Ligand binding by soluble, rGPVI isoforms. An ELISA was used to measure the binding of rsGPVIa (●) or rsGPVIb (○) to microtiter plates coated with type I collagen (A), CRP (B), CVX (C) or bovine serum albumin (data not shown). rsGPVIa or rsGPVIb was added at a final concentration of 0.5, 1, 2, 5, or 10 μg/mL (abscissa), and the plates were incubated for 90 minutes at 37°C. Unbound rsGPVI was removed, and bound rsGPVI was quantitated by addition of HRP-conjugated anti-FLAG antibody. The amount of bound antibody was determined in a colorimetric reaction, reading absorbance at 490 nm (ordinate). Each data point represents the mean ± SD for n ≥ 3. The binding of either rsGPVIa or rsGPVIb to bovine serum albumin was virtually nil (data not shown).