Activation of neutrophils and PNC formation is Sema7A dependent. Murine blood was collected from WT and Sema7A^−/− mice after LPS inhalation and analyzed by flow cytometry. (A) Representative color dot blots of PNCs (Ly6G⁺/CD42b⁺ events) in WT and Sema7A^−/− blood from NaCl (control) or LPS–inhaled mice. (B) PNC formation, platelet effector glycoprotein 2b/3a (GP2b/3a) expression (antibody clone JON/A MFI), PMN activity marker CD11b (MFI) expression, and platelet activity marker CD42b (MFI) expression were assessed by flow cytometry in the mice described in panel A. (C) Representative dot blots of PNCs (Ly6G⁺/CD42b⁺ events) in the blood of WT and Sema7A^−/− mice treated with recombinant Sema7A (recSema7A) after NaCl (control) or LPS inhalation. (D) PNC formation, platelet effector GPIIb/IIIa expression (antibody clone JON/A MFI), PMN activity marker CD11b (MFI) expression, and platelet activity marker CD42b (MFI) expression were assessed by flow cytometry in the mice described in panel C. (E) Representative dot blots of PNCs (Ly6G⁺/CD42b⁺ events) in the blood of WT mice that were untreated or treated with IgG or the Sema7A-blocking antibody (anti-Sema7A) after LPS inhalation compared with mice without conditioning (Sham). (F) PNC formation, platelet effector GPIIb/IIIa expression (antibody clone JON/A MFI), PMN activity marker CD11b (MFI) expression, and platelet activity marker CD42b (MFI) expression were assessed by flow cytometry in the mice described in panel E. Group comparisons were performed by unpaired 2-tailed Student t tests (the data are the mean ± SD). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 as indicated. IgG, immunoglobulin G; MFI, Mean Fluorescence Intensity.