Structure of the extrinsic factor Xase substrate complex with the bi-Kunitz inhibitor ixolaris

Ixolaris is a salivary protein from the tick, Ixodes scapularis, which consists of two Kunitz domains and acts similarly to TFPI by inhibiting the extrinsic factor Xase (FXase) complex. In contrast to TFPI, which recognizes FXa, ixolaris binds either FX or FXa and forms an inhibited FXase composed of TF/FVIIa-ixolaris-FX(a). Here we present a structural model for the inhibited complex generated with AlphaFold 3, which was validated using cryo-EM reconstructions of TF/FVIIa-ixolaris-FX. The AlphaFold model could be readily placed in 3D reconstructions of cryo-EM data obtained in the absence and presence of calcium chloride. The model shows the first Kunitz domain (K1) bound at the heparin-binding exosite (HBE) of FX, a subdomain of random coil and β strand made up of the N and C termini of ixolaris bound on the surface of FX and the second Kunitz domain (K2) bound at the active site of FVIIa in a substrate-like manner by means of its reactive center loop (RCL). Surface plasmon resonance experiments verify that ixolaris binds with high affinity to FX and FXa and less strongly to FVIIa. The binding of FX is enhanced in the presence of calcium ion. The formation of an apparent TF/FVIIa-ixolaris FX complex is also seen on immobilized ixolaris, which is promoted in the presence of calcium ion. This study provides the first structural model of the complete TF/FVIIa-ixolaris-FX complex and opens avenues for the development of new antithrombotic therapies.