Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, CEP 21941-902, Brazil.
Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, CEP 21941-170, Brazil.
Arch Biochem Biophys. 2023 Apr;738:109557. doi: 10.1016/j.abb.2023.109557. Epub 2023 Mar 4.
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 H 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 (IC 95 nM), collagen (IC 57 nM), and thrombin (IC 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.
整合素是一种异二聚体跨膜受体家族，将细胞外基质与细胞骨架连接起来。这些受体在许多细胞过程中发挥作用：黏附、增殖、迁移、凋亡和血小板聚集，从而调节健康和疾病中的广泛情况。因此，整合素已成为新型抗血栓药物的靶点。蛇毒液中的解体素因其能够调节整合素的活性而被识别，例如整合素 αIIbβ3，这是一种基本的血小板糖蛋白，以及表达在肿瘤细胞上的αvβ3。出于这个原因，解体素是研究整合素-基质相互作用和开发新型抗血栓药物的独特和潜在工具。本研究旨在获得重组形式的 jararacin，并评估其二级结构及其对止血和血栓形成的影响。rJararacin 在毕赤酵母（P. pastoris）表达系统中表达，并通过培养物 40mg/L 的产量纯化重组蛋白。通过质谱法确认了分子量（7722Da）和内部序列。通过圆二色性和 H 核磁共振光谱获得了结构和折叠分析。解体素结构显示出正确折叠，存在β-折叠结构。rJararacin 在静态条件下显著抑制 B16F10 细胞和血小板在纤维连接蛋白基质上的黏附。rJararacin 以剂量依赖性方式抑制 ADP（IC 95nM）、胶原（IC 57nM）和凝血酶（IC 22nM）诱导的血小板聚集。这种解体素还分别抑制血小板在连续流动下黏附纤维蛋白原和胶原的 81%和 94%。此外，rjararacin 在有效剂量（5mg/kg）下有效预防大鼠血小板体外和体内的血小板聚集和血栓闭塞。这里的数据提供了证据表明 rjararacin 具有作为 αIIbβ3 拮抗剂的潜力，能够预防动脉血栓形成。
