Characterization of the binding of bovine thrombin to isolated rat hepatocytes.

Isolated rat hepatocytes possess per cell 4,500 high-affinity binding sites for thrombin with a Kd of 30-40 pM, and 2.8 X 10(5) low-affinity sites with a Kd of 30 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labelled thrombin is achieved after 3 min at 37 degrees C and 7 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 1-2 X 10(-2) s-1 and 3-4 X 10(-4) s-1. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radioactivity migrates as intact thrombin upon sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3-treatment. Cell-associated radioactivity dissociated from the cells binds just as well in a receptor assay as tracer incubated in a conditioned medium under the same conditions, indicating the absence of a quantitatively important receptor-mediated degradation of the ligand.