Thrombin and S1P synergistically induce ERK1/2 phosphorylation, and PD98059 reduces the synergistic induction of TF. (A) ECs were treated with or without 10 nM thrombin, 4 μM S1P, or 10 nM thrombin plus 4 μM S1P for 5, 10, and 30 minutes. The cells were then harvested and processed for Western blotting with antibodies to phospho-ERK1/2. (B) The data in panel A were densitometrically quantified and are expressed as the percentage of the control (basal phospho-ERK1/2 in the absence of thrombin and S1P). The values represent the means ± SDs of 3 independent experiments. (C) ECs were pretreated for 20 minutes with indicated concentrations of PD98059 prior to incubation with thrombin (4 nM) or thrombin (1 nM) plus S1P (4 μM). These data represent the means ± SDs of 4 independent experiments with 2 points per condition.