GPVI critically contributes to local platelet–neutrophil interactions in response to LPS. Immunofluorescence staining of cryosections of the lungs of control or JAQ1-injected mice, 4 hours after NaCl or LPS treatment. Images were acquired using a Thunder Imager DMi8 (Leica Microsystems) equipped with a 20× objective (HC PL APO 20×/0.80 DRY, Leica Microsystems) and a Leica-DFC9000GT-VSC12293 camera (Leica Microsystems). (A) Representative confocal micrographs of neutrophils (Ly6G⁺, yellow), platelets (GPIX⁺, cyan), vessels (CD31⁺, magenta), and DAPI (nucleus, blue), exemplifying neutrophils directly interacting with platelets to form PNCs. The dashed line marks the region of the magnified excerpt on the right without the vessel channel. Detailed representative images were acquired with a TCS-SP8 confocal laser scanning microscope (Leica Microsystems) with a 63× objective (HC PL APO CS2 63×/1.40 OIL, Leica Microsystems). Scale bar, 10 μm. (B) Quantification of PNC numbers by manual counting. Each data point represents the average number of PNCs counted in 30 FOVs for 1 mouse. (C) Fraction of the total neutrophil number quantified in Figure 3C that was associated with platelets. Each data point represents mean value for 1 mouse. (D) Fraction of the total platelet number quantified in Figure 3D that was associated with neutrophils. Each data point represents mean value for 1 mouse. (E) Quantification of the number of platelets that associated with a neutrophil in 1 PNC. Each data point represents the average number of platelets that associated with a neutrophil in 1 PNC in 30 FOVs for 1 mouse. Bars represent mean per group ± standard deviation. The two-way ANOVA with a Šidák multiple comparisons test. ∗∗P < .01; ∗∗∗∗P < .0001.