Internalization and degradation of thrombin and up regulation of thrombin-binding sites in corneal endothelial cells.
Thrombin binds specifically to bovine corneal endothelial cells. Binding involves the formation of an apparently covalent complex between thrombin and its binding site, Mr = 77,000. The cells appear to internalize this complex by adsorptive endocytosis since there is a 10-fold greater amount of thrombin internalized than of prothrombin. Internalization proceeds at a rate of 4 ng of thrombin/1 X 10(6) cells/h and reaches a steady state by 2 h at 37 degrees C. Approximately 90% of the 125I-thrombin reappears in the extracellular media within 1 h of binding to the cells. Since this released 125I-labeled material cannot be precipitated by an anti-thrombin antibody or trichloroacetic acid, it probably represents degradation of thrombin into small peptides. Chloroquine treatment of the cells completely inhibits degradation of thrombin. This suggests that proteolysis occurs in lysosomes. Preincubation of corneal cells with physiological concentrations of thrombin for 2 to 24 h results in a concentration-dependent increase in synthesis and subsequent release into the incubation medium of thrombin binding sites. The increase in the rate of release of binding sites is proportional to the duration of pre-exposure of the cells to thrombin and reaches a maximal increase of approximately 6-fold at 24 h. 125I-thrombin binds to these soluble sites and forms a 77,000-dalton complex similar to that seen with the noninduced binding sites. This complex binds to the cells, is internalized, and then degraded. Binding, internalization, and degradation of thrombin by endothelial cells and the subsequent up regulation of binding sites may represent a mechanism for maintaining low extracellular levels of thrombin.
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002738:Chloroquine / Q000494:pharmacology; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D014357:Trypsin
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D002738:Chloroquine; D014357:Trypsin; D013917:Thrombin