High-affinity pentasaccharide abrogates the inhibitory effects of cleaved antithrombin on bFGF-induced HUVEC proliferation, wound healing, migration, and capillary tube formation. Resting HUVECs were cultured in reduced serum F-12K media and treated with bFGF (10 ng/mL) and native (N), cleaved (C), or latent (L) forms of antithrombin (AT, 20-50 μg/mL) in the presence or absence of 0.25 μM high-affinity pentasaccharide (H5*) as indicated. (A) Measurements of HUVEC proliferation after 48-hour incubation by the addition of MTT reagent and measurement of absorbance at 490 nm as in previous studies.20 Cells were unstimulated (□) or stimulated with bFGF (▪) in the absence or presence of the indicated antithrombin forms. Experiments were performed both in the absence (lanes 1-8) and presence (lanes 9-16) of the high-affinity pentasaccharide. Each value represents the mean of 3 experiments ± SD in each of which quadruplicate samples were run. (B) Wound-induced migration assay using confluent resting HUVECs wounded by scratching and further cultured with bFGF and antithrombin in the presence or absence of H5* for 4 hours. Arrows indicate the original wound edge. Magnification, × 100. The mean ± SD number of cells that had migrated into the wound area was quantitated by counting in 3 random areas in 2 independent experiments. (C) Chemotaxis assay for the migration of HUVECs cultured with antithrombin with or without H5* toward bFGF. Migrated cells appear as elongated spindle shapes (black arrow). The circular structures represent pores in the membrane (black arrow). Magnification, × 200. The mean ± SD number of migrated cells for each condition was quantitated by counting 3 independent fields at × 100 magnification. Samples were run in quadruplicate and the results reflect 2 separate experiments. (D) Subconfluent HUVECs plated on matrigel were treated as indicated. After a 16-hour incubation at 37°C, cells were stained and photographed. Magnification × 200. Images in panels B-D were obtained with a Leica Microsystems DMLB microscope and camera (Leica, Wetzlar, Germany). A water-immersion 20 ×/0.3-0.5 NA objective lens was used.
