Fig 2: Platelet activation in patients affected by COVID-19 according to the evolution of the disease. (A) Comparison of geometric mean expression of CD41 (P-selectin-PE). (B) Comparison of geometric mean expression of PAC-1 (aIIbßIII-FITC). Platelets from patients in the severe group showed higher expression of activation markers. (ANOVA, post Hoc Tukey **p < .01, *** p < .001, **** p < .0001).

Fig 3: Monocyte-platelet adhesion synapse formation drives antigen-specific immunity.(A) Top: Confocal imaging of purified human monocytes cultured with platelets overnight. Activated CD61+ (green) platelets are shown forming CD62p+ (yellow) and PSGL1+-mediated junctions (blue) with CD14+ (red) monocytes. Bottom: 3D reconstruction demonstrates punctate monocyte PSGL1 clustering around platelet CD62p. Scale bars, 5.30 µm. (B) Schematic of human experimental setup with soluble antigen (Ag). Platelet-containing (PBMC+pl+) and platelet-depleted (PBMC+pl-) PBMCs were prepared from healthy human donors. PBMC groups were incubated overnight with soluble tumor antigens, melanoma-associated antigen recognized by T cells long peptide (MART1 LP) or E7 LP, followed by washing and 3-day coculture with antigen-specific transgenic human T cells, DMF5 or E7, respectively. Supernatants were analyzed at end of culture (day 4). (C to E) Human IFNg (hIFNg) ELISA of (C) DMF5 cultured with PBMC+pl+ or PBMC+pl- pulsed with MART1 LP at varying concentrations, (D) DMF5 cultured with MART1 LP–pulsed PBMC (left) or monocytes (right) with or without platelets or recombinant human P-selectin stimulation, and (E) E7 T cells cultured with E7 LP–pulsed PBMC+pl+ or PBMC+pl- or E7 T cells only (PBMC-). (F) FACS quantitation of CD86, HLA-DR, HLA-A,B,C, and CD14 in donor PBMCs at throughout incubation (n = 5 to 7). All values are means ± SD of at least three independent experiments. (C to E) One-way ANOVA and (F) two-way ANOVA, ****P < 0.0001, **P < 0.01, *P < 0.05. (F) Each point represents data from an individual healthy blood donor.

Fig 4: Platelets interact with peripheral blood monocytes to activate antigen cross-presentation.(A and B) Platelet-containing (mono+pl+) and platelet-depleted (mono+pl-) monocytes purified from mouse PBMCs were incubated overnight with sOVA, followed by 3-day coculture with CFSE-stained OT1 T cells. (A) Flow cytometry analysis of mono+pl+, mono+pl-, and OT1 only (mono-) cultures in triggering OT1 proliferation. Shown are (left) representative CFSE dilution FACS plots and histogram and (right) quantified proliferation index of dividing OT1 cells. (B) ELISA coculture supernatant quantifications for (left) IL-2 and (right) IFNg. (C to F) Flow cytometry analysis of DC surface marker evolution in PBMCs. Representative FACS plots of (C) (left) CD11b+ cells expressing MHC I–loaded sOVA antigen, SIINFEKL-H2Kb, and (right) quantification (D) (left) SIINFEKL-H2Kb+ CD11b+ cells (orange) overlayed on CD11b and CD11c gated PBMCs (purple), and (right) quantification. (E and F) MFI of (E) (left) CD80 and (right) CD86 expression and (F) MHC II expression on CD11b+ CD11c+ cells in PBMC+pl+ (black) and PBMC+pl- (red). (G) Representative Amnis images of CD11b+ (purple) Ly6G- (blue) monocytes interacting with CD62p-expressing (yellow), CD41+ (red) platelets, quantitated at days 0 and 1 in table above. All values are means ± SD of at least three independent experiments. (A and B) One-way ANOVA and (C to F) two-way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01.

Fig 5: P-selectin on platelets triggers enhanced cross-presentation via monocyte PSGL-1.(A) Live cell confocal microscopy of platelets interacting with monocytes in overnight cultures of murine PBMC+pl+. Top: CD41+ (green) platelets expressing punctate CD62p (yellow) in association with CD11b+ (red) Ly6G- (blue) monocyte. Bottom: 3D reconstruction highlights CD62p at platelet-monocyte interface. Scale bars, 6 µm. (B to E) OT1 T cell stimulation capacity of sOVA-pulsed PBMC+pl+ or PBMC+pl- cultures treated with (B and C) varying concentrations of recombinant P-selectin (P-selectin) or (D and E) agonist anti-PSGL1 monoclonal antibody (mAb) (a-PSGL1). (B and D) Representative proliferation histograms of CFSE-stained OT1 T cells. (C and E) ELISA of (left) IL-2 and (right) IFNg. (F) Antigen-specific T cell stimulation capacity of sOVA-pulsed purified mono+pl+ or mono+pl- cultures treated with P-selectin or a-PSGL1. Representative proliferation histograms of CFSE-stained OT1 T cells are shown. All values are means ± SD of at least five independent experiments. One-way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.