Regulation of the p21Ras‐MAP kinase pathway by factor VIIa

Summary.  Background: In recent years it has become clear that factor (F)VIIa is not a passive mediator involved in the linear transduction of the coagulation cascade, but actively engages target cells to induce signal transduction and that this signal transduction fulfills critical functions in angiogenesis, arteriosclerosis and inflammatory processes. Objectives: The details of coagulation factor‐dependent signal transduction are among the least understood in biology and thus we set out to establish the molecular events responsible for MAP kinase activation induced by the interaction of FVIIa with its cellular binding partner tissue factor (TF). Methods: Two different TF‐expressing cell types, BHKTF and HaCaT cells, were assayed for p21Ras activation using a pull‐down assay that is specific for activated Ras. This activation was visualized by means of Western blotting. In addition, the upstream pathways leading to FVIIa‐induced Ras activation were characterized using phosphospecific antibodies and specific inhibitors. Results: We observed that in both BHKTF and HaCaT cells FVIIa‐induced MAP kinase activation correlates with p21Ras activation, and that this p21Ras activation is essential for FVIIa‐induced MAP kinase activation. In BHKTF cells, early p21Ras activation was mediated by the activation of protein kinase C (PKC), whereas late p21Ras activation employed alternative mechanisms. In HaCaT cells, stimulation of the Src kinase family mediated FVIIa‐dependent p21Ras activation. Finally, in both cell types, Raf activity was mandatory for MAP kinase activation. Conclusions: p21Ras activation is instrumental in FVIIa signal transduction and the FVIIa‐dependent activation of p21Ras involves either PKC or Src‐dependent mechanisms, depending on the cell type investigated.