Cytochalasin D enhances vWf-induced platelet and CHO-Ib/IX cell aggregation independent of integrin α_IIbβ₃.

(A) Washed platelets (3 × 10⁸/mL) from healthy donors were incubated with vehicle alone (0.25% Me₂SO, ░) or CD (5 μmol/L, ▪). Platelet aggregation was induced with HvWf (0.5 μg/mL) and ristocetin (1 mg/mL), in the presence of buffer alone (control), EDTA (2 mmol/L), or c7E3 Fab (20 μg/mL). Results are presented as percent change in aggregation rate (percent relative to Me₂SO control, arbitrarily defined as 100%). (B) Glanzmann thrombasthenic platelets were aggregated with HvWf (0.5 μg/mL) and ristocetin, in the absence (−CD) or presence of 5 μmol/L CD (+CD). The aggregation tracings are from one experiment performed in triplicate. (C) CHO-Ib/IX cells (1 × 10⁶/assay) were stirred for 6 minutes in the presence 5 μmol/L CD alone (CD), 10 μg/ml BvWf (BvWf), or BvWf and CD (BvWf + CD) in a platelet aggregometer. The aggregation tracings are from 1 experiment representative of 5. (D) CHO-Ib/IX cells were stirred in the presence of vehicle alone (resting), 10 μg/mL BvWf (BvWf), BvWf and 5 μmol/L CD (BvWf + CD), or BvWf in the presence of the anti-GPIb mAb, AK2 (BvWf + AK2). The cells were then fixed, stained with DiOC₆ (1 μmol/L), mounted onto glass slides and subjected to confocal microscopy (10 × objective) as described under “Materials and methods.” The images of aggregated cells were reconstructed in 3-dimension using Voxblast (Vaytek Inc). Results presented are from 1 experiment, representative of 5.