title: Expression and characterization of haemathrins, madanin-like thrombin inhibitors, isolated from the salivary gland of tick Haemaphysalis bispinosa (Acari: Ixodidae).
abstract: Saliva of hematophagous animals, such as ticks, is an excellent source of anticoagulant proteins and polypeptides. Here we describe the identification and characterization of two thrombin inhibitors named as haemathrin 1 and 2 from the salivary gland of tick Haemaphysalis bispinosa using genomic approach. Haemathrins are cysteine-less peptide anticoagulants, which share about 65-70% identity with madanins, and belong to inhibitor I53 superfamily of inhibitors of the MEROPS database. Haemathrins were overexpressed in E. coli and characterized to understand its mechanism of anticoagulant activity. Recombinant haemathrins (rHaemathrins) delayed the thrombin time, prothrombin time, activated partial thromboplastin time and fibrinogen clotting time. Selectivity screening against serine proteases of coagulation cascade reveals that rHaemathrins 1 and 2 specifically inhibit thrombin with an IC50of 46.13±0.04μM and 40.05±0.05μM respectively. Similar to madanin, rHaemathrin 1 and 2 were cleaved by thrombin and consequently lost their inhibitory function over time. Analyses of the cleavage products revealed that the first cleavage, which occurs at the C-terminal end of rHaemathrins, drastically reduced their inhibitory activity. The synthetic peptides corresponding to the cleaved fragments showed significant loss in their ability to prolong plasma clotting times and to inhibit the amidolytic activity of thrombin. Thus haemathrins are the first cleavable thrombin inhibitors characterized from the salivary glands of H. bispinosa.
authors.affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur 784028, Sonitpur District, Assam, India.
authors.forename: Rajeev Kungur
authors.initials: RK
authors.lastname: Brahma
authors.affiliation: Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Blanchet
authors.affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur 784028, Sonitpur District, Assam, India.
authors.forename: Simran
authors.initials: S
authors.lastname: Kaur
authors.affiliation: Protein Science Laboratory, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors.forename: R
authors.initials: R
authors.lastname: Manjunatha Kini
authors.affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur 784028, Sonitpur District, Assam, India. Electronic address: doley@tezu.ernet.in.
authors.forename: Robin
authors.initials: R
authors.lastname: Doley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D002417:Cattle / Q000469:parasitology; D003001:Cloning, Molecular; D005260:Female; D006041:Goats; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D026863:Ixodidae / Q000737:chemistry* / Q000235:genetics; D010314:Partial Thromboplastin Time; D011517:Prothrombin Time; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D013917:Thrombin / Q000378:metabolism
chemical_list: D000991:Antithrombins; D019476:Insect Proteins; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; D013917:Thrombin
keywords: Anticoagulant protein; Antithrombotic drugs; Salivary glands; Thrombin inhibitor; Tick proteins