title: Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin.
abstract: A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.
authors.affiliation: Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Rockville, Maryland, United States of America.
authors.forename: Willy
authors.initials: W
authors.lastname: Jablonka
authors.affiliation: Institute of Parasitology, Academy of Sciences of the Czech Republic, České Budejovice, Czech Republic.
authors.forename: Michalis
authors.initials: M
authors.lastname: Kotsyfakis
authors.affiliation: Instituto de Bioquimica Médica Leopoldo de Meis, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Daniella M
authors.initials: DM
authors.lastname: Mizurini
authors.affiliation: Instituto de Bioquimica Médica Leopoldo de Meis, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Robson Q
authors.initials: RQ
authors.lastname: Monteiro
authors.affiliation: Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America.
authors.forename: Jan
authors.initials: J
authors.lastname: Lukszo
authors.affiliation: Critical Care Medicine Department, Clinical Center; National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Steven K
authors.initials: SK
authors.lastname: Drake
authors.affiliation: Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Rockville, Maryland, United States of America.
authors.forename: José M C
authors.initials: JM
authors.lastname: Ribeiro
authors.affiliation: Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Rockville, Maryland, United States of America.
authors.forename: John F
authors.initials: JF
authors.lastname: Andersen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects*; D001780:Blood Coagulation Tests; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D016415:Sequence Alignment; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013927:Thrombosis / Q000188:drug therapy; D013987:Ticks / Q000737:chemistry*
chemical_list: D000925:Anticoagulants; D010455:Peptides; D013917:Thrombin