Figure 4: SIM Imaging of Binding of GPVI-Fc (Not Cross-Linked and Cross-Linked) and Platelets to Collagen

GPVI-Fc was incubated with anti–human-Fc antibodies in a 20:1 molar ratio (GPVI-Fc*IgG, GPVI-Fc*Fab2) or in a 1:1 molar ratio for crosslinking (GPVI-Fc*IgG-XL, GPVI-Fc*Fab2-XL). The antibodies contained 10% PE-conjugated anti–human-Fc IgG or anti–human-Fc Fab2. The mixtures were added to blood (333 nM GPVI-Fc) containing abciximab before perfusion over collagen at a shear rate of 600/s. After 4 min of flow, platelets were fixed and stained with anti-CD41 antibody and DyLight 488-conjugated secondary Ab. (Left) Structured illumination microscopy (SIM) images showing binding of PE-labeled GPVI-Fc (not cross-linked and cross-linked) (red) and platelets (green) to collagen with enlarged sections (×800) on the right. Maximum intensity projections of 0.15 μm z sections (total z 2.5 μm) are shown. (Right) Line intensity profiles (maximum intensity 100%) drawn along comparable, platelet-free segments of collagen fibers in the designated images (left). Images are representative of 5 different experiments. Binding of not cross-linked GPVI-Fc to collagen is discontinuous, showing fiber segments that are not occupied by GPVI-Fc (zero intensity in line profiles). Abbreviations as in Figures 1 and 2.