Limitations of current anticoagulant therapies have led us to develop two distinct classes of exosite peptide inhibitors for the initiator of the clotting process, the tissue factor-factor VIIa (TF.FVIIa) complex (Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522-9531). Although both peptide classes are potent and selective inhibitors of TF.FVIIa, neither showed 100% inhibition at saturating concentrations. Crystal structures of these peptides in complex with the FVII/FVIIa protease domain revealed their distinct binding sites and close proximity to the active site. The favorable orientation of the 15-mer A-site peptide A-183 (EEWEVLCWTWETCER) suggested that a C-terminal extension into the FVIIa active site could yield a chimeric inhibitor that was not only potent and selective but complete as well. A novel two-step "protease switch" approach using substrate phage display was developed by first binding all phage containing A-183 and C-terminal extension libraries to immobilized and inactive FVIIa. Upon altering pH and adding TF to switch on FVIIa enzymatic activity, only those phage released by proteolytic cleavage within the extension were propagated. This process selected for both preferred sequence and length in the extension, leading to a 27-mer peptide A-183X (EEWEVLCWTWETCERGEGVEEELWEWR) with a C-terminal 12-mer extension containing an Arg in the P1 position. A-183X was a more potent and complete inhibitor of FX activation, having a maximal extent of inhibition of approximately 99% with an IC50 of 230 pm versus A-183 which maximally inhibited to 74% with an IC50 of 1.5 nm. A-183X also had a maximal prolongation of the prothrombin time of 7.6- versus 1.9-fold for A-183, making it a more effective anticoagulant.