Cooperative PSGL-1 and CXCR2 signaling in neutrophils enhances neutrophil recruitment and NET formation in deep vein thrombi. (A) Number of Ly6G⁺ neutrophils per thrombus 24 hours after ligation in the indicated genotype, as measured by flow cytometry. (B) Normalized thrombus weight per Ly6G⁺ neutrophil. (C) Normalized fibrin level per Ly6G⁺ neutrophil. The data in panels A through C represent the mean ± SEM from 5 to 10 mice in each group. (D) Western blot of thrombus lysates probed with antibodies to fibrin, Ly6G, and citrullinated histone. The Aα, Bβ, and γ chains of fibrin are marked. The data are representative of 3 experiments. (E) Normalized densitometric ratio of fibrin Aα chain to Ly6G. (F) Normalized densitometric ratio of citrullinated histone to Ly6G. The data in panels E and F represent the mean ± SD from 3 mice in each experimental group. (G) Representative fluorescent images of isolated WT bone marrow neutrophils incubated with phorbol myristate acetate (PMA), stained with anti-citrullinated histone IgG followed by Alexa 488–conjugated anti-rabbit IgG (left), with Sytox Orange to label DNA (middle), or merged image (right). The white arrow marks extracellular staining for both citrullinated histones and DNA, indicating NET release. Scale bar, 10 μm. (H-I) Percentage of NET-forming neutrophils treated with the indicated agonist, calculated by dividing the number of cells with both extracellular citrullinated histones and Sytox Orange–positive DNA by the total number of Sytox Orange–positive cells. The data in panel H are from WT mice. The data in panel I are from mice of the indicated genotype. The data in panels H and I represent the mean ± standard deviation (SD) from 3 mice in each experimental group. *P < .05.