rJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbβ3, a fundamental platelet glycoprotein, and αvβ3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of β-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbβ3 antagonist, capable of preventing arterial thrombosis.