Head-to-Head Comparison of Tissue Factor-Dependent Procoagulant Potential of Small and Large Extracellular Vesicles in Healthy Subjects and in Patients with SARS-CoV-2 Infection

Abstract

The relative contribution of small (sEVs) and large extracellular vesicles (lEVs) to the total plasma procoagulant potential is not yet well defined. Thus, we compared total and TF^pos-sEVs and -lEVs isolated from healthy subjects and COVID-19 patients during the acute phase of the infection and after symptom remission in terms of (1) vesicle enumeration using nanoparticle tracking assay, imaging flow cytometry, and TF immunofluorescence localization in a single-vesicle analysis using microarrays; (2) cellular origin; and (3) TF-dependent Xa generation capacity, as well as assessing the contribution of the TF inhibitor, TFPI. In healthy subjects, the plasma concentration of CD9/CD63/CD81^pos sEVs was 30 times greater than that of calcein^pos lEVs, and both were mainly released by platelets. Compared to lEVs, the levels of TF^pos-sEVs were 2-fold higher. The TF-dependent Xa generation capacity of lEVs was three times greater than that of sEVs, with the latter being hindered by TFPI. Compared to HSs, the amounts of total and TF^pos-sEVs and -lEVs were significantly greater in acute COVID-19 patients, which reverted to the physiological values at the 6-month follow-up. Interestingly, the FXa generation of lEVs only significantly increased during acute infection, with that of sEV being similar to that of HSs. Thus, in both healthy subjects and COVID-19 patients, the TF-dependent procoagulant potential is mostly sustained by large vesicles.

Keywords: COVID-19; factor Xa generation; large extracellular vesicles; small extracellular vesicles; tissue factor.

Figure 1

Analysis of sEV and lEV cellular origin. (A) Correlation between circulating concentration of small and large vesicles in healthy subjects. Platelet (CD41)-, monocyte (CD14)-, granulocyte (CD66)- and endothelium (CD146)-derived small (sEVs; B) and large (lEVs; C) extracellular vesicle distributions in healthy subjects (n = 10) were evaluated using flow cytometry. Percentages of CD41^pos small (sEVs; D) and large (lEVs; E) vesicles spontaneously released in vitro from isolated washed platelets. Individual data or percentage (%) ± SD are shown.

Figure 2

Assessment of circulating tissue factor-positive plasma extracellular vesicles and analysis of their procoagulant potential. Concentration of total (Tetraspan^pos) and tissue factor-positive (TF^pos) small extracellular vesicles (sEV) from healthy subjects (n = 10) analyzed using (A) flow cytometry and (B) multiparametric peptide microarray. (C) Concentration of total (Calcein^pos) and tissue factor-positive (TF^pos) large extracellular vesicles (lEV) from healthy subjects (n = 10) assessed using flow cytometry. Percentages of tissue factor-positive (TF^pos), small (D) and large EV (E)-expressing platelet (CD41), monocyte (CD14), granulocyte (CD66) and endothelium (CD146) population markers. Factor Xa generation capacity of small (F) and large EVs (G) of healthy subjects (n = 5–10) measured in the absence or presence of neutralizing αTF or αTFPI antibody. Individual data are shown. Mean number ± SD, mean number of particles per mm² (NP) ± SD, or percentage (%) ± SD are reported. Results were analyzed using Student’s paired t-test or Mann–Whitney U test, as appropriate. * p < 0.05; **** p < 0.0001.

Figure 3

Procoagulant potential of small EVs in patients with SARS-CoV2 infection. Levels of tissue factor-positive small EVs (TF^pos-sEVs) was analyzed using (A) flow cytometry and (B) multiparametric peptide microarray in patients with SARS-CoV2 infection enrolled during the acute phase (ACUTE; n = 10; red bars) and at 6-month follow-up (FU; n = 10; blue bars). Healthy subjects (HSs; n = 10; green bars) were analyzed for comparison. (C) Factor Xa generation capacity of sEVs was measured in the absence or presence of neutralizing αTF or αTFPI antibody. The dot line indicates the mean value measured in the HSs as a reference. Individual data and mean or mean number of particles per mm² (NP) ± SD are reported. Results were analyzed using Student’s paired t-test or Mann–Whitney U test, as appropriate. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Procoagulant potential of large EVs in COVID-19 patients during acute infection and at 6-month follow-up. Levels of tissue factor-positive large EVs (TF^pos-lEVs) was analyzed using (A) flow cytometry in patients with SARS-CoV2 infection who were enrolled during the acute phase (ACUTE; n = 10; red bars) and at a 6-month follow-up (FU; n = 10; blue bars). Healthy subjects (HSs; n = 10; green bars) were analyzed for comparison. (B) Association between TF^pos-sEVs and -lEVs in acute and FU COVID-19 patients (C); factor Xa generation capacity of lEVs was measured in the absence or presence of neutralizing αTF or αTFPI antibody. The dot line indicates the mean value measured in HSs as a reference. Individual data and mean ± SD are reported. Results were analyzed using Student’s paired t-test or Mann–Whitney U test, as appropriate. * p < 0.05; ** p < 0.01.

Figure 5

Evaluation of cellular origin of tissue factor-positive sEVs and lEVs in COVID-19 patients during acute infection and at 6-month follow-up. Percentages of tissue factor-positive (TF^pos) (A,B) small and (C,D) large EV-expressing platelet (CD41), monocyte (CD14) and granulocyte (CD66) population markers during acute SARS-CoV-2 infection (A,B) and at 6-month follow-up (B,D). Data are reported as percentage ± SD.