Formation of a protein corona on the surface of extracellular vesicles in blood plasma

Abstract

In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research.

Keywords: aggregation; blood plasma; extracellular vesicles; mass spectrometry; protein corona.

FIGURE 1

Schematic illustration of the work‐flow. AnnV: Annexin V; dC: differential centrifugation; DGUC: density gradient ultracentrifugation; EVDP: extracellular vesicle‐depleted blood plasma; mEV: medium sized extracellular vesicle; MS: mass spectrometry; RA: rheumatoid arthritis; RT: room temperature; SEC: size exclusion chromatography; TEM: transmission electron microscopy; TRPS: Tunable Resistive Pulse Sensing

FIGURE 2

A protein corona is formed around nascent THP1 EVs upon incubation in blood plasma. (a): THP1 monocytes were cultured under serum free conditions, and nascent mEVs were isolated. These mEVs were next incubated in EV‐depleted blood plasma (EVDP) samples from healthy subjects (HS) and patients with rheumatoid arthritis (RA). Plasma‐incubated mEVs were subsequently separated by differential centrifugation (dC) (HS, n = 12, RA n = 10), density gradient ultracentrifugation (DGUC) (HS, n = 3) or size exclusion chromatography (SEC) (HS, n = 3). The protein content of the re‐isolated mEVs was analysed by MS. As controls, nascent mEVs were used. We identified 61 corona proteins, which were present in more than 30% of the plasma‐coated THP1 mEV samples but not in nascent mEVs. A protein was considered to be preferentially found in RA plasma‐coated THP1 mEV samples, if it was found at least 1.5‐fold more frequently in the RA coronas than in the healthy ones. The proteins that could be identified by DGUC and/or SEC besides the standard dC are indicated by symbols next to the abbreviation of the name of each human protein. Protein name abbreviations are derived from the UniProt IDs of each protein, omitting the species of origin (_HUMAN). (b): The primary protein corona list (identified in dC samples) did not include some proteins due to their presence also in nascent mEVs. These proteins are considered as members of an extended protein corona list and are indicated with asterisk. Their frequency among all samples is indicated in the figure. (c): Flow cytometry analysis of Annexin V (AnnV) positive events in density gradient ultracentrifugation fractions of THP1 mEVs (either nascent (n = 3) or corona coated (n = 3)). The density of THP1‐derived mEVs is shifted to a higher density upon incubation in EVDP samples prior to DGUC as compared to nascent mEVs. P < 0.001 and P < 0.01; multiple t‐test

FIGURE 3

Comparison of viral, nanoparticle‐ and mEV‐associated protein coronas formed in human plasma samples (a): The proteomic data of Ezzat et al. ( (Ezzat et al., 2019), marked with asterisks) on herpes simplex virus (HSV) and respiratory syncytial virus (RSV) as well as on positively (NP+) and negatively charged (NP‐) artificial nanoparticles were re‐analysed using a similar approach that we applied to identify EV corona proteins. The obtained protein lists were compared with proteins detected in ≥ 30% of the coated THP1 and platelet mEV samples (re‐isolated by differential centrifugation (dC)). On the bottom right, next to the chart, we indicated those proteins that were missing from only one dataset. (b): Schematic illustration of the Annexin V‐based affinity capture of blood plasma EV isolation from healthy samples (n = 4; individual samples are marked as 1–4 on each gel line) for Capillary Western (Wes) analysis. (c): CD63, (d): α chain of fibrinogen, (e): α chain of complement C3, (f): α chain of complement C4b, (g): ApoA1, (h): ApoE, (i): immunoglobulin G2. Molecular weights are indicated (kDa). (j): The 20 proteins that we detected in ≥ 90% of our plasma‐coated mEV samples (re‐isolated by dC) were searched in Vesiclepedia (Kalra et al., 2012). The hits in the database are shown in the diagram. Empty portions of the columns correspond to blood plasma EV‐associated protein entries, while the filled portions indicate protein entries of non‐blood plasma derived EVs. (k): The Venn diagram shows the overlaps in Vesiclepedia top EV protein hits (top 92 proteins) with proteins shared by both nascent and coated THP1 mEVs, as well as with the corona proteins and with the pellets of EV‐depleted blood plasma samples (corresponding to protein aggregates). All samples were prepared with dC. (l): Shared proteins in Vesiclepedia top hits (92 proteins) and nascent THP1 mEV samples.

FIGURE 4

Confocal microscopy of EV‐depleted blood plasma (EVDP)‐coated mEVs. DiO‐stained THP1 cell‐derived nascent mEVs were incubated with healthy EVDP, washed twice and were immuno‐stained with anti‐fibrinogen α chain and anti‐complement C3 antibodies, followed by Alexa647 and Alexa594 donkey anti‐mouse and anti‐rabbit antibodies, respectively. (a): mEV; (b): mEV with a patchy complement C3 corona; (c): mEVs with a patchy fibrinogen corona; (d): mEV with patchy fibrinogen and complement C3 deposition as well as a C3 aggregate; (e): mEV with associated fibrinogen aggregate; (f): mEV with patchy fibrinogen and complement C3 deposition as well as with a fibrinogen aggregate; (G): mEVs with associated fibrinogen and C3 aggregates; (h): aggregate of C3 and fibrinogen; (i): schematic illustration of the types of interactions of mEVs with proteins

FIGURE 5

Detection of co‐localization of corona proteins and mEV membrane proteins by immune electron microscopy. (a): Schematic illustration of the immunogold labelling. (b): THP‐1 mEVs were re‐isolated by differential centrifugation after incubation in EV‐depleted blood plasma sample of a healthy person and were immuno‐stained for the alpha chain of fibrinogen (10 nm gold particles), (c): for the alpha chain of fibrinogen and ApoA1 (10 and 5 nm gold particles, respectively), (d): for haptoglobin and CD63 (10 and 5 nm gold particles, respectively), (e): for complement C3 and CD63 (10 and 5 nm gold particles, respectively) and (f): for haptoglobin and ApoA1 (10 and 5 nm gold particles, respectively). Arrows indicate 10 nm, while arrowheads point to 5 nm gold particles

FIGURE 6

Imaging of fibrinogen‐coated THP1 mEVs. Electron micrographs of mEVs either incubated in buffer (nascent mEVs, a), or in 1 mg/mL fibrinogen (coated mEVs, b). Arrowheads point to some mEVs with ‘fluffy’ (thickened) membrane. (c): Image analysis of nascent mEVs (six independent fields, n = 596 vesicles) and fibrinogen‐coated EVs (eight independent fields, n = 838 vesicles) P < 0.0001, t‐test.

FIGURE 7

Interactions among corona proteins of mEVs and of corona proteins with the mEV surface. (a): Representation of protein‐protein interactions identified with a high confidence (interaction score ≥ 0.700) with the STRING database and web resource ( (Szklarczyk et al., 2019), https://string‐db.org). Interactions between the nascent THP1 mEV membrane proteins (with predicted membrane localisation according to the UniProt database (Bateman et al., 2021)) and corona proteins are shown. Furthermore, interactions among the corona proteins are also indicated in purple colour. Out of the 107 network nodes, 61 represent membrane proteins while 46 represent corona proteins. Out of 515 edges, 203 show corona protein interactions with membrane proteins and 312 reflect interactions among corona proteins. We found 13 corona proteins (displayed with a purple node frame) to interact with other protein corona proteins only. Non‐interacting proteins are not shown. All interaction sources (including physical and functional associations based on text mining, experiments, databases, co‑expression, neighbourhood, gene fusion and co‑occurrence) were considered in the analysis. Graph centrality measures were set based on the number of connected edges to each node. (b) and (c) show the association of fibrinogen‐FITC with nascent THP1 and platelet mEVs, respectively. EV binding of the fluorescently labelled fibrinogen decreased significantly upon exposure of the samples to high concentration salt solutions. (*P < 0.05; **P < 0.01; t‐test). (d): Representative flow cytometry dot plots show the fibrinogen‐FITC binding to platelet mEVs.

FIGURE 8

Characterization of plasma protein aggregates. (a): The Venn diagram indicates the number of proteins identified by mass spectrometry in the washed pellets of healthy EV‐depleted blood plasma (EVDP) samples (n = 3) corresponding to protein aggregates. Identical EVDP samples were processed by three different methods (differential centrifugation (dC), DGUC and SEC, indicated with symbols). (b): Word cloud illustration of the coloured section of panel A. The different colours mark the proteins detected either only with dC, or also with SEC or DGUC or with all of these methods. The font size correlates with the percentage of detection of a given protein among the samples. Asterisks indicate those proteins that were also identified in the nascent THP1 mEV samples and therefore were not included in the primary list of corona proteins. (c): Overlaps of the corona proteins with protein aggregates separated from the same three healthy EVDP samples with three different methods. (d): Representative TRPS histogram of a twice‐washed EVDP pellet separated by dC (measured before and after 0.1% Triton‐X lysis). For comparison, the insert shows the effect of the detergent lysis on a nascent THP1 mEVs. (e): Immunogold‐stained electron micrograph of a twice‐washed 12,500 g healthy EVDP pellet. The sample was stained for haptoglobin and CD63 (10 and 5 nm gold particles, respectively). Arrows indicate 10 nm gold particles (CD63 was not identified with 5 nm gold particles in the sample). (f): Confocal microscopic image of a mixed fibrinogen and complement C3 aggregate in a EVDP‐coated mEV preparation (immuno‐stained with anti‐fibrinogen α chain and anti‐complement C3 antibodies and Alexa 647 and Alexa 594 donkey anti‐mouse and anti‐rabbit antibodies respectively) (g): Healthy EVDP samples (n = 3) were subjected to serial centrifugation at 12,500 g for 40 min. Protein and lipid concentrations of the washed pellets were determined after each run. While the lipid concentration was under the detection limit throughout the analysis, proteins were detectable in the pellet even after the 6^th round of centrifugation. For comparison, the insert shows protein and lipid concentrations of nascent mEV samples (n = 3)

FIGURE 9

The effects of corona‐coated mEVs on human monocyte‐derived dendritic cells. Human monocyte‐derived dendritic cells (moDCs) were differentiated ex vivo in the presence of IL‐4 and GM‐CSF for 5 days. MoDCs were then exposed to nascent‐ or plasma protein‐coated vesicles or to pellets of EVDP samples (protein aggregates) separated with differential centrifugation. The production of TNF‐α and IL‐6 was determined by ELISA. The frequency of CD83 and CD86 positive cells as well as the mean fluorescence intensity of HLA‐DR positive cells was assessed by flow cytometry. HS: healthy subjects; RA: patients with rheumatoid arthritis; unstim.: unstimulated, cells treated with EV buffer; prot. aggregate: protein aggregate. Kruskal‐Wallis analysis with Dunn's post‐test (TNF‐α and IL‐6), One‐way ANOVA with Tukey's multiple comparisons test (CD83, CD86 and HLA‐DR), *: P < 0.05, **: P < 0.01, ***: P < 0.001