Figure 6: GPVI-Fc*Fab in Contrast to GPVI-Fc*Fab2-XL Does Not Inhibit Platelet Aggregation Induced by Plaque and Collagen Under Flow: Functional and SIM Imaging Studies

GPVI-Fc (100 μg/ml; 666 nM) was incubated with 3-fold molar excess of anti–human-Fc Fab-AlexaFluor594 (GPVI-Fc*Fab) or equimolar concentrations of anti–human-Fc Fab2 or anti-human-Fc Fab2–PE (GPVI-Fc*Fab2-XL). (A) Effect of GPVI-Fc*Fab and GPVI-Fc*Fab2-XL on the kinetic of platelet deposition onto plaque and collagen in flowing blood. Plaque homogenate and collagen coated onto coverslips were incubated with GPVI-Fc*Fab and GPVI-Fc*Fab2-XL complexes before perfusion with blood at a shear rate of 600/s. Fluorescence images of platelet deposition were continuously recorded (1 frame/5 s) and analyzed as detailed in the Methods section. Mean ± SD; n = 4. Comparison by using analysis of variance for repeated measures at full 2 to 5 min and secondary pair-wise comparisons by using Tukey correction. Significance of pair-wise comparison of GPVI-Fc*Fab2-XL to control and to GPVI-Fc*Fab was equal at all time points and is only indicated by a single line with asterisks. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (B) SIM imaging of GPVI-Fc*Fab and GPVI-Fc*Fab2-XL binding and platelet adhesion to collagen fibers. Collagen-coated coverslips were incubated with GPVI-Fc*Fab-AlexaFluor594 or GPVI-Fc*Fab2-PE-XL (red) before perfusion with blood containing abciximab 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 second Ab (green). (Left) SIM micrographs (maximum intensity projections of 0.15 μm z sections (total z 2.5 μm) with enlarged sections (×800) showing the different pattern of GPVI-Fc*Fab and GPVI-Fc*Fab2-XL binding (red) to collagen. Platelet adhesion (green) to collagen is observed in GPVI-Fc*Fab but not GPVI-Fc*Fab2-XL treated samples (left). Images are representative of 5 different experiments. (Right) Line intensity profiles (maximum intensity 100%) along comparable, platelet-free sections of collagen fibers in the designated images (left). Binding of GPVI-Fc*Fab to collagen is discontinuous with segments of fibers lacking significant binding (<10% in line profiles). Abbreviations as in Figures 1, 2, and 4.