Fig 2: Schematic outline of platelet-mediated inflammatory monocyte activation by SARS-CoV-2 spike protein.SARS-CoV-2 spike protein interacts with CD42b to activate platelets, which express CD40L via PI3K/PKC signaling and P selectin via an unknown signaling pathway. The activated platelets induce inflammatory monocyte differentiation through the engagement of P selectin/PGSL-1 and CD40L/CD40.

Fig 3: SARS-CoV-2–induced platelet-monocyte aggregation and P selectin/CD40L expression on platelets by spike protein.(A) PKH26- or CFSE-labeled platelets from healthy donors (n = 5) were mixed at 1:1 and incubated with increasing concentrations of spike protein or S-pseudovirus, and aggregates were detected by flow cytometry. Double-colored events indicated the platelet aggregation, and TRAP was used as the positive control. (B) Peripheral blood from healthy donors (n = 5) was stimulated by spike protein or S-pseudovirus at indicated concentration and analyzed by flow cytometry. Platelet-monocyte aggregation was evaluated using the percentage of CD42b+/CD14+ cells. (C) Platelet-monocyte aggregation was visualized by fluorescence microscopy; scale bar: 20 µm. (D) Purified platelets from healthy donors (n = 5) were incubated with spike protein or S-pseudovirus. P selectin, CD40L expression, and fibrinogen binding were shown by histogram and MFI. (E) Purified platelets (n = 4) were pretreated with isotype antibody (indicated as control) or anti-spike RBD before incubation with S-pseudovirus; P selectin and CD40L expression are shown. Mean with SD and P value by paired Student’s t test are displayed. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: SARS-COV-2–activated platelets mediated inflammatory monocyte activation through the engagement of P selectin/PGSL-1 and CD40L/CD40.(A) Peripheral blood from healthy donors (n = 3) was stimulated by spike protein or S-pseudovirus in the presence of anti–P selectin or anti–PSGL-1. Platelet-monocyte aggregation was evaluated using the percentage of CD42b+/CD14+ cells. (B) Anti–P selectin or anti-CD40L was added into monocyte-platelet cocultures with spike protein or S-pseudovirus. IL-1ß expression in monocytes (n = 4) was measured. (C) Purified monocytes (n = 3) were cultured with/without recombinant P selectin and/or CD40L, and their expression of IL-1ß and IL-10 is shown. Mean with SD and P value by paired Student’s t test are displayed. *P < 0.05.

Fig 5: SARS-CoV-2–activated platelets induced monocyte differentiation toward a proinflammatory phenotype.Purified monocytes from healthy donors were cocultured with purified autologous platelets in the presence of spike protein or S-pseudovirus, and then washed to remove nonadherent surplus platelets. (A) After another 48 hours in culture, the cell surface expression of CD80, CD86, CD163, CD206, and HLA-DR on monocytes was analyzed by flow cytometry (n = 3). (B–D) The expression of IL-1ß and IL-10 in monocytes was examined by intracellular cytokine staining after 12 hours in culture, and CD42b indicated platelet-monocyte adherence in cocultures (n = 4). P value by paired Student’s t test is displayed. *P < 0.05; **P < 0.01.