Intermolecular Interactions and Characterization of the Novel Factor Xa Exosite Involved in Macromolecular Recognition and Inhibition: Crystal Structure of Human Gla-domainless Factor Xa Complexed with the Anticoagulant Protein NAPc2 from the Hematophagous Nematode Ancylostoma caninum

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (Ki=8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel β-sheet formed by the segment of the polypeptide chain consisting of residues 74–80 of NAPc2 with the residues 86–93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67–73) and a turn (residues 26–29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233–243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty.