Identification of HGF Binding Domains on FN and VN and the presence of HGF-FN and HGF-VN complexes in platelet supernatants. Panel A-Plastic wells coated with various matrix proteins or BSA (10 μg/ml) were incubated with 125I-labelled HGF to equilibrium and then washed and counted in a γ-counter. Panel B-Plastic wells were coated with FN and FN derived proteolytic fragments or BSA. 125I-labelled HGF was incubated in these wells to equilibrium binding, washed and the bound HGF levels determined using γ-counter. Panels C &D-Binding of HGF to FN 70 kDa fragment in real time by SPR. Panel C-Real time binding isotherms of increasing concentrations of HGF (0, 16.5, 31.25, 62.5, 125, 188, 250, 315, 375, 500 nM)with association and dissociation phases for a single representative experiment with the arrows indicating injection start and finish. Panel D-The data from panel C is plotted as a function of HGF concentration (30–500 nM). Inset shows the data analysed by the method of Scatchard showing a single binding site with a Kdapp = 300 nM. The data could also fit equally well to a two-site model (see text for details). n = 3. Panel E-Platelet suspensions (20.0 × 108/ml) were stimulated with either saline (-) or 1 U/ml thrombin (+) for 5 min at room temperature to allow degranulation. Cellular and membranous material was cleared by centrifugation (100,000 × g) and the supernatants were immunoprecipitated with monoclonal antibodies to FN or an isotype matched control reagent (control IgG). Immune complexes were analysed by SDS-PAGE and Western blotting probing with an antibody to HGF (Santa Cruz). The blot was stripped and re-probed with antibodies to FN to confirm the specificity of the primary immunoprecipitation step. Panel F-Supernatants derived from thrombin-stimulated platelets were immunoprecipitated with antibodies with specificity for either FN, VN or an isotype matched control reagent. The immune complexes were analysed as in panel E. Lower panel shows the same blot stripped and re-probed with a monoclonal antibody to VN to confirm the primary precipitation. Blots were developed by chemiluminescence. The data shown are representative blots of experiments repeated three times.