Initiation of DNA synthesis by human thrombin: Relationships between receptor binding, enzymic activity, and stimulation of 86 Rb + influx

Stimulation of amiloride‐sensitive sodium (Na + ) influx and the subsequent activation of NA + , K + ‐ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin‐initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human α‐thrombin was converted to γ‐thrombin, nitro‐α‐thrombin, and diisopropylphospho (DIP)‐α‐thrombin. These derivatives retain either the capacity to bind cell surface α‐thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of α‐thrombin or the various thrombin derivatives, only α‐thrombin stimulates 86 Rb + influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP‐α‐thrombin that saturate the α‐thrombin recptors (up to 2 μg/ml) do not stimulate either the early or late influx of 86 Rb + , indicating that DIP‐α‐thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either γ‐thrombin or nitro‐α‐thrombin, however, stimulate both early and late 86 RB + uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86 Rb + influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.