Fig. 7. Inhibitory effect of heparin on heparanase endocytosis. (A) Human foreskin fibroblasts were incubated with 10 µg/ml 65 kDa heparanase for 120 minutes without (II) or with 30 minutes pre-incubation with 0.1 µg/ml (III) or 10 µg/ml (IV) heparin. Untreated cells (I) were used as control. After fixation and permeabilization, immunofluorescence was performed with anti-heparanase mAb 130. Cells that were incubated with heparanase and heparin showed no heparanase-containing granules. (B) Intensity (in arbitrary units) and area (in µm²) of heparanase-containing granules and (C) number of granules per cell were significantly lower in cells incubated with 1 µg/ml heparin (BIII and C) compared with control cells incubated with heparanase alone (BII and C) and with untreated cells (BI and C). (D) Western blot analysis of extracellular 65 kDa heparanase binding and processing in cells incubated for 120 minutes at 37°C with 10 µg/ml heparanase alone (lane 1) or with 10 µg/ml heparanase plus 1 µg/ml heparin (lane 2) or with 10 µg/ml heparanase at 4°C (lane 3). Note that heparanase binding was only partially inhibited by heparin, and its processing was not affected by heparin. Filled arrowhead, 65 kDa heparanase precursor; empty arrowhead, 50 kDa processed heparanase.