The ticks belong to an important group of hematophageous domestic parasites and the Amblyomma cajennense is the principal vector for different pathologies like babesiosis, Rock Montains fever and Lyme symile disease. Bloodsucker´s saliva contains several compounds which interfere in the physiological mechanisms of their hosts such as blood coagulation and platelet aggregation.
Objectives: The aim of this work is to compare the kinetic parameters of two Factor Xa inhibitors: one of them is a native protein purified from the A. cajennense saliva and the other one is a recombinant protein obtained from the cDNA library of the A. cajennense tick.
Methods: The native protein was obtained from the A. cajennense tick saliva which was collected according the modified Tachell’s method, the inhibitor was isolated by an ion exchange chromatography in a Resource-Q column (FPLC system) and the recombinant protein was cloned from the cDNA library from the salivary glands of the A. cajennense tick and the recombinant inhibitor was expressed by using the E. coli system and purified by an affinity chromatography in Ni-sepharose column. To analyze the kinetic parameters of both proteins the chromogenic substrate Z-D-Arg-Gly-Arg-pNA 2HCl (S-2765) hydrolysis was evaluated (inhibitors´concentration, substrate concentration, pre-incubation time and presence of the FX zimogen) and the data compared.
Results and conclusion: The native inhibitor shows molecular mass around 7,500 Da, a Ki = 0.04 nM, and the best pre-incubation time is 15 min, when the native inhibitor is incubated to the FX (zimogen) no interaction is observed, on the other hand to the recombinant inhibitor the Ki = 0.78 nM, the best pre-incubation time is 20 min, the obtained data suggests that the inhibitor shows a competitive pattern of inhibition, and when recombinant inhibitor is incubated with the FX (zimogen) an interaction is observed resulting in a decrease in the inhibition.
Supported by FAPESP and FINEP