Fig. 6.

Antibodies to rat or human HARE partially block heparin endocytosis by rat SECs and 190-hHARE cells. Rat liver SECs, isolated and cultured without serum as described in materials and methods and Fig. 5, were preincubated for 15 min at 37°C with medium containing no antibody (Ab) or with 60 μg/ml of purified anti-rat MAb 174, 235, or 467, or mouse IgG (as control). An equal volume of medium containing 0.1 μM ¹²⁵I-SA·b-UFH (A) or ¹²⁵I-SA·b-LMWH (B) was then added, diluting the Ab concentrations to 30 μg/ml for the duration of the experiment. Cells were incubated for 3 h at 37°C and then washed, and radioactivity and protein content were determined. Values are presented as the mean ± SE (n = 3) specific pmol/μg protein. Similar endocytosis experiments using ¹²⁵I-SA·b-UFH and rat SECs (C) or 190-hHARE cells (D) were performed to assess inhibition by total preimmune IgG or immune IgG against purified s190-hHARE. Nonspecific binding was assessed in parallel control wells incubated with ¹²⁵I-SA·biotin and subtracted from total cell associated values, presented as the mean ± SE (n = 3) specific cpm/μg protein. Symbols in A and B indicate significant differences between no Ab and test samples, as assessed by the Student's unpaired t-test; P < 0.05 (*) or P < 0.005 (‡).