P-selectin localization in platelets exposed to SFLLRN or PMA. Platelets were exposed to (A) buffer, (B) 100 μM SFLLRN, or (C) 0.2 μM PMA for 10 minutes. Platelets were then processed for immunonanogold staining by using a polyclonal rabbit antibody directed against the cytoplasmic tail of P-selectin. Label is observed primarily on α-granule surfaces (black arrowheads) in resting platelets (A) and platelets exposed to PMA (C). Label is observed mostly on plasma membrane (open arrows) in platelets exposed to SFLLRN (B). Apparent fusion of α-granules with plasma membrane could be visualized in the PMA-treated platelets (a and b). Control samples prepared by using an irrelevant primary antibody or in the absence of primary antibody did not show significant staining. (D) Expression of P-selectin in aliquots of platelet samples also used for electron microscopy was assessed by flow cytometry by using an antibody to the extracellular domain of P-selectin. Magnification of panel A is 25 000 ×;B,21 000 ×; and C, 25 000 ×.