NETs provide a scaffold for platelet adhesion and aggregation. (A) Platelets (green) bound to NETs (blue, arrows). Neutrophils (blue, arrowheads) were out of focus and did not bind platelets. (Scale bar, 20 μm.) (B) Electron micrograph of platelets (Pts) attached to a fibrous meshwork of NETs. (Scale bar, 1 μm.) (C) Numerous filopods indicated that platelets (Pt) on NETs were activated. (Scale bar, 0.5 μm.) (D–K) Time-course of platelet adhesion and aggregation on NETs. (Scale bars, 100 μm.) NETs (D and E, green) were perfused with platelets (I and J, red) in whole blood. The flow direction was from left to right. Images showed NETs and platelets after 1 min (arrows in D and I) and 10 min (E, J) of perfusion. DNase added to blood after 10 min digested NETs (F) and removed platelets (K), indicating that platelets were attached to NETs. Quantification of NETs (G) or platelets (H) in the presence (open circles) or absence of DNase (closed circles). DNase was added to untreated samples after 10 min (arrow). DNase removes NETs and inhibits platelet aggregation. A.U., arbitrary units. Data presented are representative of at least three independent experiments and are shown as mean ± SEM, n = 3.

Heparin dismantles NETs and prevents histone induced platelet aggregation. SytoxGreen staining of NETs perfused for 10 min with blood in the absence (A) or presence (B) of heparin. (Scale bars, 100 μm.) (C) Quantification of NETs after 10 min perfusion with normal (-) or heparinized (+) blood. (D) Quantification of platelets on NETs perfused for 10 min with blood before (-) and after (+) treatment with heparin. Data presented as mean ± SEM, n = 3; (Student's t test; *P < 0.05; **P < 0.01). (E) Heparin and DNase released histones from NETs. Immunodetection of histone H2B (arrow) in the culture supernatants of NETs treated with heparin or DNase (DN). A second band (arrowhead) may represent cross reactivity of the antibody or a proteolytic product. Data presented are representative of three independent experiments. (F) Aggregometry of platelets stimulated with thrombin (open circles) or human recombinant histone H3 (solid circles). EDTA (solid squares) and heparin (solid triangles) inhibited platelet aggregation by histone H3. (G) Extent of platelet aggregation 3 min after addition of histones ¹H, H2A, H2B, H3, or H4, or thrombin (Thr). Histones H3 and H4, and thrombin induced aggregation of platelets obtained from four different donors. (ANOVA; ***P < 0.001 compared with histone ¹H).

NETs provide a scaffold for RBC-rich thrombi. (A) Flow chamber coated with NETs after perfusion with blood. Light microscopy of a red thrombus (arrow) anchored on two strings (arrowheads). Figure is a composite of multiple photographs of the flow chamber. (Scale bar, 500 μm.) (B) DNA staining of thrombus (rectangle in A). Strings of DNA are seen in the thrombus (arrows). (Scale bar, 250 μm.) (C) Electron microscopy shows individual RBCs attached to NETs. (Scale bar, 5 μm.) Quantification of RBCs (D) or platelets (E) on collagen or NETs. Coated chambers were perfused with blood supplemented with DNase or not (-). (D) RBCs were detected in NETs- but not in collagen-coated flow chambers. Adhesion of RBCs to NETs could be prevented if blood was supplemented with DNase. (E) Addition of DNase had no effect on platelet adhesion to collagen but blocked platelet adhesion to NETs. A.U. arbitrary units; n.s. not significant. Data presented are representative of at least three independent experiments and presented as mean ± SEM, n = 3; (ANOVA; **P < 0.01).

Markers of NETs are abundant in baboon DVT. (A) Plasma DNA levels before (baseline: BL) or at indicated time points after initiation of DVT. Plasma DNA was elevated after 48 h (bar represents the mean value of groups; n = 3; Repeated measures ANOVA; **P < 0.01 compared with BL) and remained increased in baboon #1 at day 6. (B) Cross-section of thrombotic left iliac vein at day 6 stained for DNA. Figure is a composite of multiple micrographs of the thrombus. (Scale bar, = 500 μm.) (C–F) Immunostaining of the DNA core (arrow in B). (C) Staining for DNA shows a distinct dotted pattern indicating nuclei, as well as a diffuse pattern indicating extracellular DNA. (D) Positive staining for the DNA/H2A/H2B complex reveals that the extracellular DNA is of nuclear origin. (E) VWF strings are part of the DNA core. (F) Overlay of C, D, and E. (Scale bars C–F, 100 μm.) (G, H, and I) Immunostaining of area between DNA core and vessel wall (arrowhead in B). (G) DNA by SytoxGreen indicates nuclei (arrowhead) as well as extracellular DNA (arrow). (H) VWF in this area often colocalizes with extracellular DNA (arrow). (I) Overlay of G and H. (Scale bars G–I, 50 μm.) (J) Nuclei stained by SytoxGreen in an area free of extracellular DNA (arrowhead in B). (K) Extracellular histone H3 is abundantly present in this area although DNA is no longer detected. (L) Overlay of J and K. (Scale bars J–L, 20 μm.)