MAP kinase phosphorylation in response to Xa and Ecarin in the presence of a defined amount of prothrombin.

Serum-starved HeLa cells were preincubated for 20 minutes with prothrombin (10 nM) and activated for 10 minutes with increasing concentrations of Xa (A) or Ecarin (B). Where indicated, the cells were also preincubated for 20 minutes with factor Va (2 nM) or hirudin (100 nM) before the addition of the proteases. Cells were lysed in SDS-sample buffer followed by SDS-PAGE and Western blotting and ECL to detect phosphorylated MAP kinases p44/42. Autoradiographs were analyzed by laser densitometry, and the fold induction of phosphorylated MAP kinase was calculated (mean ± SEM; n = 3).