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The action of Amblyomin-X in the blood clotting process. 

Branco VG, Alvarez-Flores MP, Sciani JM, Iqbal A, Chudzinski-Tavassi AM

Laboratório de Bioquímica e Biofísica, Instituto Butantan, Brasil

Introduction: Hemostasis is a biological event, responsible for the smooth blood flow in blood vessels, made up of related elements such as coagulation, fibrinolysis and endothelium. Amblyomin-X is a recombinant protein characterized as an inhibitor of the Kunitz type, expressed in the prokaryotic system (E. coli) and the eukaryote system (P. pastoris) and also capable of inhibiting FXa in blood coagulation. FXa is a protein essential for triggering the clotting mechanism. Once in its active form (FXa) causes a thrombin generation and, finally, the formation of the fibrin clot.  Objectives: In this study, we evaluated the effect of Amblyomin-X in human and rabbit plasmas. Methods: Amblyomin-X was injected (1 mg / kg) on the marginal ear vein of rabbits and blood samples were collected at different times (30 min, 2, 4 and 24 h), and were submitted to analysis of activated partial thromboplastin time (aPTT) and prothrombin time (PT). In parallel, human blood from healthy donors treated with Amblyomin-X were subjected to the same tests.  Results and Discussion: The results showed that Amblyomin-X was able to prolong the PT and APTT in rabbit’s plasma, and APTT but not PT in human plasma. Furthermore, the kinetics of enzyme inhibition by Amblyomin-X was also evaluated Amblyomin-X inhibited different enzymes (Plasmin, FXa, and Trypsin) in a non-competitive manner. The Ki valves for plasmin, FXa and trypsin inhibition by Amblyomin-X were 3.8, 2.2 and 3.0 µM respectively. A 55% inhibition on the tenase complex was observed and also a 44% inhibition of the Factor Xa, and in both cases the inhibitor concentration was the same (3.0 µM). These results indicate an anti-coagulant, or anti-thrombotic, activity of Amblyomin-X. Therefore, Amblyomin-X might be a promising novel candidate for hemostatic disorders.

Supported by BNDES, Capes, CNPq, FAPESP

2 - BIOCHEMISTRY 17th Annual Scientific Meeting of Instituto Butantan.