title: Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions.
abstract: Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin 19 that is characterised by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterisation reveals that the ubiquitously transcribed A. americanum serpin 19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris-expressed recombinant (r) A. americanum serpin 19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, recombinant A. americanum serpin 19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of recombinant A. americanum serpin 19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14-29%. In anti-hemostatic functional assays, recombinant A. americanum serpin 19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250s, and up to 40s in the activated partial thromboplastin time assay. Given A. americanum serpin 19 high cross-tick species conservation, and specific reactivity of recombinant A. americanum serpin 19 with antibodies to A. americanum tick saliva proteins, we conclude that recombinant A. americanum serpin 19 is a potential candidate for development of a universal tick vaccine.
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
authors.forename: Tae Kwon
authors.initials: TK
authors.lastname: Kim
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA; Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Lucas
authors.initials: L
authors.lastname: Tirloni
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
authors.forename: Zeljko
authors.initials: Z
authors.lastname: Radulovic
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: Lewis
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
authors.forename: Mariam
authors.initials: M
authors.lastname: Bakshi
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.
authors.forename: Creston
authors.initials: C
authors.lastname: Hill
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Itabajara
authors.initials: I
authors.lastname: da Silva Vaz
authors.affiliation: Laboratório de Sanidade Animal, Laboratório de Química e Função de Proteínas e Peptídeos and Unidade de Experimentação Animal, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
authors.forename: Carlos
authors.initials: C
authors.lastname: Logullo
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
authors.forename: Carlos
authors.initials: C
authors.lastname: Termignoni
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA. Electronic address: amulenga@cvm.tamu.edu.
authors.forename: Albert
authors.initials: A
authors.lastname: Mulenga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000378:metabolism; D000933:Antifibrinolytic Agents / Q000378:metabolism*; D015945:Factor XIa / Q000037:antagonists & inhibitors*; D065427:Factor Xa Inhibitors / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D026863:Ixodidae / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism*
chemical_list: D000925:Anticoagulants; D000933:Antifibrinolytic Agents; D065427:Factor Xa Inhibitors; D015843:Serpins; D014361:Trypsin Inhibitors; D015945:Factor XIa
keywords: Amblyomma americanum; Tick anti-haemostatic functions; Tick conserved protein; Tick saliva serpin