vWf induces actin polymerization in CHO-Ib/IX and CHO-IbΔ569 cells.
(A) Surface expression of GPIbα on CHO-Ib/IX and CHO-IbΔ569 cells was examined by FACS analysis using the anti-GPIb mAb, ALMA12 (filled histogram), as detailed under “Materials and methods.” (B) CHO-Ib/IX and CHO-IbΔ569 cells (3 × 106/mL) were stirred in the presence of buffer (resting) or BvWf (10 μg/mL) for 20 minutes. Where indicated, cells were also incubated with an anti-GPIb mAb, AK2 (5 μg/mL), prior to the initiation of aggregation. The cells were then lysed and F-actin contents in the whole lysates determined using the DNaseI inhibition assay. Results are the mean ± SE from 3 experiments, performed in duplicate. (C) CHO-Ib/IX cells (1 × 106/mL) were fixed in suspension (preadherent) with 3.7% formaldehyde for 10 minutes, prior to adhesion to poly-l-lysine (100 μg/mL)–coated coverslips. Alternatively, CHO-Ib/IX or CHO-IbΔ569 cells were adhered to a HvWf matrix (10 μg/mL) in the presence of 1 μg/mL botrocetin and 2 mmol/L EDTA for 60 minutes (adherent). Adherent cells were fixed, permeabilized, stained with fluorescein isothiocyanate-conjugated phalloidin and subjected to confocal fluorescence microscopy (100 × objective) as described under “Materials and methods.” The images presented were reconstructed using VoxBlast software and are representative of 5 independent experiments.
