Characterization of the cellular binding domain and the effects of monoclonal antibodies and thrombin inhibitors on the binding and internalization of the antithrombin-III--thrombin complex by cultured cells.
Antithrombin III (AT) binds to cultured cells mainly as a complex with thrombin or other serine proteases rather than in its free form. This implies that, upon complex formation, a new determinant appears on the AT molecule which is recognized by the cells. Fragmentation of AT by cyanogen bromide exposes this determinant and an 8-kDa fragment is recognized by cultured cells. The binding of this fragment to cultured cells is inhibited by antithrombin-III-thrombin (AT-T) complex, but not by free AT. The putative cellular binding domain of AT-T is located over amino acid residues 253-314 of AT, in the large loop close to the carboxy-terminus of the molecule. The cell-associated AT-T is internalized and degraded, forming the thrombin-modified AT (ATM). This process is inhibited by lysosomal degradation inhibitors, such as chloroquine or benzamidine. Thus, the appearance of ATM in cells is not a result of its binding to the cell surface, but probably a result of lysosomal degradation of cell-associated AT-T. Another degradation product, with a molecular mass of 43 kDa, appears in cells along with the appearance of ATM. Hirudin, a specific inhibitor of thrombin, inhibits the cellular internalization of AT-T complexes, indicating a possible role for thrombin activity in the internalization process of complexes. Monoclonal antibodies (mAb) against AT, whose epitopes on the AT molecule have been located, affect the binding of AT-T to cultured cells. Two mAb, A10 whose epitope is found outside of the cellular binding site, and B108 whose epitope may overlap this sequence, totally inhibit the binding of the complex to cells. Inhibition of binding results either from steric hindrance or induced changes in the binding site.
Knoller
S
S
Maurice and Gabriela Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel-Aviv University, Israel.
Savion
N
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000990:Antithrombin III / Q000378:metabolism*; D001550:Benzamidines / Q000494:pharmacology*; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology*; D004220:Disulfides / Q000032:analysis; D004728:Endothelium, Corneal / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D011487:Protein Conformation; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001550:Benzamidines; D004220:Disulfides; D002738:Chloroquine; D000990:Antithrombin III; D013917:Thrombin; C032157:benzamidine