Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate.
Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I-thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.
Frost
G H
GH
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Thompson
W C
WC
Carney
D H
DH
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004261:DNA Replication / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011956:Receptors, Cell Surface / Q000276:immunology / Q000502:physiology*; D018179:Receptors, Thrombin; D012867:Skin / Q000166:cytology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013917:Thrombin / Q000502:physiology*
D000911:Antibodies, Monoclonal; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate