Title: Procoagulant activity of extracellular vesicles in plasma of patients with SARS-CoV-2 infection
Abstract: Extracellular vesicles (EVs) have been of special interest in recent years. Emerging evidence suggests that EVs play a key role in health and disease [[1]Ratajczak M.Z. Ratajczak J. Extracellular microvesicles/exosomes: discovery, disbelief, acceptance, and the future?.Leukemia. 2020; 34: 3126-3135Crossref PubMed Scopus (129) Google Scholar]. Released by all cell types, EVs circulate freely, are present in all body fluids and mediate intercellular communication locally and systemically. Further, EVs reprogram functions of circulating and tissue-bound recipient cells in physiological and pathological conditions [[2]Kalluri R. LeBleu V.S. The biology, function, and biomedical applications of exosomes.Science. 2020; 367Crossref PubMed Scopus (3574) Google Scholar]. EVs represent a heterogenous population of differently sized nanovesicles with distinct biogenesis that carry diverse molecular and genetic cargo and deliver it to recipient cells [[1]Ratajczak M.Z. Ratajczak J. Extracellular microvesicles/exosomes: discovery, disbelief, acceptance, and the future?.Leukemia. 2020; 34: 3126-3135Crossref PubMed Scopus (129) Google Scholar,[2]Kalluri R. LeBleu V.S. The biology, function, and biomedical applications of exosomes.Science. 2020; 367Crossref PubMed Scopus (3574) Google Scholar]. Numbers of circulating EVs increase in inflammatory or infectious diseases, and their molecular/genetic content changes with disease progression. Therefore, EVs are being intensively evaluated as non-invasive liquid biomarkers of disease onset, progression and/or outcome [[3]Whiteside T.L. Proteomic analysis of plasma-derived exosomes in defining their role as biomarkers of disease progression, response to therapy and outcome.Proteomes. 2019; 7Crossref PubMed Google Scholar]. EVs are prominently involved in SARS-Cov-2 infection [[4]Elrashdy F. Aljaddawi A.A. Redwan E.M. Uversky V.N. On the potential role of exosomes in the COVID-19 reinfection/reactivation opportunity.J Biomol Struct Dyn. 2020; (Online ahead of print): 1-12https://doi.org/10.1080/07391102.2020.1790426Crossref PubMed Scopus (47) Google Scholar]. A proteomic analysis of EVs isolated from plasma of COVID-19 patients identified several molecules involved in the immune response, inflammation, and activation of the coagulation and complement pathways, which are the main mechanisms of COVID-19-associated tissue damage and multiple organ dysfunctions [[5]Barberis E. Vanella V.V. Falasca M. Caneapero V. Cappellano G. Raineri D. et al.Circulating exosomes are strongly involved in SARS-CoV-2 infection.Front Mol Biosci. 2021; 8632290Crossref PubMed Scopus (110) Google Scholar]. SARS-CoV-2, unlike other related viruses, shows tropism for alveolar epithelial cells and endothelial cells, which express the human counter-receptor, angiotensin converting enzyme 2 (ACE2). The virus replicates within infected cells causing a severe respiratory syndrome accompanied by systemic inflammation, which may result in multi-organ damage and death [[6]Oxford A.E. Halla F. Robertson E.B. Morrison B.E. Endothelial cell contributions to COVID-19.Pathogens. 2020; 9Crossref PubMed Scopus (14) Google Scholar]. The vascular and endothelial cell dysfunction associated with increased mortality appears to involve the coagulation pathway. Although procoagulant activity of circulating EVs has been known since 1967 [[7]Wolf P. The nature and significance of platelet products in human plasma.Br J Haematol. 1967; 13: 269-288Crossref PubMed Scopus (1200) Google Scholar], the role these EV play in the COVID-19-induced pathology has not been defined. A report by Balbi and colleagues [[8]Balbi C.B.J. Bolis S. Lazzarini E. Biemmi V.;. et al.Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infection.EBioMedicine. 2021; https://doi.org/10.1016/j.ebiom.2021.103369Summary Full Text Full Text PDF PubMed Scopus (51) Google Scholar] shows that tissue factor (TF, CD142), a key initiator of the coagulation pathway, is present on the surface of circulating EVs in patients with SARS-CoV-2 infection. While surface TF expression in circulating EVs accumulating in high procoagulant pathological states was previously described [[9]Zwicker J.I. Liebman H.A. Neuberg D. Lacroix R. Bauer K.A. Furie B.C. et al.Tumor-derived tissue factor-bearing microparticles are associated with venous thromboembolic events in malignancy.Clin Cancer Res. 2009; 15: 6830-6840Crossref PubMed Scopus (427) Google Scholar], the Balbi et al. study, identifies TF as a prominent component of the antigenic signature characteristic for circulating EVs in COVID-19 patients and a potentially significant contributor to thrombotic episodes commonly seen in SARS-CoV-2 infections. EVs in sera of 33 COVID(-) and 34 COVID(+) patients were immunocaptured using a cocktail of 37 colored beads, each coated with an antibody specific for one of the 37 target antigens. Fluorescein-labeled antibodies specific for CD9/CD63/CD81 (i.e., tetraspanins) were used for detection of the EV-associated antigens by flow cytometry. A panel of 7/37 EV-associated antigens (CD142, CD133/1, CD209, CD86, CD69, CD49e and CD20) with the highest scores in EVs of COVID(+) patients was identified. This antigen profile discriminated COVID(+) from COVID(-) patients. Among the 7 antigens in the profile, TF (CD142) had the highest discriminating score. Importantly, expression levels of CD142 on the EV surface correlated with increased serum levels of TNF-alpha in COVID(+) patients. Further, EV-associated TF was biologically active in an assay measuring amidolytic activity of the TF/FVIIa complex, and antibodies neutralizing TF activity significantly reduced procoagulant activity of these EVs. The identification of the EV associated protein signature that reliably discriminates COVID(+) from COVID(-) patients is a significant achievement: TF in EVs emerges as a potential noninvasive biomarker of COVID-19 infection. Even more significant is the finding that TF in EVs from sera of COVID(+) patients was bioactive in ex vivo assays. Another observation, linking the TF scores and activity of EVs in COVID(+) patients with serum levels of an inflammatory cytokine, TNF alpha, adds special significance to this study. It is known that TF expressed on cell surfaces is "cryptic" and has a low procoagulant activity. To acquire the full-fledged procoagulant activity, membrane-associated TF is "decripted" by an oxidoreductase, protein disulfide isomerase (PDI) [[10]Reinhardt C. von Bruhl M.L. Manukyan D. Grahl L. Lorenz M. Altmann B. et al.Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation.J Clin Invest. 2008; 118: 1110-1122PubMed Google Scholar]. In COVID (+) patients with elevated serum levels of IL-6, IL-8 and TNF-alpha, a "cytokine storm" results in vascular injury and endothelial cell (EC) damage. Activated platelets adhering to damaged ECs release PDI, which enhances the TF decription, inducing a massive release from ECs of TF(+) EVs with high procoagulant activity. Balbi et al. demonstrated significant elevations in soluble TNF-alpha levels and in the concentration of circulating TF(+) EVs with strong procoagulant activity in COVID (+) relative to COVID(-) patients. The authors hypothesized that TNF-alpha in sera of COVID(+) patients binds to TNF receptors on the EC surface and induces activated ECs to release TF(+)EVs with strong pro-thrombotic activity. These TF-enriched EVs derived from virus-infected ECs might play a major role in vascular injury that characterizes COVID-19 infection. In this study, Balbi et al. did not show that TF(+)EV derived from ECs directly contribute to vascular thrombosis in COVID-19 infection. Neither do the authors convincingly show that ECs rather than e.g., platelets, which are known to carry TF, are the source of TF(+) EVs. To do so, selective immune capture of EVs derived from ECs or from platelets would be necessary. The multiplex flow cytometry analysis of EVs from COVID(+) patients indicated that immune as well as non-immune cell types contributed to the total immunocaptured EV population. This emphasizes tremendous heterogeneity of the examined EVs. Based on expression of cell type-associated antigens, the majority of captured EVs originated from ECs or platelets. However, it remains unclear how many EV originating from ECs were TF(+)EVs. Nevertheless, multiplex flow cytometry on beads discriminated COVID(+) from COVID(-) patients by the EV protein profile that was significantly enriched in biologically-active TF only in COVID+ patients. Further, circulating EVs in COVID(+) patients with poor outcome carried significantly higher levels of CD142. The results suggest that TF(+)EVs not only have a diagnostic potential, but might also qualify as a non-invasive prognostic biomarker in SARS-CoV-2 infections. Future studies are necessary to confirm these promising results. Also, Balbi et al. provide a rationale for a future strategy of therapeutically targeting EVs in body fluids of COVID-19 patients. A depletion of pro-thrombotic TF(+)EVs and a pharmacologic blockade of TF activity in these EVs represent a potentially promising future therapeutic strategies. The author has no conflicts of interest to disclose. Partial support was provided by the NIH grant 1U01-DE029759 to TLW. Circulating extracellular vesicles are endowed with enhanced procoagulant activity in SARS-CoV-2 infectionIn 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. Full-Text PDF Open Access