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

SIMPLE SUMMARY: Procoagulant extracellular vesicle (EV) concentrations have been found to increase in several diseases, but the relative contribution of small (sEVs) and large (lEVs) EVs to plasma prothrombotic potential is poorly defined. Our study shows for the first time that the concentration of tissue factor (TF)(pos) sEVs is significantly higher than that of lEVs. Despite this, the TF-dependent procoagulant potential is primarily sustained by lEVs, although sEVs may also contribute to factor Xa generation when TF pathway inhibitor (TFPI) activity is reduced. Also, in thromboinflammatory conditions, such as COVID-19, the enhanced procoagulant potential that characterizes the infection is predominantly supported by lEVs, although both TFpos-sEVs and -lEVs increase during the acute phase of the disease and return to normal levels with infection remission. Therefore, circulating large EVs, instead of small ones, may be identified as a promising target for a future strategy aiming at reducing the procoagulant potential of blood. 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.