Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex

Abstract

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.

Disruption of Ixodes scapularis anticoagulation by using RNA interference

“…Characterizations of tick salivary antigens have, therefore, been based on their ability to (i) induce skin hypersensitivity reaction on tick-resistant animals, (ii) react with anti-tick immune serum, (iii) modulate host immune responses, and (iv) thwart host hemostatic mechanisms (28). Although these approaches have identified several tick salivary proteins (11,14,16,23,(28)(29)(30)(31), an effective vaccine against I. scapularis ticks remains elusive. Recently, random sequencing of clones from an adult I. scapularis salivary gland cDNA library identified Ϸ100 I. scapularis genes (17).…”

“…Ixolaris, an inhibitor of the factor X-tissue factor VIIIa complex (11), and Salp14, an inhibitor of factor Xa (12), represent two of the prototypic anticoagulants that have been identified in I. scapularis saliva. Proteins that modulate various host immune responses have been characterized also from ixodid tick saliva (13)(14)(15)(16).…”

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

“…In order to counteract host response to injury, salivary glands (SGs) of blood-sucking arthropods express a number of inhibitors of blood coagulation, platelet aggregation, host immunity, inflammation, angiogenesis, neutrophil function, wound healing, and vasodilation (1, 8 -16). Among anticoagulants, inhibitors targeting FVIIa/tissue factor, thrombin, FXa, FIXa, FXIIa, and high molecular weight kininogen have been reported (17)(18)(19)(20). Of note, members of different protein families, including Kunitz-, ascaris-, and antistasin-like, and SALP have been identified as FXa inhibitors (17,21).…”

Targeting exosites on blood coagulation proteases

“…Ixolaris is a novel recombinant two Kunitz tick salivary gland inhibitor that is homologous to a physiological plasma inhibitor named tissue factor pathway inhibitor (TFPI) (Francischetti et al 2002). Similarly to TFPI, ixolaris is a potent inhibitor of the extrinsic tenase complex (FVIIa/tissue factor).…”