The GPIbα intracellular domain is dephosphorylated after platelet adhesion and shape change on a VWF matrix. Resting platelets were either fixed and allowed to adhere to a polylysine matrix (A,D), or allowed to adhere to a VWF matrix in the presence (B,E) or absence (C,F) of EDTAby incubation with the matrix for 20 minutes in the presence of botrocetin followed by washing and fixation. GPIbα was labeled with ALMA.12-Cy3 (red), and phosphorylated GPIbα was labeled with pSGPIb1 (A-C) or pSer609 (D-F), which was revealed with a secondary FITC-coupled antibody (green). Samples were analyzed by dual-label confocal microscopy. (A,D) Discoid platelets appeared predominantly yellow after staining with either pSGPIb1 or pSer609, indicating that GPIbα was mainly phosphorylated in resting platelets. (B,E) When platelets adhered to a VWF matrix and integrin activation was blocked with EDTA and ReoPro, they extended numerous filopodia. The staining of the cell body remained yellow, whereas the filopodia appeared in red, suggesting a lack of recognition by pSer609 or pSGPIb1 and dephosphorylation of pSer609 and pSer587/590 within the membrane extensions. (C,F) In the absence of ReoPro and EDTA, platelets spread on the VWF matrix and the labeling appeared as a mixture of yellow and red over the entire cell surface, pointing to the existence of separate pools of phosphorylated and dephosphorylated GPIbα. Shown is 1 representative experiment of 3 performed. Scale bars indicate 10 μm.