Vania G Branco1, Asif Iqbal2, Miryam P Alvarez-Flores2, Juliana M Sciani1, Sonia A de Andrade1, Leo K Iwai3, Solange M T Serrano3, Ana M Chudzinski-Tavassi4. 1. Laboratory of Biochemistry and Biophysics, Butantan Institute, Sao Paulo, SP, Brazil. 2. 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. 3. Special Laboratory of Applied Toxinology, Butantan Institute, São Paulo, SP, Brazil. 4. 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.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. GENERAL SIGNIFICANCE: 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.
BACKGROUND: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. METHODS: 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. RESULTS: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. CONCLUSION: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 disulfideCys(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. GENERAL SIGNIFICANCE: 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: Asif Iqbal; Mauricio Barbugiani Goldfeder; Rafael Marques-Porto; Huma Asif; Jean Gabriel de Souza; Fernanda Faria; Ana Marisa Chudzinski-Tavassi Journal: Sci Rep Date: 2017-05-03 Impact factor: 4.379
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