title: Penthalaris, a novel recombinant five-Kunitz tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick vector of Lyme disease, Ixodes scapularis.
abstract: Tick saliva is a rich source of molecules with antiinflammatory, antihemostatic and immunosupressive properties. In this paper, a novel tick salivary gland cDNA with sequence homology to tissue factor pathway inhibitor (TFPI) and coding for a protein called Penthalaris has been characterized from the Lyme disease vector, Ixodes scapularis. Penthalaris is structurally unique and distinct from TFPI or TFPI-like molecules described so far, including Ixolaris, NAPc2, TFPI-1 and TFPI-2. Penthalaris is a 308-amino-acid protein (35 kDa, pI 8.58) with 12 cysteine bridges and 5 tandem Kunitz domains. Recombinant Penthalaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor(TF)-induced factor X (FX) activation with an IC50 of approximately 100 pM. Penthalaris tightly binds both zymogen FX and enzyme FXa (exosite), but not FVIIa, as demonstrated by column gel-filtration chromatography. At high concentrations, Penthalaris attenuates FVIIa/TF-induced chromogenic substrate (S2288) hydrolysis and FIX activation. In the presence of DEGR-FX or DEGR-FXa, but not des-Gla-DEGR-FXa as scaf-folds, tight and stoichiometric inhibition of FVIIa/TF was achieved. In addition, Penthalaris blocks cell surface-mediated FXa generation by monomer (de-encrypted), but not dimer (encrypted) TF in HL-60 cells. Penthalaris may act in concert with Ixolaris and other salivary anti-hemostatics in order to help ticks to successfully feed on blood. Penthalaris is a novel anticoagulant and a tool to study FVIIa/TF-initiated biologic processes.
authors.affiliation: Medical Entomology Section, Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA. ifrancischetti@niaid.nih.gov
authors.forename: Ivo M B
authors.initials: IM
authors.lastname: Francischetti
authors.forename: Thomas N
authors.initials: TN
authors.lastname: Mather
authors.forename: José M C
authors.initials: JM
authors.lastname: Ribeiro
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018884:Ixodes / Q000737:chemistry*; D008074:Lipoproteins; D008193:Lyme Disease; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012469:Salivary Glands / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013925:Thromboplastin / Q000037:antagonists & inhibitors*
chemical_list: D019476:Insect Proteins; D008074:Lipoproteins; D012471:Salivary Proteins and Peptides; C051928:lipoprotein-associated coagulation inhibitor; C494203:penthalaris protein, Ixodes scapularis; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D015951:Factor Xa