Ligand binding by membrane-associated GPVI in stably transfected Dami cell lines. (A) Coprecipitation of FcRγ with GPVIa or GPVIb. Equal amounts of rGPVIa (samples 1, 3, 5, and 7) or rGPVIb (samples 2, 4, 6, and 8) solubilized from transfected Dami cells were incubated with LJ6.5 (samples 1-4) or rabbit anti-FcRγ IgG (samples 5-8) for 2 hours at ambient temperature, and protein G-Sepharose (Pierce Chemical) was then added. After incubation for 1 hour, the beads were washed, and the proteins were eluted and separated by NuPAGE. Proteins separated in the polyacrylamide gel were transferred electrophoretically to a PVDF membrane, and the membrane was then probed with either LJ6.5 (samples 1-4) to visualize rGPVI (60 kDa) or rabbit anti–FcRγ IgG (samples 5-8) to visualize the FcRγ chain (18 kDa). Bound probes were visualized by enhanced chemiluminescence using HRP-conjugated secondary reagents. Relative protein concentrations loaded on the NuPAGE gel were as follows: 2× (samples 1, 2, 5, 6) and 1× (samples 3, 4, 7, 8). (B-D) Ligand binding by membrane-associated full-length GPVI isomers. This figure depicts the binding of Dami cells stably transfected with rGPVIa or rGPVIb to microtiter plates coated with type I collagen (B), CRP (C), or CVX (D). Bound cells were detected by a colorimetric reaction, and the extent of binding is directly proportional to the optical density plotted on the ordinate: a, represents cells transfected with rGPVIa; b, those transfected with rGPVIb. (B) The binding to collagen was conducted in the presence of either 1 mM EDTA (left) or 60 μg/mL monoclonal antibody 6F1 (right) to minimize the potential contribution of endogenous Dami cell integrin α₂β₁ to adhesion.