Hepatocyte receptors for antithrombin III-proteinase complexes

Abstract

The in vivo clearance of antithrombin III‐proteinase complexes occurs via a specific and saturable pathway located on hepatocytes. We now report studies of the catabolism of antithrombin III‐proteinase complexes in vitro using rat hepatocytes in primary culture. Antithrombin III‐thrombin and trypsin complexes were prepared and purified to homogeneity. Ligand uptake by hepatocytes was concentration, temperature, and time dependent. Initial rate studies were performed to characterize the maximum rate of uptake, V, and apparent Michaelis constant K_app. These studies yielded a V of 12.8 fmol/mg cell protein/min and a K_app of 144 nM for antithrombin‐trypsin complexes. Competition experiments with antithrombin III, antithrombin III‐proteinase complexes, α2‐macroglobulin‐methylamine, asialoorosomucoid and the neoglycoproteins, fucosyl‐bovine serum albumin (BSA), N‐acetylglucosammyl‐BSA, and mannosyl‐BSA indicated that only antithrombin III‐proteinase complexes were recognized by the hepatocyte receptor. Uptake studies were performed at 37°C with ¹²⁵I‐antithrombin III‐trypsin and analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in conjunction with autoradiography. These studies demonstrate time‐dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time‐dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions.