title: Amblyomin-X having a Kunitz-type homologous domain, is a noncompetitive inhibitor of FXa and induces anticoagulation in vitro and in vivo.
abstract: Cancer has long been associated with thrombosis and many of the standard chemotherapeutics used to treat cancer are pro-thrombotic. Thus, the identification of novel selective anticancer drugs that also have antithrombotic properties is of enormous significance. Amblyomin-X is an anticancer protein derived from the salivary glands of the Amblyomma cajennense tick.
  
  
  
  In this work, we determined the inhibition profile of Amblyomin-X and its effect on activated partial thromboplastin time (aPTT) and prothrombin time (PT), using various approaches such as, kinetic analyses, amidolytic assays, SDS-PAGE, and mass spectrometry.
  
  
  
  Amblyomin-X inhibited factor Xa, prothrombinase and tenase activities. It was hydrolyzed by trypsin and plasmin. MS/MS data of tryptic hydrolysate of Amblyomin-X suggested the presence of Cys(8)-Cys(59) and Cys(19)-Cys(42) but not Cys(34)-Cys(55) disulfide bond. Instead of Cys(34)-Cys(55), two noncanonical Cys(34)-Cys(74) and Cys(55)-Cys(74) disulfide bonds were identified. Furthermore, when Amblyomin-X (1mg/kg) injected in rabbits, it prolonged aPTT and PT.
  
  
  
  Amblyomin-X is a noncompetitive inhibitor (Ki=3.9μM) of factor Xa. It is a substrate for plasmin and trypsin, but not for factor Xa and thrombin. The disulfide Cys(34)-Cys(55) bond probably scrambles with interchain seventh free cysteine residues (Cys(74)) of Amblyomin-X. The prolongation of PT and aPTT is reversible.
  
  
  
  In term of anticoagulant property, this is structural and functional characterization of Amblyomin-X. All together, these results and previous findings suggest that Amblyomin-X has a potential to become an anticancer drug with antithrombotic property.
authors.affiliation: Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil.
authors.forename: Vania G
authors.initials: VG
authors.lastname: Branco
authors.affiliation: Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil; Centre of Excellence for New Target Discovery (CENTD), Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Asif
authors.initials: A
authors.lastname: Iqbal
authors.affiliation: Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil; Centre of Excellence for New Target Discovery (CENTD), Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Miryam P
authors.initials: MP
authors.lastname: Alvarez-Flores
authors.affiliation: Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil.
authors.forename: Juliana M
authors.initials: JM
authors.lastname: Sciani
authors.affiliation: Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil.
authors.forename: Sonia A
authors.initials: SA
authors.lastname: de Andrade
authors.affiliation: Special Laboratory of Applied Toxinology, Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Leo K
authors.initials: LK
authors.lastname: Iwai
authors.affiliation: Special Laboratory of Applied Toxinology, Butantan Institute, São Paulo, SP, Brazil.
authors.forename: Solange M T
authors.initials: SM
authors.lastname: Serrano
authors.affiliation: Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil; Centre of Excellence for New Target Discovery (CENTD), Butantan Institute, São Paulo, SP, Brazil. Electronic address: ana.chudzinski@butantan.gov.br.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Chudzinski-Tavassi
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D060829:Arthropod Proteins; D001777:Blood Coagulation / Q000187:drug effects*; D001780:Blood Coagulation Tests / Q000379:methods; D015951:Factor Xa / Q000378:metabolism*; D065427:Factor Xa Inhibitors / Q000494:pharmacology*; D006801:Humans; D008297:Male; D000072417:Protein Domains; D011517:Prothrombin Time / Q000379:methods; D011817:Rabbits; D012469:Salivary Glands / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000378:metabolism / Q000494:pharmacology*; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism; D013927:Thrombosis / Q000178:diet therapy; D013987:Ticks / Q000378:metabolism
chemical_list: C559369:Amblyomin-X protein, Amblyomma cajennense; D000925:Anticoagulants; D000970:Antineoplastic Agents; D060829:Arthropod Proteins; D065427:Factor Xa Inhibitors; D012471:Salivary Proteins and Peptides; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa
keywords: Amblyomin-X; Anticancer; Anticoagulation; Factor Xa; Inhibition kinetics