作者: Santos, Yasmyn Fernandes Silva ; Frattani, Flávia Serra ; Wermelinger, Luciana Serrão ; David, Victor ; Zingali, Russolina Benedeta ; Lima, Antonio Gilclêr Ferreira ; Mourão, Paulo Antônio de Souza ; Almeida, Fabio Ceneviva Lacerda ; Kurtenbach, Eleonora

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.

2020-04-01·International Journal of Biological Macromolecules1区 · 化学

Delineating the venom toxin arsenal of Malabar pit viper (Trimeresurus malabaricus) from the Western Ghats of India and evaluating its immunological cross-reactivity and in vitro cytotoxicity

1区 · 化学

Article

作者: Raveendran, Dileepkumar ; Nair, Sudarslal Sadasivan ; Shaji, Sanu Korumadathil ; Vanuopadath, Muralidharan ; Nair, Bipin Gopalakrishnan

The venom protein components of Malabar pit viper (Trimeresurus malabaricus) were identified by combining SDS-PAGE and ion-exchange chromatography pre-fractionation techniques with LC-MS/MS incorporating Novor and PEAKS-assisted de novo sequencing strategies. Total 97 proteins that belong to 16 protein families such as L-amino acid oxidase, metalloprotease, serine protease, phospholipase A₂, 5'-nucleotidase, C-type lectins/snaclecs and disintegrin were recognized from the venom of a single exemplar species. Of the 97 proteins, eighteen were identified through de novo approaches. Immunological cross-reactivity assessed through ELISA and western blot indicate that the Indian antivenoms binds less effectively to Malabar pit viper venom components compared to that of Russell's viper venom. The in vitro cell viability assays suggest that compared to the normal cells, MPV venom induces concentration dependent cell death in various cancer cells. Moreover, crude venom resulted in chromatin condensation and apoptotic bodies implying the induction of apoptosis. Taken together, the present study enabled in dissecting the venom proteome of Trimeresurus malabaricus and revealed the immuno-cross-reactivity profiles of commercially available Indian polyvalent antivenoms that, in turn, is expected to provide valuable insights on the need in improving antivenom preparations against its bite.

2019-12-01·Cell Communication and Signaling2区 · 生物学

Recombinant RGD-disintegrin DisBa-01 blocks integrin αvβ3 and impairs VEGF signaling in endothelial cells

2区 · 生物学

ArticleOA

作者: Danilucci, Taís M ; Pisani, Graziéle F D ; Selistre-de-Araujo, Heloisa S ; Pachane, Bianca C ; Altei, Wanessa F ; Santos, Patty K ; Casali, Bruna C ; Lino, Rafael L B

BACKGROUNDIntegrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between α_vβ₃ integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to α_vβ₃ integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with α_vβ₃/VEGFR2 cross-talk and its downstream pathways.METHODSHuman umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of α_vβ₃/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy.RESULTSDisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of α_vβ₃/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and β₃ integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with α_vβ₃ integrin and VEGFR2 in treated cells.CONCLUSIONSDisintegrin inhibition of α_vβ₃ integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis.

100 项与 Disintegrin 相关的药物交易

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