Effect of platelet GPIIb on melanoma metastasis formation.

<p><b>a)-c)</b> B16-D5 melanoma cells were injected intravenously into GPIIb^+/+ or GPIIb^-/- littermate mice. After 10 days, metastasis formation was analyzed. <b>a)</b> Photomicrographs show representative lung images. Bars, 0.5 cm. <b>b)</b> Quantification of metastatic nodules in the lung (n = 5; *P<0.05). Boxes indicate median, whiskers indicate min and max values. <b>c)</b> Quantification of Pmel mRNA expression. Individual Pmel data normalized to beta-actin is indicated as 2^(-ΔCt) (n = 5; *P<0.05). Boxes indicate median, whiskers indicate min and max values. <b>d-e)</b> Pulmonary melanoma proliferation was determined by analyzing Ki67 expression in HMB-45-positive melanoma cells 10 days after intravenous seeding (n = 5; *P<0.05). Photomicrographs show fluorescence microscopy images of mouse lung tissue stained with antibodies directed against melanoma HMB-45 (green) and Ki67 (red); nuclei were stained with DAPI (blue). Images show 3μm optical sections. Left image, overview of lung tissue section; right image, magnification. Bars, 100μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x and 40x objective. <b>f)</b> Effect of platelets on M21 mitogenesis <i>in vitro</i>. Serum-starved M21 cells were grown in the absence or presence of increasing concentrations of washed human platelet and BrdU uptake was quantified (*P<0.05, **P<0.01). <b>g-h)</b> Effect of platelet GPIIb on subcutaneous melanoma growth. <b>g)</b> B16-D5 cells were injected subcutaneously in the right flank of wildtype and GPIIb^-/- littermate mice and tumor growth surveyed using a digital scaliper. <b>h)</b> 21 days after tumor cell seeding tissues were explanted. Tumor weight was measured indicating no difference in subcutaneous tumor growth (P = n.s.).</p

Platelet-tumor-aggregate formation <i>in vivo</i>.

<p><b>a)</b> GFP-transfected B16-D5 melanoma cells were injected intravenously into wildtype mice. After 1 hour, lung tissue was obtained for immunofluorescence analysis. Photomicrographs show mouse lung tissue stained with antibodies directed against platelet GPIIb (CD41, red) and B16-D5 (GFP, green); nuclei were stained with DAPI (blue). Bars, 40μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x objective. <b>b-d)</b> Arrest of DCF-tagged B16-D5 melanoma cells was visualized in the pulmonary vasculature by intravital confocal videofluorescence microscopy (IVM) in GPIIb^+/+ and GPIIb^-/- littermate mice immediately and after 1 hour. <b>b)</b> Number of metastatic events was quantified. Results are given as percentage of firmly adherent B16-D5 in GPIIb^-/- mice compared to its WT littermate (n = 5–6 experiments per group; *<i>P</i><0.01 acute; **<i>P</i><0.001 after 1 hour). <b>c)</b> Size of B16-aggregates was quantified. Results are given as percentage of B16-aggregate size in GPIIb^-/- mice compared to its WT littermate (n = 5–6; *<i>P</i><0.05 after 1 hour). <b>d)</b> Photomicrographs show representative IVM images obtained in GPIIb^+/+ and GPIIb^-/-. In GPIIb^+/+ mice, arrest of large multicellular aggregates is frequently observed (left). Arrest of DCF-tagged B16-D5 is visualized in precapillary vessels (right). Bars, 20μm. <b>d e-f)</b> GPIIb^+/+ (WT) or GPIIb^-/- platelets were injected into GPIIb^-/- mice just prior to administration of DCF-labeled B16-D5. IVM was performed immediately and after 1 hour. <b>e)</b> Number of metastatic events was quantified. Results are given as percentage of firmly adherent B16-D5 in GPIIb^-/- littermates receiving GPIIb^-/- platelets (n = 4; *<i>P</i><0.05). <b>f)</b> Size of B16-aggregates was quantified. Results are given as percentage of B16-aggregate size in GPIIb^-/- littermates receiving GPIIb^-/- platelets (n = 3; P = n.s.).</p

Platelet-tumor-endothelium interaction under flow.

<p><b>a)</b> Photomicrographs show platelet and M21 aggregate formation under flow (1000/s). Co-perfusion of platelets and M21 cells induced formation of aggregates which did not firmly adhere and are therefore only partly focused (middle picture; arrows indicate platelet-M21 aggregates). Aggregation is virtually abolished in the absence of platelets (left picture) or the presence of c7E3 Fab (right picture). Bars 0.2mm. Images were taken using a Zeiss Axiovert 100 microscope, 20x objective. <b>b)</b> HUVEC (resting or TNF-α/IFN-γ-stimulated) were perfused with M21 or αv-deficient M21L melanoma cells, that lack the expression of both αvβ3 and αvβ5 integrins, in the absence and presence of platelet-rich plasma (10min, shear rate 1000/s). Results are shown as number of firmly adherent M21 and M21L (n = 5; *<i>P</i><0.05 for M21 vs. M21+PRP on activated HUVEC). <b>c)</b> Frequency of M21 and M21L aggregates in the absence and presence of platelets and c7E3 antibody. Results are given as number of melanoma cell-aggregates in % of all perfused tumor cells (n = 5; *<i>P</i><0.001 for M21 in the absence vs. presence of platelets). <b>d)</b> Size distribution of M21 aggregates in the absence and presence of platelets and c7E3. <b>e-g)</b> M21 melanoma cells were incubated for 15 minutes in the absence and presence of platelet-rich plasma. <b>e)</b> Scanning electron microscopy indicates single melanoma cells in the absence (left picture) and melanoma cell aggregates in the presence of platelets (right picture). Bars, 5μm. <b>f)</b> Transmission electron microscopy indicates direct platelet-melanoma interaction. Bar, 1μm. <b>g)</b> Photomicrographs show representative fluorescence microscopy images of M21-platelet aggregates stained with phalloidin (red) and anti-GPIIb (CD41) mAb (green). Bars, 20μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x objective. <b>h-i)</b> Analysis of mouse GPIIb^-/- or WT platelet interaction with B16 melanoma under flow conditions (1000/s). <b>h)</b> Percentage of platelet-positive B16 cells (n = 4; *<i>P</i><0.05). <b>i)</b> Representative FACS contour plots indicate CD42-staining on B16 cells in the absence (control) and presence of GPIIb^+/+ or GPIIb^-/- platelets.</p

Histological comparison of arterial thrombi in mice and men and the influence of Cl-amidine on thrombus formation

<div><p>Aims</p><p>Medical treatment of arterial thrombosis is mainly directed against platelets and coagulation factors, and can lead to bleeding complications. Novel antithrombotic therapies targeting immune cells and neutrophil extracellular traps (NETs) are currently being investigated in animals. We addressed whether immune cell composition of arterial thrombi induced in mouse models of thrombosis resemble those of human patients with acute myocardial infarction (AMI).</p><p>Methods and results</p><p>In a prospective cohort study of patients suffering from AMI, 81 human arterial thrombi were harvested during percutaneous coronary intervention and subjected to detailed histological analysis. In mice, arterial thrombi were induced using two distinct experimental models, ferric chloride (FeCl₃) and wire injury of the carotid artery. We found that murine arterial thrombi induced by FeCl₃ were highly concordant with human coronary thrombi regarding their immune cell composition, with neutrophils being the most abundant cell type, as well as the presence of NETs and coagulation factors. Pharmacological treatment of mice with the protein arginine deiminase (PAD)-inhibitor Cl-amidine abrogated NET formation, reduced arterial thrombosis and limited injury in a model of myocardial infarction.</p><p>Conclusions</p><p>Neutrophils are a hallmark of arterial thrombi in patients suffering from acute myocardial infarction and in mouse models of arterial thrombosis. Inhibition of PAD could represent an interesting strategy for the treatment of arterial thrombosis to reduce neutrophil-associated tissue damage and improve functional outcome.</p></div

Accumulation of fibrinogen/fibrin in human and mouse arterial thrombi.

<p>(A) Representative immunohistochemical staining of mouse and human thrombi for fibrinogen/fibrin (red) and control stainings. Nuclei were counterstained with Hoechst (including controls). Bars: 50μm (top left and right), 200μm (bottom left and right), 300μm (top and bottom middle). (B) Fibrinogen/fibrin-covered area in the thrombus (human thrombi n = 6, mouse thrombi n = 3). Data are shown as mean ± SD.</p

Characteristics of mouse arterial thrombi induced by FeCl₃ injury or wire denudation in mice.

<p>(A) Immunohistological images of platelet aggregate area (red) in arterial thrombi (n = 3/group) and control stainings. Bars, 100μm. Control (isotype) or secondary antibody alone. (B) Comparison of leukocyte recruitment to the mouse carotid artery 3h after FeCl₃ exposure or wire denudation (n = 3/group). Representative images show immunohistochemical staining for leukocytes (CD45, green) and their subsets, as distinguished by expression of neutrophil elastase (NE, red) for neutrophils and CD68 (red) for blood monocytes. Nuclei were counterstained with Hoechst (including controls). Bars, 10μm. Control (isotype) or secondary antibody alone. (C) Association between number of leukocytes and thrombus age (n = 3/group). Mean ± SD. (D) Quantification of monocyte and neutrophil subsets within mouse thrombi 3h after FeCl₃ exposure (n = 3/group). Mean ± SD.</p

List of antibodies used for immunohistochemistry.

<p>List of antibodies used for immunohistochemistry.</p

Patients’ baseline characteristics.

<p>Patients’ baseline characteristics.</p

Cl-amidine inhibits arterial thrombosis in mice.

<p>(A) Representative intravital microscopy images 5, 10 and 20min after FeCl₃ injury in mice treated with Cl-amidine or vehicle. Platelets were labeled in vivo (green). Bars, 200μm. (B) Time until occlusion (left) and duration of vessel occlusion (right) after FeCl₃ exposure in mice treated with vehicle (n = 8) or Cl-amidine (n = 8). (C) Left: Representative histological images (Ly6G in red, cit H3 in green, DAPI in blue) of NETs in mice treated with vehicle or Cl-amidine (n = 5/group). Bars, 5μm. Arrowhead, NET fiber. Middle: Quantification of NETs per 100 neutrophils (n = 5/group). Right: Quantification of leukocytes (left axis) and neutrophils (right axis) in murine arterial thrombi.</p

Cl-amidine reduces myocardial ischemia-reperfusion injury.

<p>(A) Representative masson-trichrome stainings of myocardial sections from mice 7 days after myocardial ischemia-reperfusion injury treated with vehicle (left) or Cl-amidine (right). Mice treated with Cl-amidine show a decrease in fibrotic tissue compared to vehicle. Bars 2mm. (B) Infarct size 7 days after myocardial ischemia-reperfusion injury in mice treated with vehicle (n = 10) and Cl-amidine (n = 7). (C) Myocardial function was evaluated by measuring ejection fraction (in %) and (D) cardiac output (in μl/min) 7 days after myocardial injury in mice treated with vehicle (n = 5) and Cl-amidine (n = 6). Dots represent individual experiments, lines indicate mean values for each group.</p