Aggregation by lectins as an approach for exosome isolation from biological fluids: Validation for proteomic studies

Shtam, Tatiana; Burdakov, Vladimir; Landa, Sergey; Naryzhny, Stanislav N.; Bairamukov, V. Yu.; Malek, Anastasia; Orlov, Yu. N.; Филатов, М. В.

doi:10.1134/s1990519x17020043

Cited by 8 publications

(7 citation statements)

References 30 publications

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“…3) Выделение экзосом, основанное на седиментации их лектиновых агрегатов, проводили, следуя методике, описанной ранее [14,19]. В качестве лектина, связывающего сахарные остатки на поверхности ВМВ использовали Конконавалин А (Кон-А).…”

Section: выделение экзосом из кондиционированной культуральной среды unclassified

“…Оба метода достаточно эффективны и обеспечивают удаление белковых молекул и белковых комплексов невезикулярной природы. В случае использования метода Кон-А, белок растительного происхождения Конконавалин-А может быть удален из препарата без влияния на результаты последующих 2D электрофореза и масспектрометрии [19].…”

Section: оценка состава белков и рнкunclassified

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Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods

Shtam¹,

Samsonov²,

Volnitskiy

et al. 2018

BIOMED KHIM

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Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by "horizontal" exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional features of the EV is being commonly studies in in vitro condition. Several methods of EV isolation from cell culture medium are established, however selection of method might influence on obtained results. The choice of the optimal method depends usually from the amount of medium and the aims of the research while is still challenging issue. We performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with a 30% sucrose/D2O "cushion", precipitation with plant proteins and immune-affinity capturing. EV isolated by different approaches were compared in terms of following parameters: size, concentration, morphology of EV, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, RNA, and several glioma-associated miRNAs. Applied methods included nano-patricle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. On the base of obtained results, we developed practical recommendations that may help researchers to make a best choice of EV isolation method.

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Section: выделение экзосом из кондиционированной культуральной среды unclassified

Section: оценка состава белков и рнкunclassified

Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods

Shtam¹,

Samsonov²,

Volnitskiy

et al. 2018

BIOMED KHIM

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“…For our comparative analysis we used a preparative technique for isolation of EVs based on their ability to aggregate in the presence of lectins. The method for lectin-based isolation of exosomes from different biological fluids was tested earlier for exosome-based protein or miRNA biomarker researches [ 12 , 19 , 20 ]. In the comparative analysis of the methods for isolating vesicles that we conducted in this study, the isolation of EVs by precipitation of lectin aggregates proves significantly inferior to all other methods in terms of purity and quantity of the final sample.…”

Section: Discussionmentioning

confidence: 99%

“…Exosome isolation based on sedimentation of their lectin aggregates was carried out by the procedure described earlier [ 19 , 20 ]. Concanavalin A (Con-A) was used as a lectin binding sugar residue on the EV surface.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of immune and chemical precipitation methods for plasma exosome isolation

et al. 2020

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Exosomes are a type of extracellular vesicles (EVs) secreted by multiple mammalian cell types and involved in intercellular communication. Numerous studies have explored the diagnostic and therapeutic potential of exosomes. The key challenge is the lack of efficient and standard techniques for isolation and downstream analysis of nanovesicles. Conventional isolation methods, such as ultracentrifugation, precipitation, filtration, chromatography, and immune-affinity-based approaches, rely on specific physical properties or on surface biomarkers. However, any of the existing methods has its limitations. Various parameters, such as efficacy, specificity, labor input, cost and scalability, and standardization options, must be considered for the correct choice of appropriate approach. The isolation of exosomes from biological fluids is especially challenged by the complex nature and variability of these liquids. Here, we present a comparison of five protocols for exosome isolation from human plasma: two chemical affinity precipitation methods (lectin-based purification and SubX™ technology), immunoaffinity precipitation, and reference ultracentrifugation-based exosome isolation method in two modifications. An approach for the isolation of exosomes based on the phenomenon of binding and aggregation of these particles via clusters of outer membrane phosphate groups in the presence of SubX™ molecules has been put forward in the present study. The isolated EVs were characterized based upon size, quantity, and protein content.

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“…To investigate whether the anticoagulatory effect is caused by promoting aggregation of MVs, and thereby reducing the procoagulant surface, we assessed the size profile of MVs after incubation with LR04 or concanavalin A, which has been previously described as causing MV aggregation. 57 Although concanavalin A caused a significant shift in the size profile of MVs, no measurable shift occurred after incubation with LR04 (Figure 2H; supplemental Figure 2G). We next tested whether LR04 affects the interaction between MVs and components of the common coagulation pathway.…”

Section: Endogenous Igm Antibodies Affect Mv-dependent Tgmentioning

confidence: 95%

Natural IgM antibodies inhibit microvesicle-driven coagulation and thrombosis

Obermayer

Afonyushkin

Göderle

et al. 2021

Blood

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Thrombosis and the complications associated with it are a major cause of morbidity and mortality worldwide. Microvesicles (MVs), a class of extracellular vesicles, are increasingly recognized as mediators of coagulation and biomarkers of thrombotic risk. Thus, identifying factors targeting MV-driven coagulation may help in the development of novel antithrombotic treatments. We have previously identified a subset of circulating MVs that is characterized by the presence of oxidation-specific epitopes and bound by natural IgM antibodies targeting these structures. Here, we investigated whether natural IgM antibodies, which are known to have important anti-inflammatory house-keeping functions, inhibit the procoagulatory properties of MVs. We found that the extent of plasma coagulation is inversely associated with the levels of both free and MV-bound endogenous IgM. Moreover, the oxidation epitope-specific natural IgM antibody LR04, which recognizes malondialdehyde adducts, reduced MV-dependent plasmatic coagulation and whole blood clotting without affecting thrombocyte aggregation. Intravenous injection of LR04 protected mice from MV-induced pulmonary thrombosis. Of note, LR04 competed the binding of coagulation factor X/Xa to MVs, providing a mechanistic explanation for its anticoagulatory effect. Thus, our data identify natural IgM antibodies as hitherto unknown modulators of MV-induced coagulation in vitro and in vivo and their prognostic and therapeutic potential in the management of thrombosis.

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Aggregation by lectins as an approach for exosome isolation from biological fluids: Validation for proteomic studies

Cited by 8 publications

References 30 publications

Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods

Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods

Evaluation of immune and chemical precipitation methods for plasma exosome isolation

Natural IgM antibodies inhibit microvesicle-driven coagulation and thrombosis

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