Fluorescent imaging of platelet GPs.

Washed platelets from healthy volunteers (A-F) and the patient with BSS (G-L) were plated onto glass slides precoated with fibrinogen (20 μg/mL) and allowed to adhere for 1 hour at room temperature. Platelets were then either stained directly (A-C and G-I) or permeabilized with ice-cold methanol and acetone (D-F and J-L) before staining. Slides were dual stained for GPIbα with a rabbit anti-GC polyclonal antibody and for GPIIb-IIIa with a mouse mAb, LYP18. Platelets were imaged on a Zeiss Axioplan II confocal microscope with a 63 × oil immersion lens (1.4 n/a). The platelets were permeabilized in the images shown in A to C and G to I to allow antibody probes to react with cytoplasmic epitopes. Data are presented as triplets of images separated to show GPIbα staining with an Alexa 546–conjugated anti-rabbit antibody (red: A, D, G, and J), GPIIb-IIIa staining with Alexa 488 antimouse antibody (green: B, E, H, and K), or the confocal image showing both colors (C, F, I, and L). One image is shown for each treatment; this is representative of 3 independent experiments in which up to 50 platelets were analyzed. GPIbα was present in permeabilized and nonpermeabilized normal platelets (A, D) and permeabilized BSS platelets (J) but absent from the surface of nonpermeabilized preparations of the BSS platelets (G). In contrast, the platelet integrin GPIIb-IIIa was present on the surface of both normal and BSS platelets, regardless of permeabilizing treatment of the cell membrane (B, E, H, and K).