Production and Functional Analysis of Echis coloratus Disintegrins in Platelet Aggregation

Snake venom disintegrins are a group of small, non-enzymatic proteins that act by binding to integrins on platelet surfaces and other cells, preventing the interactions required for cell adhesion and platelet aggregation. We expressed and purified three disintegrins from Echis coloratus, containing different integrin-binding motifs: Dis_VGD (VGD motif), Dis_KGD (KGD motif), and Dis_RGD (RGD motif). The expression was carried out using the E. coli expression system, and the purification steps included immobilised metal affinity, anion exchange, and size exclusion chromatography. The ability to inhibit platelet aggregation was evaluated by plate-based aggregometry, using 2-MeS-ADP and CRP-XL as agonists. Dis_VGD presented no effect on platelet aggregation under the tested conditions, while Dis_RGD and Dis_KGD were capable of inhibiting platelet aggregation in a non-competitive and dose-dependent manner. When the platelet aggregation was induced by 2-MeS-ADP, Dis_RGD presented almost complete inhibition at 250 nM, while Dis_KGD presented only 20% of inhibition at the same concentration. The effect on platelet aggregation induced by CRP-XL was less pronounced, with Dis-RGD and Dis-KGD reaching 70% and 10% of inhibition at 500 nM, respectively. The VGD motif binds to integrin α5β1, a fibronectin receptor involved in platelet adhesion and spreading, but not in aggregation. In contrast, the RDG and KGD motifs interact with the αIIbβ3 integrin, a key mediator of platelet aggregation, and are therefore expected to inhibit this process. Our study confirmed that the expressed E. coloratus disintegrins are functional and present the expected effect on platelet aggregation. Further analyses are underway to evaluate their impact on cell adhesion and to gain a more comprehensive understanding of their broader activity.