Binding uptake and degradation of antithrombin III · protease complexes by cultured corneal endothelial cells

Interaction of ¹²⁵I-labeled human antithrombin III (¹²⁵I-AT III) · protease complexes with bovine corneal endothelial cells has been studied in tissue culture. ¹²⁵I-AT III does not bind to endothelial cells, but its complexes with either thrombin or trypsin bind specifically to the cultures. The binding of ¹²⁵I-AT III · protease complexes is not via the moiety of the free antithrombin III (AT III) or the free protease, since neither AT III nor thrombin compete on the binding of ¹²⁵I-AT III · thrombin complexes. Only unlabeled AT III · thrombin complexes compete on the binding of the iodinated ligand. ¹²⁵I-AT III · trypsin complexes bind with a K_D of 1.4 × 10^-7 M to high affinity-binding sites present on the cell surface of corneal endothelial cells. Saturation of binding to the cell surface is observed at a concentration of 2.5 × 10^-7 M ¹²⁵I-AT III · trypsin complexes and the number of binding sites per cell is about 4 × 10⁴. The cell surface binding reaches a maximum by 15 min and then decreases with time. The cells, when incubated at 37 °C, appear to internalize the bound complexes by adsorptive endocytosis which proceeds at a rate of 0.5-0.8 pmole/1 × 10⁶ cells/h. The internalization process of ¹²⁵I-AT III · protease complexes is saturated at a concentration of 2.5 × 10^-7 M. Since the cells release ¹²⁵I-labeled material into the extracellular media which cannot be precipitated by trichloroacetic acid (TCA), it probably represents degradation of ¹²⁵I-AT III · protease complexes into small fragments at a linear rate of about 0.5 pmole/1 × 10⁶ cells/h. The described process of AT III · protease complexes binding, internalization and subsequent degradation by corneal endothelial cells may represent a clearing mechanism for extracellular AT III · protease complexes formed under pathological conditions.