title: Avathrin: a novel thrombin inhibitor derived from a multicopy precursor in the salivary glands of the ixodid tick,Amblyomma variegatum.
abstract: Tick saliva is a rich source of antihemostatic compounds. We amplified a cDNA from the salivary glands of the tropical bont tick (Amblyomma variegatum) using primers based on the variegin sequence, which we previously identified as a novel thrombin inhibitor from the same tick species. The transcript encodes a precursor protein comprising a signal peptide and 5 repeats of variegin-like sequences that could be processed into multiple short peptides. These peptides share 31 to 34% identity with variegin. Here, we structurally and functionally characterized one of these peptides named "avathrin." Avathrin is a fast, tight binding competitive inhibitor with an affinity of 545 pM for thrombin and is 4 orders of magnitude more selective towards thrombin than to the other serine proteases of the coagulation cascade. The crystal structure of thrombin-avathrin complex at 2.09 Å revealed that avathrin interacts with the thrombin active site and exosite-I. Although avathrin is cleaved by thrombin, the C-terminal cleavage product continues to exert prolonged inhibition. Avathrin is more potent than hirulog-1 in a murine carotid artery thrombosis model. Such precursor proteins that could be processed into multiple thrombin inhibiting peptides appear to be widespread among Amblyomminae, providing an enormous library of molecules for development as potent antithrombotics.-Iyer, J. K., Koh, C. Y., Kazimirova, M., Roller, L., Jobichen, C., Swaminathan, K., Mizuguchi, J., Iwanaga, S., Nuttall, P. A., Chan, M. Y., Kini, R. M. Avathrin: a novel thrombin inhibitor derived from a multicopy precursor in the salivary glands of the ixodid tick,Amblyomma variegatum.
authors.affiliation: Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, Singapore.|Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Janaki Krishnamoorthy
authors.initials: JK
authors.lastname: Iyer
authors.affiliation: Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Cho Yeow
authors.initials: CY
authors.lastname: Koh
authors.affiliation: Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: Maria
authors.initials: M
authors.lastname: Kazimirova
authors.affiliation: Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: Ladislav
authors.initials: L
authors.lastname: Roller
authors.affiliation: Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Chacko
authors.initials: C
authors.lastname: Jobichen
authors.affiliation: Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.affiliation: The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Mizuguchi
authors.affiliation: The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan.
authors.forename: Sadaaki
authors.initials: S
authors.lastname: Iwanaga
authors.affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Nuttall
authors.affiliation: Yong Loo Lin School of Medicine, National University of Singapore, Singapore.|Department of Cardiology, National University Heart Centre, Singapore.
authors.forename: Mark Y
authors.initials: MY
authors.lastname: Chan
authors.affiliation: Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, Singapore; dbskinim@nus.edu.sg.|Singapore Eye Research Institute, Singapore.
authors.forename: R Manjunatha
authors.initials: RM
authors.lastname: Kini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D002341:Carotid Artery Thrombosis / Q000139:chemically induced / Q000188:drug therapy; D002417:Cattle; D002712:Chlorides / Q000633:toxicity; D003001:Cloning, Molecular; D005260:Female; D005290:Ferric Compounds / Q000633:toxicity; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D026863:Ixodidae / Q000378:metabolism*; D007610:Kallikreins / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009758:Nymph; D010455:Peptides / Q000494:pharmacology*; D012469:Salivary Glands / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014357:Trypsin / Q000378:metabolism
chemical_list: D060829:Arthropod Proteins; D002712:Chlorides; D005290:Ferric Compounds; D010455:Peptides; C000622537:avathrin, Amblyomma variegatum; D005340:Fibrinogen; D007610:Kallikreins; D014357:Trypsin; D013917:Thrombin; C024555:ferric chloride
keywords: blood-feeding arthropods; blood-sucking animals; direct thrombin inhibitor; exosite-I inhibitor; thrombin-inhibitor complex