PIRB regulates GPVI-mediated platelet activation. (A) Binding of Alexa 488-Fg to washed WT and PIRB-TM platelets stimulated with 0.5 μg/mL CRP. (B) Binding of Alexa 488-Fg to washed WT and PIRB-TM platelets stimulated with 100 μM PAR4 agonist peptide AYPGKF. (C) Washed WT and PIRB-TM platelets at a concentration of 3 × 10⁷/mL were incubated with FITC-conjugated JAQ1 and FITC-conjugated rat IgG control at 25°C for 30 minutes. The expression levels of GPVI were detected using a flow cytometer. (D) The expression levels of LAT, SLP76, PLCγ2, Shp1, and Shp2 in WT and PIRB-TM platelets were examined by western blotting. GAPDH was used to verify equal gel loading. (E) The goat anti-mouse PIRB polyclonal antibody or goat IgG control was used to immunoprecipitate PIRB from lysate of washed WT platelets stimulated with 0.25 μg/mL CRP for indicated times. The immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted with anti-PIRB, anti-Shp1, and anti-Shp2 antibodies for detection of Shp1/2 association with PIRB. IgG was used as a loading control. (F) The immunoprecipitations of tyrosine phosphorylated proteins from the lysates of WT and PIRB-TM platelets were treated with 0.25 μg/mL CRP for the indicated times, followed by the detections of tyrosine phosphorylation of LAT, SLP76, and PLCγ2. IgG was used as a loading control. (G) The time courses of phosphorylation levels of Shp1 Y564 and Shp2 Y580 in washed WT and PIRB-TM platelets in response to 0.25 μg/mL CRP. GAPDH was used to verify equal loading.