A novel method for measuring cell surface-bound thrombin: detection of iodination-induced changes in thrombin-binding affinity

• Published in: The Journal of biological chemistry Vol. 257; no. 2; pp. 850 - 858
• Main Authors: LOW, D. A, CUNNINGHAM, D. D
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 01.01.1982
• Subjects: Animals; assays; Biological and medical sciences; Blood coagulation. Blood cells; Cell Line; Cell Membrane - metabolism; Embryo, Mammalian; fibroblasts; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; hirudin; Hirudins - metabolism; iodination; Kinetics; Methods; Mice; Molecular and cellular biology; Receptors, Cell Surface - analysis; Receptors, Cell Surface - metabolism; Receptors, Thrombin; thrombin; Thrombin - analysis; Thrombin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)68275-X

A method is presented for measuring picogram amounts of thrombin bound to the cell surface of normal mouse fibroblasts. After incubation of cells with hirudin, virtually all cell-bound thrombin is released into the medium. Importantly, over 90% of cell-dissociated thrombin is in complex with hirudin as shown by native gel electrophoresis. Therefore, by incubating cells with super(125)I-hirudin and precipitating super(125)I-hirudin-thrombin complexes with an antibody to thrombin, the authors can quantitatively measure the amount of cell-bound thrombin. Comparison of the binding of super(125)I-thrombin and unlabeled thrombin to mouse cells using the hirudin assay shows that super(125)I-thrombin detects only about one-half of the binding sites that thrombin does. The discrepancies between super(125I-thrombin detects only about one-hal The discrepancies between ) super(1)25-)thrombin and thrombin binding to mouse cells are caused, at least in part, by the presence of a large fraction of super(125)I-thrombin containing diiodotyrosine (DIT). super(125)I-thrombin containing monoiodotyrosine binds to mouse cells with a 2- to 3-fold higher affinity than thrombin.