MAP kinase phosphorylation in response to Xa is mediated by PAR-1 but does not involve intermediate thrombin generation.

HeLa cells were serum-starved, and, where indicated, the cells were incubated with 100 nM hirudin, 2 nM factor Va, or monoclonal antibodies to PAR-1 (10 μg/mL ATAP2, 25 μg/mL WEDE15) for 20 minutes before the addition of 50 nM Xa or 0.5 U/mL Ecarin for 10 minutes. Cells were lysed in SDS-sample buffer; this was followed by SDS-PAGE and Western blotting with ECL detection of phosphorylated MAP kinases p44/42. Autoradiographs were analyzed by laser densitometry, and the fold induction of phosphorylated MAP kinase was calculated (mean ± SEM; n = 3-5).