Factor VII activating protease (FSAP) is a circulating serine protease implicated in thrombosis, atherosclerosis, stroke, and cancer. Using an overexpression strategy, we have systematically investigated the role of protease activated receptors (PAR)‐1, ‐2, ‐3, and ‐4 on FSAP‐mediated signaling in HEK293T and A549 cells. Cleavage of PAR‐reporter constructs and MAPK phosphorylation was used to monitor receptor activation. FSAP cleaved PAR‐2 and to a lesser degree PAR‐1, but not PAR‐3 or PAR‐4 in both cell types. Robust MAPK activation in response to FSAP was observed after PAR‐2, but not PAR‐1 overexpression in HEK293T. Recombinant serine protease domain of wild type FSAP, but not the Marburg I isoform of FSAP, could reproduce the effects of plasma purified FSAP. Canonical cleavage of both PARs was suggested by mass spectrometric analysis of synthetic peptide substrates from the N‐terminus of PARs and site directed mutagenesis studies. Surprisingly, knockdown of endogenous PAR‐1, but not PAR‐2, prevented the apoptosis‐inhibitory effect of FSAP, suggesting that PAR1 is nevertheless a direct or indirect target in some cell types. This molecular characterization of PAR‐1 and ‐2 as cellular receptors of FSAP will help to define the actions of FSAP in the context of cancer and vascular biology.
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