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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0237.
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Blood, 15 May 2002, Vol. 99, No. 10, pp. 3602-3612
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
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
Ivo M. B. Francischetti,
Jesus G. Valenzuela,
John F. Andersen,
Thomas N. Mather, and
José M. C. Ribeiro
From the Section of Medical Entomology, Laboratory of
Parasitic Diseases, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD, and Center for
Vector-Borne Disease, University of Rhode Island, Kingston.
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% (IC50) 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  -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.
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