Factor Xa is the activated form of the coagulation factor thrombokinase, known eponymously as Stuart-Prower factor. Factor X is an enzyme, a serine endopeptidase, which plays a key role at several stages of the coagulation system. Factor X is synthesized in the liver. Factor Xa has emerged as an attractive target for novel anticoagulants for its key position in the coagulation cascade and its limited roles outside of coagulation. The most commonly used anticoagulants in clinical practice, warfarin and the heparin series of anticoagulants and fondaparinux, act to inhibit the action of Factor Xa in various degrees. As a result, the past decade has witnessed an explosion of research into small-molecule, oral, direct Factor Xa inhibitors, and several are now in clinical development.
Blood vessel trauma initiates a proteolytic cascade of serine proteases that culminates in the cross-linking of the clotting protein fibrin and the formation of blood clots. The coagulation cascade is composed of two interconnected pathways: the extrinsic and the intrinsic, which converge in the activation of zymogen factor X (FX) to functional factor Xa (FXa). This activation occurs either by the factor VIIa(FVIIa)-tissue factor complex (extrinsic pathway) or by the factor IXa (FIXa)-factor VIII complex (intrinisic pathway). Mature FXa consists of two disulfide linked polypeptide chains: a light chain composed of an N-terminal γ-carboxyglutamate (Gla) containing Gla domain, followed by two epidermal-growth-factor-like (EGF) domain, and a heavy chain harboring a trypsin-like serine protease domain. In the presence of its cofactor, factor Va (FVa), FVa cleaves the zymogen prothrombin to generate active thrombin protease.
Due to its dual role as both the final enzyme of the cascade and a positive feedback regulator of the intrinsic pathway, thrombin has historically been the major protease target of the blood coagulation cascade in the development of anti-coagulant therapies. However, because thrombin is so ubiquitous in the blood coagulation response, many thrombin inhibitors have had low safety to efficacy profiles in clinical trials, with their usage often associated with an increased risk of bleeding complications. The search for alternative targets has led to FXa. Like thrombin, it is strategically place to regulate both the intrinsic and extrinsic pathway, but because of its more restricted activity, it is thought that inhibiting the upstream FXa would be more efficient and less likely to elicit the side-effects seen with thrombin inhibitors. However, similarities of structure and substrate selection among the serine proteases of the blood coagulation cascade have challenged the design of specific inhibitors against FXa. Interactions between FXa and its inhibitors have not been investigated as thoroughly as interactions between thrombin and its inhibitors. X-ray crystallography has been used to define several FXa-inhibitor complexes. However, no structure of FXa with a canonically bound macromolecular inhibitor showing the substrate-like interactions at both sides of the scissile bond has been determined. Because of the importance of extended interactions in FXa substrate specificity, such a complex may prove to be especially useful in inhibitor design.
The E. coli macromolecular protease inhibitor ecotin inhibits a broad range of serine proteases of the chymotrypsin fold including trypsin, chymotrypsin, and collagenase. Previous structural analyses showed that inhibition by ecotin occurs by the formation of a tetrameric complex consisting of a domain swapped ecotin dimer binding two protease molecular. Each ecotin molecular binds its target at two distinct sites on the protease: a primary site and a secondary site. The primary site interaction is characterized by substrate-like binding of the 80’s loop of ecotin to the active site cleft of the protease. Binding at the secondary site involves less specific interactions involving the distally located 60’s loop of ecotin with a flat, hydrophobic patch on the protease.
Ecotin is the most potent reversible inhibitor of FXa, with an inhibition constant (Ki) of 54 pM. An M84R mutation in the P1 residue of ecotin further increases inhibition by five fold to 11 pM. We have crystallized and determined the x-ray structure of human FXa in complex with ecotin M84R. The canonical binding mode of the ecotin 80’s loop provides valuable insights into the role of extended interactions in FXa substrate recognition. Comparisons with the existing structure of thrombin-M84R reveal how ecotin adapts to different proteases to achieve high affinity binding.
Hur, E. (2006). Structure and Plasticity of Protein-protein Interfaces in Factor Xa and the Androgen Receptor. University of California, San Francisco.