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Contribution of neutrophil elastase to the lysis of obliterative thrombi in the context of their platelet and fibrin content.

Rábai G, Szilágyi N, Sótonyi P, Kovalszky I, Szabó L, Machovich R, Kolev K - Thromb. Res. (2010)

Bottom Line: Leukocytes invade newly formed thrombi through interactions with platelets and fibrin and later contribute to the removal of fibrin deposits mainly through the action of neutrophil elastase.The digitalized fluorescent microscopic images were decomposed according to the color channel of each thrombus constituent.Association between NE-FDP and leukocyte content of thrombi is evidenced by a significant Pearson correlation coefficient of 0.71 (p=0.00002).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

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Fibrin, NE-digested fibrin and leukocyte content of arterial thrombi. Sections of surgically removed human thrombi were double-immunostained for fibrin (green) and NE-digested fibrin (red) as well as with a DNA-dye, DAPI (blue) as described in Methods. Images were taken at original magnification of × 20 (A) and × 60 (B). The pixel values of the three color channels (C: red, NE-FDP; D: green, fibrin; E: blue, nuclei) of an image taken as illustrated in panels A and B were decomposed according to the space coordinates of the thrombus section and their intensity is presented in a color-coded scale.
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fig1: Fibrin, NE-digested fibrin and leukocyte content of arterial thrombi. Sections of surgically removed human thrombi were double-immunostained for fibrin (green) and NE-digested fibrin (red) as well as with a DNA-dye, DAPI (blue) as described in Methods. Images were taken at original magnification of × 20 (A) and × 60 (B). The pixel values of the three color channels (C: red, NE-FDP; D: green, fibrin; E: blue, nuclei) of an image taken as illustrated in panels A and B were decomposed according to the space coordinates of the thrombus section and their intensity is presented in a color-coded scale.

Mentions: After surgery, the removed thrombus samples were frozen immediately at − 70 °C and stored until examination. Cryosections (6 μm thickness) of thrombi were attached to silane-coated slides. Sections were fixed in acetone at 4 °C for 10 min and air-dried for 5 min at room temperature. After further fixation in 4 w/v% paraformaldehyde (pH 7.0) at 4 °C for 10 min, the sections were washed in 10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl (TBS) for 5 min, followed by incubation in 100 mM Na-phosphate 100 mM NaCl pH 7.5 buffer (PBS) containing 5 w/v% bovine serum albumin to eliminate nonspecific binding of antibodies. For double immunostaining, sections were first incubated overnight at 4 °C in 2 μg/mL mouse anti-human NE-digested FDP monoclonal antibody IF-123 (Mitsubishi Kagaku Iatron, Tokyo, Japan) [16] in TBS. Following washing with PBS, sections were treated with Alexa Fluor 555 (excitation 555 nm, emission 565 nm) carboxylic acid, succinimidyl ester conjugated donkey anti-mouse immunoglobulin antibody (Invitrogen, Oregon, USA) at 1:100 dilution to detect the IF-123. Subsequently slides were washed in PBS 3 times and blocked with 5 w/v% bovine serum albumin in PBS, then incubated with 2 μg/mL mouse anti-human fibrin monoclonal antibody ADI-350 (American Diagnostica, Pfungstadt, Germany) or mouse anti-human CD41 (platelet GPIIb/IIIa) antibody (Biodesign International, Saco, ME) for 30 min at 37 °C followed by detection of the second immune reaction with Alexa Fluor 488 (excitation 494 nm, emission 519 nm) 5-carboxamido-(propargyl), bis(triethylammonium salt)) *5-isomer* conjugated goat anti-mouse antibody (Invitrogen, Oregon, USA) at 1:100 dilution. After repeated washing with PBS, sections were mounted with 4’,6-diamidino-2-phenylindole (DAPI), which recognizes DNA, and images were taken with Eclipse E600 fluorescent microscope (Nikon, Japan) (Fig. 1).


Contribution of neutrophil elastase to the lysis of obliterative thrombi in the context of their platelet and fibrin content.

Rábai G, Szilágyi N, Sótonyi P, Kovalszky I, Szabó L, Machovich R, Kolev K - Thromb. Res. (2010)

Fibrin, NE-digested fibrin and leukocyte content of arterial thrombi. Sections of surgically removed human thrombi were double-immunostained for fibrin (green) and NE-digested fibrin (red) as well as with a DNA-dye, DAPI (blue) as described in Methods. Images were taken at original magnification of × 20 (A) and × 60 (B). The pixel values of the three color channels (C: red, NE-FDP; D: green, fibrin; E: blue, nuclei) of an image taken as illustrated in panels A and B were decomposed according to the space coordinates of the thrombus section and their intensity is presented in a color-coded scale.
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Related In: Results  -  Collection

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fig1: Fibrin, NE-digested fibrin and leukocyte content of arterial thrombi. Sections of surgically removed human thrombi were double-immunostained for fibrin (green) and NE-digested fibrin (red) as well as with a DNA-dye, DAPI (blue) as described in Methods. Images were taken at original magnification of × 20 (A) and × 60 (B). The pixel values of the three color channels (C: red, NE-FDP; D: green, fibrin; E: blue, nuclei) of an image taken as illustrated in panels A and B were decomposed according to the space coordinates of the thrombus section and their intensity is presented in a color-coded scale.
Mentions: After surgery, the removed thrombus samples were frozen immediately at − 70 °C and stored until examination. Cryosections (6 μm thickness) of thrombi were attached to silane-coated slides. Sections were fixed in acetone at 4 °C for 10 min and air-dried for 5 min at room temperature. After further fixation in 4 w/v% paraformaldehyde (pH 7.0) at 4 °C for 10 min, the sections were washed in 10 mM Tris-HCl, pH 7.4, containing 150 mM NaCl (TBS) for 5 min, followed by incubation in 100 mM Na-phosphate 100 mM NaCl pH 7.5 buffer (PBS) containing 5 w/v% bovine serum albumin to eliminate nonspecific binding of antibodies. For double immunostaining, sections were first incubated overnight at 4 °C in 2 μg/mL mouse anti-human NE-digested FDP monoclonal antibody IF-123 (Mitsubishi Kagaku Iatron, Tokyo, Japan) [16] in TBS. Following washing with PBS, sections were treated with Alexa Fluor 555 (excitation 555 nm, emission 565 nm) carboxylic acid, succinimidyl ester conjugated donkey anti-mouse immunoglobulin antibody (Invitrogen, Oregon, USA) at 1:100 dilution to detect the IF-123. Subsequently slides were washed in PBS 3 times and blocked with 5 w/v% bovine serum albumin in PBS, then incubated with 2 μg/mL mouse anti-human fibrin monoclonal antibody ADI-350 (American Diagnostica, Pfungstadt, Germany) or mouse anti-human CD41 (platelet GPIIb/IIIa) antibody (Biodesign International, Saco, ME) for 30 min at 37 °C followed by detection of the second immune reaction with Alexa Fluor 488 (excitation 494 nm, emission 519 nm) 5-carboxamido-(propargyl), bis(triethylammonium salt)) *5-isomer* conjugated goat anti-mouse antibody (Invitrogen, Oregon, USA) at 1:100 dilution. After repeated washing with PBS, sections were mounted with 4’,6-diamidino-2-phenylindole (DAPI), which recognizes DNA, and images were taken with Eclipse E600 fluorescent microscope (Nikon, Japan) (Fig. 1).

Bottom Line: Leukocytes invade newly formed thrombi through interactions with platelets and fibrin and later contribute to the removal of fibrin deposits mainly through the action of neutrophil elastase.The digitalized fluorescent microscopic images were decomposed according to the color channel of each thrombus constituent.Association between NE-FDP and leukocyte content of thrombi is evidenced by a significant Pearson correlation coefficient of 0.71 (p=0.00002).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.

Show MeSH
Related in: MedlinePlus