PEAR1 in the platelet membrane and in α-granules. (A) Western blot of lysate from resting platelets, revealing PEAR1 by antibodies against the extracellular (PEAR1-EC Ab, upper lanes) and intracellular (PEAR1-IC Ab, lower lanes) domains, for the indicated amounts of loaded protein. (B) Flow cytometric measurement as mean fluorescence intensity (MFI) of PEAR1 and P-selectin (CD62-P) on the membrane of resting washed platelets and platelets stimulated with thrombin, PAR4-activating peptide (PAR4) and ADP, at the indicated concentrations. Data shown represent one experiment, representative of at least 3 analyses. (C) Confocal immunofluorescence microscopy of human platelets spread over a fibrinogen matrix; left panel: stained with rhodamin-phalloidin (F-actin, red), and labeled for PEAR1 (green), and VWF (blue) and for the colocalization of PEAR1/VWF (cyan), PEAR1/actin (yellow), and PEAR1/VWF/actin (white). Bar, 10 μm. Original magnification ×630; right panel: human platelets (control and uncharacterized-secretion defective patient platelets) spread over a fibrinogen matrix, labeled for β3 integrin (red) and for PEAR1 (green). Original magnification ×400.