Detection of FKN and VWF binding to GPIbα by ELISA. (A) Glycocalicin was immobilized in microtiter plates, and FKN at the indicated concentration was allowed to bind before VWF and ristocetin addition. Bound VWF (open bars) or FKN (closed bars) was detected with the appropriate antibodies. No binding of VWF to GPIbα was seen in the absence of ristocetin. The experiment was performed twice, and results presented here are derived from one representative experiment and are presented as mean ± SEM for that experiment performed in triplicate. O.D. indicates optical density. (B) Concentration-dependent binding of FKN to GPIbα. Bound FKN was detected by ELISA as described in panel A. Shown is the mean ± SEM of 3 independent experiments. (C) Mapping of the FKN ligand binding site in GPIbα. Effect of diverse mAbs, with known GPIba binding site, on FKN binding. Untreated control (labeled) was set to 100%, and the inhibition of the corresponding mAb is shown. Results are shown as mean ± SEM of 3 independent experiments. (D) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of FKN and glycocalicin protein interaction pull-down experiments: recombinant his-tagged FKN was allowed to bind to Ni-NTA agarose and incubated with glycocalicin or buffer. Bound proteins were eluted with imidazole and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot analysis with the anti-GPIbα mAb 27A10 to detect the presence of glycocalicin.