Investigation of procoagulant activity in extracellular vesicles isolated by differential ultracentrifugation

Abstract

Tissue factor (TF) is the main initiator of coagulation and procoagulant phospholipids (PPL) are key components in promoting coagulation activity in blood. Both TF and PPL may be presented on the surface of extracellular vesicles (EVs), thus contributing to their procoagulant activity. These EVs may constitute a substantial part of pathological hypercoagulability that is responsible for triggering a higher risk of thrombosis in certain patients. The aim of this study was to describe a model system for the isolation of EVs required for investigating their effect on coagulation. Differential ultracentrifugation (DUC) with and without a single washing step was used to isolate and evaluate the procoagulant capacity of EVs from healthy volunteers through analysis of thrombin generation and PPL activity. Ultracentrifugation at 20,000 × g and 100,000 × g resulted in pellets containing larger vesicles and smaller vesicles, respectively. Isolation yield of particle concentration was assessed by nanoparticle tracking analysis. Immunoelectron microscopy and western blotting revealed vesicles positive for the commonly used EV-marker CD9. Plasma proteins and lipoproteins were co-isolated with the EVs; however, application of a washing step clearly diminished the amount of contaminants. The isolated EVs were capable of enhancing thrombin generation, mainly due to PPL predominantly present in pellets from 20,000 × g centrifugation, and correlated with the activity measured by a PPL activity assay. Thus, DUC was proficient for the isolation of EVs with minimal contamination from plasma proteins and lipoproteins, and the setup can be used to study EV-associated procoagulant activity. This may be useful in determining the procoagulant activity of EVs in patients at potentially increased risk of developing thrombosis, e.g. cancer patients. Abbreviations: TF: Tissue factor; PL: Phospholipids; EVs: Extracellular vesicles; FXa: Activated coagulation factor X; TGA: Thrombin generation assay; PPL: Procoagulant phospholipids; DUC: Differential ultracentrifugation; NTA: Nanoparticle tracking analysis; TEM: Transmission electron microscopy; SPP: Standard pool plasma; CTI: Corn trypsin inhibitor; 20K: 20,000 × g; 100K: 100,000 × g; FVIII: Coagulation factor VIII.

Keywords: Electron microscopy; extracellular vesicles; isolation; microvesicles; procoagulant phospholipids; thrombin generation; tissue factor.

Figure 1.

NTA performed on pellets. (A) Concentration of particles and (B) size determination of particles in each pellet group. *P < 0.05. **P < 0.01. ***P < 0.001. (C) Size distribution of the different pellets divided into three fractions, i.e. <100 nm, 100–200 nm, and >200 nm. EV suspensions were diluted to obtain a particle count per frame within the recommended limits. Capture duration was 5 × 30 s at camera level 10 and the videos were analysed at detection threshold 2 with 9 × 9 blur.

Figure 2.

TEM and western blotting was applied to verify the presence of EV subpopulations in both washed and unwashed pellets. TEM showing EV-characteristics, i.e. shape and size in (A) unwashed and (B) washed pellets. Immunogold labelling with anti-CD9 (clone M-L13) bound to EVs in unwashed (C) and washed (D) pellets confirming presence of CD9-positive subpopulations. (E) Small (<50 nm) vesicular structures adhering to or residing in proximity to larger vesicles. (F) Western blot with anti-CD9, anti-CD142 (clone HTF-1), and anti-apolipoprotein B (clone F2C9) on a pellet pool for each pellet type showing the presence of EV marker CD9 and the removal of apolipoprotein B after washing in PBS.

Figure 3.

Thrombograms (mean ± SEM) depicting the effect on thrombin generation of EV-containing pellets “spiked” into SPP with (n = 6) or without (n = 12) collected with CTI. PBS was added to the standard pool plasma that was not spiked with EVs (control). Thrombograms were generated with different trigger either MP, PRP, or PPPlow reagent or without addition of trigger reagents (depicted vertically). The effect of EVs on thrombin generation seems to mainly be driven by PPL. The use of CTI is depicted in the horizontally.

Figure 4.

EV-associated PPL activity measured by PPL activity assay. (A) EVs reduce the PPL clotting time of SPP, mainly those in 20K pellets, thus increasing the PPL activity. The blue and dotted lines represents the reference PPL clotting time (mean ± SD, n = 12) of SPP without addition of EVs. (B) The 20K pellets showed tendency towards larger particles correlating with increased PPL activity, although this was not significant. (C) A significant correlation between PPL clotting time and thrombin generation using PRP reagent (containing no PL) was observed, but this disappeared when using PPPlow reagent (containing PL). *P < 0.001.

Figure 5.

Loss of procoagulant activity from plasma from healthy donors after DUC and EV removal. (A) Thrombin generation (using PRP reagent) decreased in the donor plasma as result of EV-removal during DUC. Dotted lines represent mean ± SEM. (B) PPL activity in the donor plasma was reduced significantly after DUC and EV-removal. *P < 0.05; **P < 0.01; ***P < 0.001.