Desintegrinas de Bothrops alternatus: biologia molecular, estudos in vitro, in vivo e bioinformática estrutural.

Abstract

Disintegrins are toxins frequently found in snake venoms. Their biological effects are due to their binding to cell receptors known as integrins. Aiming to isolate new disintegrins from Bothrops alternatus snake, a cDNA library was constructed upon the transcripts expressed in the venom gland from one specimen. Two disintegrins, called DisBa-01 and DisBa-02 were cloned and sequenced. The amplification of other medium-size disintegrins and metalloprotease-disintegrins was also achieved. The recombinant toxin, DisBa-01, was expressed in an optimized bacterial system (Escherichia coli BL21(DE3) pET28a+DisBa-01) and tested in several biological assays. The toxin inhibited the platelet aggregation (IC50= 235nM) induced by collagen in rabbit s platelets rich plasma (PRP). Using ADP as inductor, the platelet aggregation inhibition was also observed in PRP from rabbits (IC50= 124nM) and human (IC50= 475nM), as well as in washed platelets from mice (IC50= 25nM). Competition assays for the &#945;IIb&#946;3 integrin using specific ligands, fibrinogen and a specific antibody against activated &#945;IIb&#946;3, revealed that the toxin strongly binds to this receptor (respectively IC50=18nM and IC50=70nM). Furthermore, the toxin inhibited the proliferation of cells that representatively express &#945;V&#946;3 integrin, HMEC-1 and B16F10-2B8, in a dose/time dependent manner and the adhesion of B16F10-2B8 to vitronectin (IC50= 225nM). In vivo, the toxin significantly inhibited the thrombus formation in arterioles (Treated: 58,4 ± 1,6 min.; Control: 24,3 ± 2,8 min., p<0,002) and venules (Treated: 46,6 ± 6,8min.; Control: 27,1 ± 1,3min., p<0,002), prolonged the bleeding time (Treated: 989 ± 140 sec.; Control: 202 ± 22 sec.; p<0,008) and inhibited the pulmonary mestastasis of B16F10-2B8 (~92%, 12 days after the tumor cells injection). These results, as well as structural bioinformatics studies strongly suggest the binding of the toxin with &#945;IIb&#946;3 and &#945;Vb3 integrins, what motivates further studies aiming the therapeutic/laboratorial application of DisBa-01 or its derivatives.