 U/mL) and indomethacin (10μM). (C) After spreading, platelets were lysed and analyzed by western blotting with antibodies directed against either the antigen or phosphorylated forms of FcγRIIa, Syk, and PLCγ2. (D) Quantitation of the ratio of phosphorylated of Syk and PLCγ2 relative to total expression level. Results represent the mean ± SEM (n = 3 per group). Note that FcγRIIapos platelets show enhanced activation of Syk and PLCγ2 compared with FcγRIIaneg platelets. (E-F) Washed platelets from FcγRIIapos mice were plated on BSA- or fibrinogen-coated coverslips and allowed to spread for 45 minutes in the presence or absence of 10 μg/mL IV.3 Fab fragments. (E) FcγRIIa (top 2 panels) was analyzed after immunoprecipitation with mAb IV.3, while Syk and PLCγ2 (bottom 4 panels) were detected by western blot analysis of whole platelet detergent lysates. (F) Ratio of phosphorylated of Syk and PLCγ2 to total protein expression. Results represent the mean ± SEM (n = 3 per group). Note strong activation of FcγRIIa, Syk, and PLCγ2 after platelet binding to immobilized fibrinogen, and inhibition of this outside-in signaling circuit by IV.3 Fab. **P < .01, *P < .05.

Ectopic expression of FcγRIIa in murine platelet enhances outside-in signaling initiated by platelet adhesion to immobilized fibrinogen. (A) Washed platelets from WT (FcγRIIaneg) and FcγRIIa transgenic (FcγRIIapos) mice were plated on BSA or fibrinogen-coated coverslips in the presence of apyrase (0.25 U/mL) and indomethacin (10μM) and allowed to spread for 45 minutes in the presence or absence of 10 μg/mL IV.3 Fab. After spreading, platelets were fixed, permeabilized, and stained with rhodamine-phalloidin to visualize F-actin. Images are representative of 3 independent experiments. (B) Platelet spreading was quantified using Metamorph software, with each bar representing the mean micrometers-squared ± SEM of at least 300 platelets. Note that FcγRIIapos platelets exhibit enhanced αIIbβ3-dependent spreading on fibrinogen, and preincubation of platelets with IV.3 Fab significantly inhibits FcγRIIapos platelet spreading on immobilized fibrinogen. (C-D) Washed platelets from WT and FcγRIIa transgenic mice were plated on BSA or fibrinogen-coated dishes and allowed to spread for 15, 30, or 45 minutes in the presence of apyrase (0.25 U/mL) and indomethacin (10μM). (C) After spreading, platelets were lysed and analyzed by western blotting with antibodies directed against either the antigen or phosphorylated forms of FcγRIIa, Syk, and PLCγ2. (D) Quantitation of the ratio of phosphorylated of Syk and PLCγ2 relative to total expression level. Results represent the mean ± SEM (n = 3 per group). Note that FcγRIIapos platelets show enhanced activation of Syk and PLCγ2 compared with FcγRIIaneg platelets. (E-F) Washed platelets from FcγRIIapos mice were plated on BSA- or fibrinogen-coated coverslips and allowed to spread for 45 minutes in the presence or absence of 10 μg/mL IV.3 Fab fragments. (E) FcγRIIa (top 2 panels) was analyzed after immunoprecipitation with mAb IV.3, while Syk and PLCγ2 (bottom 4 panels) were detected by western blot analysis of whole platelet detergent lysates. (F) Ratio of phosphorylated of Syk and PLCγ2 to total protein expression. Results represent the mean ± SEM (n = 3 per group). Note strong activation of FcγRIIa, Syk, and PLCγ2 after platelet binding to immobilized fibrinogen, and inhibition of this outside-in signaling circuit by IV.3 Fab. **P < .01, *P < .05.

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