A strategy to design potent antagonists of human coagulation factor VIIa (FVIIa) by linking two proteins that independently inhibit activity and bind at separate, nonoverlapping sites is presented. A bifunctional inhibitor (KDTF5), comprising a Kunitz-type domain engineered to inhibit the FVIIa active site and a soluble tissue factor (TF) variant that is defective as a cofactor for factor X (FX) activation, was developed from structure-based modeling of a ternary FVIIa-Kunitz domain-TF complex. KDTF5 inhibited FVIIa-dependent FX activation with a K-i* of 235 +/- 45 pM, a 193-fold and 398-fold increase in potency compared to the TF Variant and Kunitz domain individually. Similarly, KDTF5 was a more potent anticoagulant in vitro compared to either inhibitory domain alone. The results demonstrate the harnessing of a macromolecular chelate effect by fusing two inhibitory Ligands that bind a target at spatially distinct sites.
