Interaction Features Between the Recombinant disintegrin, Jararacin, with Platelets: NMR studies and In vivo Activities.

Disintegrins are small cysteine-rich peptides found in snake venom, capable of modulating a broad range of transmembrane receptors known as integrins, heterodimeric receptors that have a key role in mediating physiological and pathological processes, such as platelet aggregation and thrombosis. Integrins, thus, have become a therapeutical target and disintegrins can be used as tools to provide new information about physiopathologies related to this receptor. The objective of this study was to express and analyze the effect of recombinant disintegrin, jararacin (rJarc), upon platelets. For this, we expressed the disintegrin on a Pichia pastoris system. The yeast was transformed with the vector pPIC9 containing the synthetic gene of jararacin. The disintegrin was expressed and secreted in the cultured media and then purified by molecular exclusion chromatography. Also, 15N-rJarc was expressed and purified. We confirmed the molecular mass by mass spectrometry and their correct folding by 1H nuclear magnetic resonance spectra and circular dichroism. NMR studies, in the presence or absence of platelets, revealed changes in rJarc structure and the residues involved in the platelet-desintegrin interaction. We, then, generate dockings based on AlphaFold and compared it with our findings in NMR, to generate in the future experimental models that corroborate the interaction mechanism between rJarc and the Integrins. These models will help us to a better understanding how this disintegrin interacts with platelets integrins, simulating the interaction in a more real way. Following this data we evaluate the ability of rJarc on an arterial thrombosis model, ex vivo platelet aggregation and bleeding assays revealed the ability to prevent thrombus formation and impaired platelet hemostasis at 5mg/kg dose. In conclusion, rJarc was expressed and can be used to get more information on the inhibition mechanism of integrins.