Cleaved and latent forms of antithrombin down-regulate perlecan protein expression. HUVECs were cultured in serum-free media with or without 10 μg/mL of the different forms of antithrombin in the presence and absence of 10 ng/mL bFGF as indicated for 72 hours. The conditioned media were applied to a nitrocellulose membrane in the indicated amounts, and perlecan protein was detected by enhanced chemiluminescence using a primary antibody to domain III of perlecan followed by a secondary enzyme-conjugated antibody as detailed in “Materials and methods.”