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. 2020 Aug 25;4(16):3971–3976. doi: 10.1182/bloodadvances.2020001786

Figure 1.

Figure 1.

Seropositive platelets efficiently neutralize viral infection in vitro via their IgG. (A) Human umbilical vein endothelial cells (HUVECs) were infected with CMV (5 hours) and incubated with buffer (control), undiluted plasma, or platelets for 30 minutes. Viral load was quantified 72 hours after infection via qPCR by determining relative CMV immediate early (IE) expression (n = 8-14). (B) Blood donors in panel A were stratified according to their anti-CMV IgG serostatus in seronegative (neg.) and seropositive (pos.) donors (n = 7). (C) The relative number of infected HUVECs was quantified by immunofluorescence 72 hours after infection (right panel). Representative images show noninfected HUVECs (Non-inf.) CMV-infected HUVECs incubated with buffer (CMV [control]), or platelets of a seronegative (CMV+PLT neg.) or seropositive (CMV+PLT pos.) donor (left panels). Blue, DAPI; yellow, IE (CMV); red, CD61 (platelets). (D) Surface and intracellular IgG levels of human platelets were determined via flow cytometry (n = 16). (E) Total IgG levels in human citrate-theophyllin-adenosine-dipyridamole (CTAD) plasma and platelet releasate (PLTR) were examined by ELISA (n = 10). (F) Plasma/PLTR ratio of total IgG (n = 10). (G) As in panel A, but seropositive platelets were optionally treated for 20 minutes with ticagrelor (Tica), aspirin (ASA), compstatin (Comp), or P-selectin blocking antibody (P-sel) before incubation with CMV-infected HUVECs (n = 7-13). (H) As in panel A, but CMV-infected HUVECs were incubated with buffer (control) or PLTR of seronegative (neg.) or seropositive (pos.) donors (n = 4-5). (I) As in panel G, but PLTR was optionally treated for 20 minutes with Comp. Alternatively, CMV-infected cells were incubated with IgG-depleted seropositive PLTR (IgG-depl), isolated IgG of seropositive PLTR (IgG only), or seronegative PLTR reconstituted with isolated seropositive IgG (+IgG) (n = 4-5). (J) Total IgG levels in naive murine plasma and PLTR were examined by ELISA (n = 8). (K) Plasma/PLTR ratio of total IgG (n = 8). (L) MDCK cells were infected with IAV (2 hours) and incubated with buffer (control) or platelets from anti-IAV IgG seronegative (neg.) or seropositive (pos.) mice for 30 minutes. Viral load was quantified 72 hours after infection via qPCR by determining relative IAV hemagglutinin (HA) expression (n = 24-26). (M) As in panel L, but seropositive platelets were optionally treated for 20 minutes with Tica, ASA, Comp, or P-sel before incubation with IAV-infected MDCK cells (n = 12-26). (N) Platelets of Tica- or control-treated mice were stimulated for 15 minutes with IAV or convulxin (CVX), and released IgG was quantified by ELISA (n = 5). (O) As in panel L, but IAV-infected MDCK cells were incubated with buffer (control) or PLTR of seronegative (neg.) or seropositive (pos.) donor mice (n = 15-17). *P < .05, **P < .01, ***P < .001, ****P < .0001 between indicated groups; #P < .05, ##P < .01, ###P < .001, ####P < .0001 vs control. HPRT, hypoxanthine guanine phosphoribosyl transferase; ns, not significant; PBS, phosphate-buffered saline; -, no additional treatment.