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Acoustic trapping based high throughput isolation and characterization of pathogen activated platelet derived extracellular vesicles from plasma

Broman, Axel LU ; Palm, Frida LU ; Malmström, Johan LU orcid ; Marcoux, Geneviève LU ; Semple, John W LU ; Laurell, Thomas LU and Shannon, Oonagh LU (2023) 27th International conference on miniaturized systems for chemistry and life sciences, MicroTAS 2023
Abstract
We present the use of a high capacity and high throughput acoustic trapping platform for phenotypic characterization and functional studies of extracellular vesicles (EVs) from pathogen activated platelets. Platelet rich plasma was stimulated with bacterial M1 protein isolated from S. Pyogenes, which is known to activate platelets. The subsequently released platelet EVs were isolated from 400 μL plasma by acoustic trapping at a flowrate of 500 μL/min. We have previously reported on the acoustic trapping platform, which can process milliliter sized samples in minutes1. The EVs were then compared to EVs released by platelets stimulated with endogenous platelet activator (Thrombin) and negative control (HEPES buffer).
A schematic of the... (More)
We present the use of a high capacity and high throughput acoustic trapping platform for phenotypic characterization and functional studies of extracellular vesicles (EVs) from pathogen activated platelets. Platelet rich plasma was stimulated with bacterial M1 protein isolated from S. Pyogenes, which is known to activate platelets. The subsequently released platelet EVs were isolated from 400 μL plasma by acoustic trapping at a flowrate of 500 μL/min. We have previously reported on the acoustic trapping platform, which can process milliliter sized samples in minutes1. The EVs were then compared to EVs released by platelets stimulated with endogenous platelet activator (Thrombin) and negative control (HEPES buffer).
A schematic of the sample processing can be seen in Fig. 1. Human plasma from healthy donors was incubated with HEPES buffer, Thrombin or M1 protein to stimulate platelets and induce EV release. The platelets were then removed by centrifugation, leaving EVs in plasma. The EVs were isolated and enriched by acoustic trapping and the protein content was analyzed using mass spectrometry. The EVs were also analyzed by immunoblotting, as well as immunogold labelling against CD42b and M1 protein and imaged by transmission electron microscopy (TEM). Additionally, isolated EVs were incubated with whole blood from healthy donors to investigate functional immunomodulatory effects compared to known platelet agonists (Thrombin, M1).
The mass spectrometry data showed a clear distinction between isolated vesicles and plasma, Fig. 2A, with one protein cluster enriched for vesicles and one enriched for plasma samples. There was also a clear distinction between EVs from activated platelets (Thrombin, M1) and resting platelets (HEPES), Fig. 2B. Interestingly, the bacterial M1 protein was enriched in the vesicle fraction, Fig. 3A, suggesting that M1 protein binds to platelet EVs.
To confirm that M1 binds to EVs, trapped samples and centrifuged samples (both pellet and supernatant) were analyzed with immunoblot against M1 protein, Fig. 3B. Clear bands are present around 54 kDa, in accordance with M1 mass, in samples containing enriched EVs. This further confirms that M1 protein binds to EVs. The TEM images showed isolated EVs for all samples, Fig. 3C. Vesicles from platelets stimulated with thrombin were found positive for CD42b. Pathogen activated platelet EVs were found positive for both CD42b and M1 protein, showing that the bacterial protein binds to platelet EVs. Although no CD42b positive EVs were found in HEPES stimulated samples in the TEM analysis, we observed a wealth of them in cytometry data.
The whole blood assay showed that isolated platelet EVs stimulated platelet-neutrophil complex formation, compared to resting state, Fig. 4A. Additionally, platelet EVs stimulated IL-8 cytokine release from monocytes, Fig. 4B, suggesting functionally intact vesicles.
We have demonstrated rapid isolation and enrichment of platelet EVs from plasma samples by acoustic trapping. The isolated vesicles were functionally intact, and it was possible to perform several downstream analyses, including whole blood stimulation. We found that bacterial M1 protein from S. Pyogenes binds to platelet EVs and is transported with them, a mechanism which could contribute to the rapid infectious progress in sepsis. (Less)
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Contribution to conference
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conference name
27th International conference on miniaturized systems for chemistry and life sciences, MicroTAS 2023
conference location
Katowice
conference dates
2023-10-15 - 2023-11-19
language
English
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yes
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be24a825-1202-4d65-b75a-6185f7b3254d
date added to LUP
2023-11-29 13:57:40
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2024-03-14 10:11:34
@misc{be24a825-1202-4d65-b75a-6185f7b3254d,
  abstract     = {{We present the use of a high capacity and high throughput acoustic trapping platform for phenotypic characterization and functional studies of extracellular vesicles (EVs) from pathogen activated platelets. Platelet rich plasma was stimulated with bacterial M1 protein isolated from S. Pyogenes, which is known to activate platelets. The subsequently released platelet EVs were isolated from 400 μL plasma by acoustic trapping at a flowrate of 500 μL/min. We have previously reported on the acoustic trapping platform, which can process milliliter sized samples in minutes1. The EVs were then compared to EVs released by platelets stimulated with endogenous platelet activator (Thrombin) and negative control (HEPES buffer).<br/>A schematic of the sample processing can be seen in Fig. 1. Human plasma from healthy donors was incubated with HEPES buffer, Thrombin or M1 protein to stimulate platelets and induce EV release. The platelets were then removed by centrifugation, leaving EVs in plasma. The EVs were isolated and enriched by acoustic trapping and the protein content was analyzed using mass spectrometry. The EVs were also analyzed by immunoblotting, as well as immunogold labelling against CD42b and M1 protein and imaged by transmission electron microscopy (TEM). Additionally, isolated EVs were incubated with whole blood from healthy donors to investigate functional immunomodulatory effects compared to known platelet agonists (Thrombin, M1).<br/>The mass spectrometry data showed a clear distinction between isolated vesicles and plasma, Fig. 2A, with one protein cluster enriched for vesicles and one enriched for plasma samples. There was also a clear distinction between EVs from activated platelets (Thrombin, M1) and resting platelets (HEPES), Fig. 2B. Interestingly, the bacterial M1 protein was enriched in the vesicle fraction, Fig. 3A, suggesting that M1 protein binds to platelet EVs.<br/>To confirm that M1 binds to EVs, trapped samples and centrifuged samples (both pellet and supernatant) were analyzed with immunoblot against M1 protein, Fig. 3B. Clear bands are present around 54 kDa, in accordance with M1 mass, in samples containing enriched EVs. This further confirms that M1 protein binds to EVs. The TEM images showed isolated EVs for all samples, Fig. 3C. Vesicles from platelets stimulated with thrombin were found positive for CD42b. Pathogen activated platelet EVs were found positive for both CD42b and M1 protein, showing that the bacterial protein binds to platelet EVs. Although no CD42b positive EVs were found in HEPES stimulated samples in the TEM analysis, we observed a wealth of them in cytometry data.<br/>The whole blood assay showed that isolated platelet EVs stimulated platelet-neutrophil complex formation, compared to resting state, Fig. 4A. Additionally, platelet EVs stimulated IL-8 cytokine release from monocytes, Fig. 4B, suggesting functionally intact vesicles.<br/>We have demonstrated rapid isolation and enrichment of platelet EVs from plasma samples by acoustic trapping. The isolated vesicles were functionally intact, and it was possible to perform several downstream analyses, including whole blood stimulation. We found that bacterial M1 protein from S. Pyogenes binds to platelet EVs and is transported with them, a mechanism which could contribute to the rapid infectious progress in sepsis.}},
  author       = {{Broman, Axel and Palm, Frida and Malmström, Johan and Marcoux, Geneviève and Semple, John W and Laurell, Thomas and Shannon, Oonagh}},
  language     = {{eng}},
  month        = {{10}},
  title        = {{Acoustic trapping based high throughput isolation and characterization of pathogen activated platelet derived extracellular vesicles from plasma}},
  year         = {{2023}},
}