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.
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.
Francischetti
Ivo M B
IM
Section of Medical Entomology, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA.
Valenzuela
Jesus G
JG
Andersen
John F
JF
Mather
Thomas N
TN
Ribeiro
José M C
JM
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D001779:Blood Coagulation Factors / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018884:Ixodes / Q000033:anatomy & histology / Q000737:chemistry*; D008074:Lipoproteins / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012469:Salivary Glands / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013925:Thromboplastin / Q000378:metabolism
D000925:Anticoagulants; D001779:Blood Coagulation Factors; D019476:Insect Proteins; C455900:Ixolaris protein, Ixodes scapularis; D008074:Lipoproteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C051928:lipoprotein-associated coagulation inhibitor; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa