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 Kapp. These studies yielded a V of 12.8 fmol/mg cell protein/min and a Kapp of 144 nM for antithrombin-trypsin complexes. Competition experiments with antithrombin III, antithrombin III-proteinase complexes, alpha 2-macroglobulin-methylamine, asialoorosomucoid and the neoglycoproteins, fucosyl-bovine serum albumin (BSA), N-acetylglucosaminyl-BSA, and mannosyl-BSA indicated that only antithrombin III-proteinase complexes were recognized by the hepatocyte receptor. Uptake studies were performed at 37 degrees 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.

Hepatocyte uptake of α1‐proteinase inhibitor‐trypsin complexes in vitro: Evidence for a shared uptake mechanism for proteinase complexes of α1‐proteinase inhibitor and antithrombin III

“…In addition, the uptake of ATIII-trypsin complexes was accompanied by the formation of a disulfide interchange product between the ligand and a cellular protein [18]. A similar observation has been made with insulin, which undergoes disulfide interchange with the insulin receptor [ 191. In the present report, the catabolism of alPI-trypsin complexes was studied in vitro using rat hepatocytes in primary culture.…”

Role of the Proposed Serpin-Enzyme Complex Receptor Recognition Site in Binding and Internalization of Thrombin-Heparin Cofactor II Complexes by Hepatocytes

“…Subsequent studies demonstrated uptake of radiolabeled serpin-enzyme complexes by hepatocytes (24,25). Cross-competition experiments in vivo suggested that complexes containing ␣1-antitrypsin, ␣1-antichymotrypsin, antithrombin, or heparin cofactor II are cleared by the same hepatic receptor (26,27), whereas ␣2-antiplasmin-protease complexes are eliminated by a different mechanism (28).…”

C1 Inhibitor-C1¯sComplexes Are Internalized and Degraded by the Low Density Lipoprotein Receptor-related Protein

“…Cells were then washed in 0.1% BSA-PBS supplemented with 1 mM CaCl 2 . Primary rat hepatocytes were isolated and cultured as detailed previously (29). PMN and monocytes used for flow cytometric and radioreceptor binding experiments were prepared, cultured, and harvested as described previously (26).…”

“…Tetrameric C1-INH⅐C1 s⅐C1 r⅐C1-INH complexes were prepared using a procedure described by Ziccardi and Cooper (28). Complexes of ␣ 1 -AT-trypsin or ␣ 1 -AT-elastase were prepared and purified as described (29 (32). Immunostaining was performed with a polyclonal antibody against the COOH-terminal C1-INH peptide and alkaline phosphatase-conjugated polyclonal goat IgG antibodies against rabbit IgG (Sigma), followed by a color reaction using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chloride.…”

“…Nonspecific binding was determined in the presence of a 50-fold molar excess of unlabeled complexes (reaction mixture). Uptake assays with primary rat hepatocytes and C1-INH⅐ 125 I-C1 s were performed as described previously (29).…”