VPB0609 - Characterization of TF positive small extracellular vesicles in SARS-CoV-2 infection

Background: Coronavirus-2 (SARS-CoV-2) infection causes a sustained prothrombotic state driven by a massive Tissue Factor (TF) expression in circulating platelets, leukocytes and microvesicles (MVs). Whether also circulating small extracellular vesicles (sEVs), in addition to large MVs (MVs), can contribute to this hypercoagulable scenario through TF expression is not yet known, mainly due to methodological issues in detecting and sizing the smallest vesicles.

Aims: To characterize TF expression and activity in circulating sEVs, compared to that of MVs, of COVID-19 patients during acute phase infection and after symptom remission.

Methods: MVs and sEVs were isolated by plasma differential centrifugation from 10 COVID-19 patients enrolled at acute phase infection (T0) and at six-month-follow-up (T1). Ten healthy subjects (HS) were analyzed as controls. sEVs were counted by Nano Tracking Assay. In sEVs TF expression was analyzed within CD9/CD63/CD81pos events by imaging flow cytometry (IFC) and ExoView™ microarray, while TF activity by FXa generation assay. TF expression and activity in MVs were evaluated for comparison.

Results: By IFC analysis COVID-19 patients at T0 have about 1.5- and 4-fold higher number of TFpos-sEVs and -MVs, respectively, compared to HS, with a trend toward reduction to physiological levels at T1. By microarray analysis sEVs behaved similarly (36±12 and 25±10 TFpos-spots at T0 and T1, respectively; p=0.0281).
sEVs-associated TF is functionally active thus able to partially support FXa formation as sEVs preincubation with TF-neutralizing antibody reduced FXa generation by 30%. However, although sEVs number is significantly higher compared to that measured for MVs (~600-fold in HS), functional activity of sEV is one-third lower compared to that of MVs.

Conclusion(s): These data suggest that, in COVID-19 patients, the altered procoagulant phenotype could also be supported by TF carried by sEVs, although their functional activity is significantly lower than that of MVs.