Initiation of proliferative events by human α‐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 γ‐thrombin stimulates ion fluxes without binding to high‐affinity sites, whereas proteolytically inhibited DIP‐α‐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‐α‐thrombin and γ‐thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native α‐thrombin. This combinatory effect is dose‐dependent for both γ‐thrombin and DIP‐α‐thrombin in the same concentration range as α‐thrombin alone. Thus, these same concentrations of α‐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 α‐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.