Figure 1
Endocytic degradation of extravascular fibrin in vivo. (A) Fluorescent fibrin gels placed in subcutaneous space undergo degradation. Alexa Fluor 488–labeled fibrin gels were placed in the dermis of mice. The gels were extracted at the indicated time points, and gel weight (blue circles) and total fluorescence (red diamonds) were determined. Data are expressed as percent residual weight and residual fluorescence, as compared with control gels stored for the identical time period in the dark at 37°C in PBS with sodium azide. Triplicate readings for each sample were made and divided by the mean reading of the control gels to yield percent remaining fluorescence. (B) Fibrin implantation causes cell recruitment. Total cell number in control (left bar), sham-operated (middle bar), and fibrin-implanted (right bar) mice at 24 hours, as determined by the counting of nuclei in serial z-stacks from the implantation zone. Data are shown as mean ± standard deviation. N = 7 mice for control, 7 for sham operated, and 12 for fibrin-implanted mice. Significance was determined by 1-way ANOVA. (C-G) Fibrin implanted to subcutaneous space undergoes cellular endocytosis. (C-F) Representative images of mouse dermis 2, 6, 12, and 24 hours postinjection show progressive accumulation of cells that present with fluorescently labeled fibrin (green, white in top insets) in perinuclear vesicles sometimes colocalizing with the Alexa Fluor 647 dextran lysosomal marker (red, white in bottom insets). (G) High magnification of a single fibrin-internalizing cell showing the colocalization of fibrin (green, white in top inset) and dextran (red, white in bottom inset) in perinuclear vesicles (yellow), indicative of lysosomal routing of endocytosed fibrin. (H) Fibrin uptake (green) by cells 24 hours after fibrin implantation in Gt(ROSA)26Sortm4 (ACTB-tdTomato,-EGFP)Luo+/0 transgenic mice expressing plasma membrane–localized tomato fluorescent protein (red). Cross hairs mark a cell with multiple fibrin-containing vesicles completely circumscribed by plasma membrane. N.S., not significant.

Endocytic degradation of extravascular fibrin in vivo. (A) Fluorescent fibrin gels placed in subcutaneous space undergo degradation. Alexa Fluor 488–labeled fibrin gels were placed in the dermis of mice. The gels were extracted at the indicated time points, and gel weight (blue circles) and total fluorescence (red diamonds) were determined. Data are expressed as percent residual weight and residual fluorescence, as compared with control gels stored for the identical time period in the dark at 37°C in PBS with sodium azide. Triplicate readings for each sample were made and divided by the mean reading of the control gels to yield percent remaining fluorescence. (B) Fibrin implantation causes cell recruitment. Total cell number in control (left bar), sham-operated (middle bar), and fibrin-implanted (right bar) mice at 24 hours, as determined by the counting of nuclei in serial z-stacks from the implantation zone. Data are shown as mean ± standard deviation. N = 7 mice for control, 7 for sham operated, and 12 for fibrin-implanted mice. Significance was determined by 1-way ANOVA. (C-G) Fibrin implanted to subcutaneous space undergoes cellular endocytosis. (C-F) Representative images of mouse dermis 2, 6, 12, and 24 hours postinjection show progressive accumulation of cells that present with fluorescently labeled fibrin (green, white in top insets) in perinuclear vesicles sometimes colocalizing with the Alexa Fluor 647 dextran lysosomal marker (red, white in bottom insets). (G) High magnification of a single fibrin-internalizing cell showing the colocalization of fibrin (green, white in top inset) and dextran (red, white in bottom inset) in perinuclear vesicles (yellow), indicative of lysosomal routing of endocytosed fibrin. (H) Fibrin uptake (green) by cells 24 hours after fibrin implantation in Gt(ROSA)26Sortm4 (ACTB-tdTomato,-EGFP)Luo+/0 transgenic mice expressing plasma membrane–localized tomato fluorescent protein (red). Cross hairs mark a cell with multiple fibrin-containing vesicles completely circumscribed by plasma membrane. N.S., not significant.

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