Amblyomma cajenneserecombinant Kunitz type inhibitor: effect in the blood clotting and B16F10 melanoma cells

Batista, IFC; Simons, SM, Maria, DA and Chudzinski-Tavassi, AM

Laboratório de Bioquímica e Biofísica - Instituto Butantan, Sao Paulo, SP, Brasil

In the bloodsuckers saliva several compounds, which interfere in the physiological mechanisms of their hosts, among them we can point out inhibitors and enzymes whose interfere in the blood coagulation and platelet aggregation. The Amblyommacajennense is a tick, and in previous studies its crude saliva was used to purify a blood clot and a platelet aggregation inhibitor); the Expressed Sequence Tag (ESTs) profile of its salivary glands was partially established and a Kunitz type inhibitor was cloned and expressed. The recombinant inhibitor is a 13,5 kDa protein which presents 40% homology to the Ixolaris (a recombinant inhibitor obtained from Ixodes scapularis tick), and is able to inhibit the Factor Xa activity using either chromogenic or fluorogenic substrates. The aim of this work is to check out the effects of the recombinant inhibitor in the plasma clotting time (using phospholipids and CHO cells apoptotic bodies), evaluate the treatment of B16F10 tumor cells with the inhibitor, verify the citotoxicity and analyze the inhibitor effects on cycle phase. The recombinant inhibitor delays the clotting time (40% phospholipides and 60 % apoptotic bodies) and some citoplasmatic alterations (the inhibitory effect in the cell adhesion and multi-cellular aggregation) are observed as citotoxic effect. The cell adhesion and the burden tumor decrease whith the inhibitor treatment without causing an haemorragic effect, and the analysis of tumor cell cycle showed a huge anti-proliferative activity, demonstrated by the arrestment in the phase cell cycle (apoptosis and G2/M). In conclusion, we can suggest that the inhibitor has a potent antitumor activity and this fact indicates that it can be used as a tool as anticoagulant in coagulation abnormalities, including those induced by tumor process.

Supported by FAPESP and CAT/CEPID – COINFAR

Correspondence to: simonemsimons@yahoo.com.br