Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, has a repertoire of compounds that counteract host defenses. Following sequencing of an I scapularis salivary gland complementary DNA (cDNA) library, a clone with sequence homology to tissue factor pathway inhibitor (TFPI) was identified. This cDNA codes for a mature protein, herein called Ixolaris, with 140 amino acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant Ixolaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor (TF)-induced factor X (FX) activation with an inhibitory concentration of 50% (IC(50)) in the picomolar range. In nondenaturing gel, Ixolaris interacted stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a fast-and-tight ligand of the exosites of FXa and gamma-carboxyglutamic acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity. At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as assessed by recombinant factor IX (BeneFIX) activation assays. This indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that Ixolaris blocks FXa generation by endothelial cells expressing TF. Ixolaris may be a useful tool to study the structural features of FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.