Characterization of the Binding of Bovine Thrombin to Isolated Rat Hepatocytes

• Published in: Thrombosis and haemostasis Vol. 60; no. 3; pp. 419 - 427
• Main Authors: Weyer, Britta, Petersen, Torben E, Sonne, Ole
• Format: Journal Article
• Language: English
• Published: Stuttgart Schattauer GmbH 22.12.1988
• Subjects: Animals; Cattle; Cells, Cultured; Endocytosis; Hydrogen-Ion Concentration; Liver - cytology; Liver - metabolism; Male; Original Article; Rats; Receptors, Cell Surface - metabolism; Receptors, Thrombin; Thrombin - metabolism; High; Rat hepatocyte; Receptor; Thrombin; binding; binding site; affinity
• ISSN: 0340-6245; 2567-689X
• DOI: 10.1055/s-0038-1646983

Summary Isolated rat hepatocytes possess per cell 4,500 high-affinity binding sites for thrombin with a K d of 30-40 pM, and 2.8 × 10 5 low-affinity sites with a K d of 30 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125 I-labelled thrombin is achieved after 3 min at 37¸ C and 7 min at 4¸ C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 1—2 × 10 −2 s −1 and 3—4 × 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 aswell 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 ofthe ligand.