High-affinity pentasaccharide abolishes the effects of cleaved anti-thrombin on perlecan gene expression. Electrophoretic mobility shift assay of a 32P-labeled DNA oligonucleotide probe of the TGF-β binding element of the perlecan gene promoter after incubation with nuclear extracts obtained from resting HUVECs pretreated with native or cleaved forms of antithrombin (20 μg/mL) and bFGF (10 ng/mL) in the presence or absence of 0.25 μM natural pentasaccharide (H5) or the high-affinity pentasaccharide (H5*) for 48 hours. TGF-β1 or TGF-α (both 1 ng/mL; Sigma) was added to the cells at 4 hours before harvesting as positive and negative controls for the induction of perlecan gene expression. The identity of the NF-1–probe complex band was confirmed from the supershifting of the complex band when anti–NF-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was included in the incubation.
