Covalent Binding of Thrombin to Specific Sites on Corneal Endothelial Cells

J. D. Isaacs, N. Savion, D. Gospodarowicz, M. A. Shuman*, J. W. Fenton

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Binding of 125I-labeled human a-thrombin to endothelial cells derived from bovine corneas was studied in tissue culture. Specific and saturable binding to the cell surface occurred at 37 °C but to a much smaller extent at 4 °C. Binding of [125I]thrombin to a specific site on these cells with formation of a 77 000-dalton complex was demonstrated by NaDodS04 (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis. Binding of [125I] thrombin was blocked by a 100-fold excess of unlabeled a-thrombin and by the thrombin inhibitor, hirudin. There are ~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 NaDodS04-stable complex as shown by the inability of diisopropyl phosphorofluoride inactivated thrombin to form stable complexes with these cells. The complex was dissociated in NaDodS04 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 ɳ 90 000) on gel electrophoresis, and its formation was not enhanced by heparin. Additional thrombin-cell complexes {M, < 77 000) were also identified; however, they represent a small fraction of the total thrombin bound to the cells. These observations demonstrate that a-thrombin is capable of reacting specifically with corneal endothelial cells to form a NaDod- SO4-stable complex which requires the catalytically active enzyme.

Original languageEnglish
Pages (from-to)398-403
Number of pages6
JournalBiochemistry
Volume20
Issue number2
DOIs
StatePublished - Jan 1981
Externally publishedYes

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