Figure 3: Cross-Linking of GPVI-Fc With Anti–Human-Fc IgG or Anti–Human-Fc Fab2 Increases Its Inhibition of Platelet Aggregation Stimulated by Collagen or Plaque Under Flow

Buffer (control), GPVI-Fc, or GPVI-Fc (50 μg/ml; 333 nM) premixed with equimolar anti–human-Fc IgG or anti–human-Fc Fab2 antibodies for crosslinking was added to blood containing DiOC6 for platelet visualization and perfused over plaque homogenate at a shear rate of 600/s. (A) Representative micrographs display platelet coverage of plaque at 2, 5, and 9 min after start of blood flow. (B) Effect of buffer, GPVI-Fc, GPVI-Fc*IgG-XL, or GPVI-Fc*Fab2-XL on the kinetics of platelet deposition from flowing blood onto plaque and collagen. Measurements are each second. Mean (solid line) ± SD (shaded area); n = 4 to 6. Comparison at 2, 5, and 9 min by using repeated measures analysis of variance or if inappropriate by repeated measures analysis of variance on ranks (only 2 min). Significance of secondary pair-wise comparisons by Tukey correction is indicated by bars. ∗p < 0.05. (C) Kinetics of not cross-linked and cross-linked GPVI-Fc binding to collagen. (Videos 3 and 4 present additional details.) GPVI-Fc pre-incubated with PE-labeled anti–human-Fc Fab2 (20:1 mol/mol) (GPVI-Fc*Fab2) or with an equimolar mixture of PE-labeled and unlabeled anti–human-Fc Fab2 (1:10) for cross-linking (GPVI-Fc*Fab2-XL), was added to blood (333 nM GPVI-Fc final concentration) containing abciximab and perfused over collagen at a shear rate of 600/s. Binding of PE-labeled GPVI-Fc (cross-linked or not cross-linked) to collagen was quantified every second by fluorescence microscopy using a 10× objective. Top: Time course of GPVI-Fc*Fab2 and GPVI-Fc*Fab2-XL binding to collagen (area coverage). Mean (solid line) ± SD (shaded area); n = 4. Bottom: Fluorescence intensity surface plots of GPVI-Fc*Fab2 and GPVI-Fc*Fab2-XL bound to collagen at 3 min after start of blood flow. Color gradients indicate increase of fluorescence intensity. Abbreviations as in Figures 1 and 2.