The
serine protease factor VIIa (FVIIa) in complex with its cellular cofactor
tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in
thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of
cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor
G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM).
G17905 did not appreciably inhibit 12 of the 14 examined
trypsin-like
serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.
G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other
serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193
peptide bond. Macromolecular substrate activation assays demonstrated that
G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless,
G17905 effectively inhibited
thrombus formation in a baboon arterio-venous shunt model, reducing platelet and
fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of
G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.