Table 1.
List of antibodies used for immunohistochemistry.
Fig 1.
Characteristics of human arterial thrombi.
(A) Pie chart shows the distribution of thrombus age. 50 out of 81 patients described the precise onset of AMI symptoms, which allowed the calculation of thrombus age following its removal during PCI. The majority of human thrombi (with precise onset of symptoms) was younger than 24h. (B) Leukocyte accumulation in human thrombi. Representative images of HE staining (n = 3). Bars, 200μm (top image) and 50μm (bottom image). (C) Immunohistochemical visualization of leukocytes (CD45, green, n = 3), neutrophils (NE, red, n = 81) and monocytes (CD14, green, n = 11). Nuclei are counterstained with Hoechst (including controls). Control (isotype) or secondary antibody alone. Bars, 10μm. (D) The graph shows the quantification of monocytes (n = 11) and neutrophils (n = 81) in human thrombi. Results are shown as mean ± SD. (E) Correlation between human thrombi younger than 12h and the number of leukocytes (n = 33).
Table 2.
Patients’ baseline characteristics.
Fig 2.
Characteristics of mouse arterial thrombi induced by FeCl3 injury or wire denudation in mice.
(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.
Fig 3.
Accumulation of fibrinogen/fibrin in human and mouse arterial thrombi.
(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.
Fig 4.
NETs in arterial thrombi of mice and humans.
(A) Representative illustration of NETs stained for NE and DNA (DAPI) in the early phase of arterial thrombosis. Human and mouse thrombi showed comparable morphology after 3, 6 or 12h. Extracellular DNA originates from NE+ neutrophils. Bars, 10μm. Arrows, nuclei; arrowheads, NET fibers. (B) Quantification of NETs per 100 neutrophils in human thrombi (<12h) (n = 10) and experimental thrombosis (FeCl3) (3–6h) (n = 5). Dots represent individual experiments; lines indicate mean values for each group. (C) Association between thrombus age and number of NETs in mice and humans.
Fig 5.
Cl-amidine inhibits arterial thrombosis in mice.
(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.
Fig 6.
Cl-amidine modulates thrombus composition.
(A) Immunofluorescence analysis of fibrinogen (1st row), tissue factor (2nd row) and factor XII (3rd row) in arterial thrombi of mice treated with vehicle or Cl-amidine. Nuclei were counterstained with Hoechst. Controls were stained with isotype and secondary antibody antibody together, and Hoechst, Bars: 50μm. (B) Immunofluorescence staining of coagulation factors in % of whole thrombus area. Left: Fibrinogen-covered thrombus area (vehicle n = 5, Cl-amidine n = 5). Middle: Tissue factor (vehicle n = 5, Cl-amidine n = 4). Right: Factor XII (vehicle n = 6, Cl-amidine n = 5). Results are mean ± SD.
Fig 7.
Cl-amidine reduces myocardial ischemia-reperfusion injury.
(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.