High-affinity pentasaccharide attenuates the inhibitory effects of cleaved antithrombin on bFGF-induced MAP kinase and FGFR-1 phosphorylation in HUVECs. Resting HUVECs (5 × 105) were incubated with 20 μg/mL native or cleaved forms of antithrombin in the presence and absence of 0.25 μM H5* in reduced FBS medium for 2 hours at 37°C. Then, 20 ng/mL bFGF was applied to stimulate the cells for 10 minutes at 37°C. (A) Cell extracts were prepared and resolved by 10% SDS-PAGE and then immunoblotted with either an anti–phosphospecific MAPK IgG (p42/44-MAPK) or an anti–pan-MAPK IgG (42/44-MAPK). The ratio of phosphorylated to total MAPK was quantified from the band intensities with a Kodak Image Station 440-CF (Kodak, Rochester, NY). (B) Equal amounts (200 μg) of cell extract protein were immunoprecipitated with a phospho-tyrosine–specific antibody and were subsequently immunoblotted with an antibody specific for FGFR-1. The results shown are from 1 of 3 experiments that all gave similar results.
