Initiation of proliferative events by human alpha-thrombin requires both receptor binding and enzymic activity.
To determine the role of thrombin high-affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active gamma-thrombin stimulates ion fluxes without binding to high-affinity sites, whereas proteolytically inhibited DIP-alpha-thrombin which binds to high-affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP-alpha-thrombin and gamma-thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native alpha-thrombin. This combinatory effect is dose-dependent for both gamma-thrombin and DIP-alpha-thrombin in the same concentration range as alpha-thrombin alone. Thus, these same concentrations of alpha-thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native alpha-thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.
D000818:Animals; D002455:Cell Division / Q000187:drug effects*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004622:Embryo, Mammalian; D006801:Humans; D051379:Mice; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D004247:DNA; D013917:Thrombin; C027962:diisopropylphosphoryl-thrombin