Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infection

Abstract

Background: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents.

Methods: We analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serum-derived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-α levels were measured by ELISA.

Findings: Profiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-α serum levels.

Interpretation: In SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity.

Funding: Cardiocentro Ticino Institute, Ente ospedaliero Cantonale, Lugano-Switzerland.

Keywords: Coagulation; Extracellular vesicles; Pneumonia; SARS-CoV-2; Tissue factor.

Graphical abstract

Fig. 1

Profiling of EV surface antigens The molecular signature derived by EV surface antigen expression allowed the discrimination of patients with or without infection by SARS-CoV-2 (n = 67) compared to healthy controls (HC; n = 16). (a) Schematic illustration of the protocol. (b) Median fluorescence intensity (MFI, expressed as arbitrary unit, a.u.) for CD9, CD63, CD81 and for the mean of CD9-CD63-CD81. Dot plots show median and interquartile range; levels of MFI were compared by Kruskal–Wallis test; *P < 0.05; **P < 0.01; ***P < 0.001 (statistics is reported in Supplementary Table S1-S2). (c) Heat map showing MFI after normalization by the average MFI of CD9-CD63-CD81 (nMFI; %) for EV surface antigens evaluated by flow cytometry. Blue/red = low/high fluorescence. (d) Principal component analysis displaying patient clustering according to EV antigen expression (CD49e, CD209, CD86, CD133/1, CD69, CD142, CD20) and diagnosis of SARS-CoV-2 infection. Each patient is indicated by a point and diagnoses are represented by color (HC, green; COVID [-], blue; COVID [+]). The principal components 1 and 2 are calculated by weighted linear combinations of the 7 EV markers differentially expressed in patients with or without SARS-CoV-2 infection. The ellipses include patients which falls within the mean +/- SD (principal components 1 and 2 +/- SD). Confusion matrix reports real and predicted diagnosis, accuracy, sensitivity and specificity; *sensitivity and specificity were calculated considering COVID (+) patients vs. COVID (-) and HC grouped together.

Fig. 2

EV surface antigens associated with infection by SARS-CoV-2 EV surface antigens in patients with or without infection by SARS-CoV-2 (n = 67) compared to healthy controls (HC; n = 16). Median fluorescence intensity (MFI) was analyzed after normalization by the average MFI of CD9-CD63-CD81 (normalized MFI; nMFI, %). Dot plots show median and interquartile range; levels of MFI were compared by Kruskal–Wallis test; *P < 0.05; **P < 0.01; ***P < 0.001 (statistics is reported in Supplementary Tables S1-4). (a) nMFI (%) for the 7 EV surface antigens differentially expressed in patients with or without infection by SARS-CoV-2, compared to HC. (b) Association of EV surface antigens with a positive nose-pharyngeal swab after correction for age, sex and diagnosis of pneumonia. Odd ratios (ORs) are shown together with their 95% confidence intervals. Squares were used to indicate significant associations. (c) Performance of EV surface antigens in the discrimination of patients COVID [+] vs. COVID [-]. ROC curves are reported for each marker and for a compound EV marker calculated by linear combination of all the others (dashed black line).

Fig. 3

EV surface antigens associated to pneumonia by SARS-CoV-2 EV surface antigens in patients diagnosed as pneumonia, with or without infection by SARS-CoV-2 (n = 28). Median fluorescence intensity (MFI) was analyzed after normalization by the average MFI of CD9-CD63-CD81 for each patient (normalized MFI; nMFI, %). Dot plots show median and interquartile range; levels of MFI were compared by Mann-Whitney test *P < 0.05; **P < 0.01; ***P < 0.001 (statistics is reported in Supplementary Tables S5-6). (a) MFI (arbitrary unit, a.u.) for CD9, CD63, CD81 and for the mean of CD9-CD63-CD81. (b) nMFI (%) for the 7 EV surface antigens differentially expressed in patients with or without infection by SARS-CoV-2. (c) Association of EV surface antigens with a positive nose-pharyngeal swab after correction for age and sex. Odd ratios (ORs) are showed together with their 95% confidence intervals. Squares were used to indicate significant associations. (d) Principal component analysis displaying patient clustering according to EV antigen expression (CD86, CD133/1, CD69, CD142, CD20) and diagnosis of SARS-CoV-2 infection; 26 of 28 patients were correctly classified by the model (accuracy 92.9%).

Fig. 4

In-vitro analysis of tissue factor The activity of extracellular vesicles (EV)- tissue factor (TF) CD142 was evaluated by ELISA in EV obtained from serum samples from patients with or without infection by SARS-CoV-2: all patients (plein boxes; n = 67) vs. patients with pneumonia (dashed boxes; n = 28). CD142 carried by EV was also quantified by western blot. Dot plots show median and interquartile range; CD142 quantification and activity were compared by Mann-Whitney test; levels of TNF-α were compared by T-student test for independent samples; *P < 0.05; **P < 0.01; ***P < 0.001 (statistics is reported in Supplementary Tables S7-8). (a) CD142 activity normalized for particle number at NTA (pM per 10⁹ particle). (b) The specificity of tissue factor activity assay for EV-bearing CD142 was validated by the use of a blocking antibody (anti-CD142); fold change reduction equal to 2.2. (c) The specificity of CD142 binding with FVIIa was validated by the pre-treatment with recombinant FVIIa. CD142 nMFI decrease by 1.9 fold compare to untreated EV. (d) Representative blots; CD142 is reported together with EV specific markers (CD81, Syntenin-1, TSG101) and potential contaminants (Apolipoprotein -B48, and -A1). (e) CD142 quantification by western blot (arbitrary unit, a.u., after normalization for TSG101 expression) in patients diagnosed with pneumonia. (f) TNF-α (pg/mL) in serum. (g) Correlation between CD142 activity per particle (pM) at tissue factor activity assay and normalized median fluorescence intensity for CD142 (nMFI; %) at flow-cytometric analysis. (h) Correlation between CD142 activity per particle (pM) at tissue factor activity assay and CD142 quantification (a.u.) at western blot (CD142-WB) in patients with pneumonia. (i) Correlation between mean TNF-α (pg/mL) and CD142 (nMFI; %) at flow-cytometric analysis. (j) Correlation between mean TNF-α (pg/mL) and CD142 quantification (a.u.) at western blot (CD142-WB) in patients with pneumonia. Regression lines are reported together with their 95% confidence interval, Spearman's Rho coefficient and level of significance.