Immunofluorescence confocal microscopic analysis of GPIbα, F-actin, and tissue factor in perfusion-fixed tethers and microparticles. Whole blood was perfused over immobilized dVWFA1 at γw of 30 000 and 40 000 s–1. (A-B) Two fields of view were visualized by RICM (top), confocal immunofluorescence microscopy after staining with an anti-GPIbα antibody (A) or phalloidin (B) to visualize F-actin (middle), and their composite (bottom). White arrows indicate microparticles and tethers; arrowheads show platelets. Phycoerythrin (PE) immunofluorescence staining for GPIbα (A) reveals abundant receptor exposure with a few unlabeled tether segments and microparticles (black arrows). This finding indicates a heterogeneous receptor distribution suggestive of clustering and corresponds well with the immuno-EM data in Figures 3 and 4. PE immunofluorescence staining for F-actin with phalloidin, despite the relatively low intensity (B), shows a complete congruence with the RICM image in the tether ends and microparticles. (C-D) Cytochalasin D–treated platelets clearly exhibit GPIbα labeling (Cy3), as do tethers and microparticles originating from them (C), whereas the phalloidin staining for F-actin (rhodamin) can be seen in platelets but is absent in tethers and microparticles (D). (E) The specific monocyte marker CD14 could not be detected on microparticles, tethers, and platelets, which exhibit only low background fluorescence. (F) Differential interference contrast (DIC) image of the field of view shown in panels E, G, and H. Comparative fluorescence staining (fluorescein) for GPIbα and tissue factor (TF) on microparticles, tethers, and platelets. Note the more intense staining with the anti-GPIbα antibody (G) as compared with the anti-TF antibody (H). All scale bars shown = 5 μm.
