Covalent binding of thrombin to specific sites on corneal endothelial cells.
Binding of 125I-labeled human alpha-thrombin to endothelial cells derived from bovine corneas was studied in tissue culture. Specific and saturable binding to the cell surface occurred at 37 degrees C but to a much smaller extent at 4 degrees C. Binding of [125I]thrombin to a specific site on these cells with formation of a 77000-dalton complex was demonstrated by NaDodSO4 (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Binding of [125I]thrombin was blocked by a 100-fold excess of unlabeled alpha-thrombin and by the thrombin inhibitor, hirudin. There are approximately 100000 of these thrombin binding sites on the cell surface. Formation of the complex could be detected as early as 15 s, increased rapidly over the next 20-30 min, and then continued at a slower rate for the next 2.5 h. The catalytically active site of the enzyme was required for formation of the NaDodSO4-stable complex as shown by the inability of diisopropyl phosphorofluoride inactivated thrombin to form stable complexes with these cells. The complex was dissociated in NaDodSO4 with 1.0 M hydroxylamine, suggesting an acyl linkage of the enzyme to the cellular binding site. The thrombin-endothelial cell complex was distinct from the thrombin-antithrombin III complex (Mr approximately 90000) on gel electrophoresis, and its formation was not enhanced by heparin. Additional thrombin-cell complexes (Mr less than 77000) were also identified; however, they represent a small fraction of the total thrombin bound to the cells. These observations demonstrate that alpha-thrombin is capable of reacting specifically with corneal endothelial cells to form a NaDod-SO4-stable complex which requires the catalytically active enzyme.
D000818:Animals; D002417:Cattle; D003315:Cornea / Q000378:metabolism*; D004727:Endothelium / Q000378:metabolism; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013696:Temperature; D013917:Thrombin / Q000378:metabolism*
D006898:Hydroxylamines; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013917:Thrombin