Disrupting the physical link between GPIbα and the cytoskeleton abolishes the ability of CD to enhance vWf-induced cell aggregation.

(A) CHO-Ib/IX and CHO-IbΔ569 cells (3 × 10⁶/mL) were incubated with vehicle alone (−) or 5 μmol/L CD (+). The cells were then aggregated in a platelet aggregometer using BvWf (5 μg/mL). The aggregation traces are from 1 experiment, representative of 5 independent experiments. (B) CHO-Ib/IX and CHO-IbΔ569 cells (3 × 10⁶/mL) were incubated with vehicle alone or 5 μmol/L CD, then stirred for 5 minutes in the presence of control buffer (resting) or BvWf (5 μg/mL). Cells were then fixed, stained, mounted onto glass slides, and subjected to confocal microscopy (10 × objective) as described for Figure 4D. Images were obtained from 1 experiment, representative of 5. (C) Cells from 8 random fields (10 × objective) were subjected to volumetric analysis, and the extent of cell aggregation was expressed as the fold increase in the volume of cell aggregates over that detected for Me₂SO-treated control cells. Results are the mean ± SE from 5 experiments.