Summary
Isolated rat hepatocytes possess per cell 4,500 high-affinity binding sites for thrombin
with a Kd of 30-40 pM, and 2.8 × 105 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¸ 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.
Keywords
Thrombin - Receptor - Thrombin - binding site - High - affinity - binding - Rat hepatocyte
