Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Expression of fibrinolytic system components in 24 h and 7 days old venous thrombi. Panels A-F show the expression (red) of (A-B) tPA, (C-D) uPA, (E-F) PAI-1, (G-H) plasminogen (Pg), (I-J) α2AP, and (K-L) their quantification in 8-µm cryosections of 24 hours and 7 days thrombi as labeled. Red color (Alexa Fluor 555) represents each component as labeled and blue color shows 4′,6-diamidino-2-phenylindole–stained nuclei. For comparison, IVC from sham mice were immunostained and quantified for each antibody under the same color range in the histogram mode of Image Pro-Plus software. The total immunostained area (arbitrary units; AU/mm2) for each protein was measured in 500 μm (original magnification ×4) images of an IVC sample. (N = 4-5 per group, mean ± SEM). *P < .05, **P < .01, ***P < .001.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Expressing, Labeling, Staining, Software

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on early thrombus formation. (A) Representative IVCs in different groups as labeled. (B) Vein thrombus weight milligram/gram of body weight. Each symbol represents an animal used in each group (N = 33). The bar graph shows the statistical significance between different groups by one-way ANOVA and Newman-Keuls post hoc test. ****P < .0001; ***P < .001; ns, nonsignificant. (C-F) Formalin-fixed paraffin-embedded (5 µm) sections of IVC thrombi were stained with Martius scarlet blue kit (scale bar = 500 µm). Representative image of N = 5 for each group. (C) Thrombus formation in the IVC of α2AP+/+ mice 5 hours after ligation (500 µm). (D) The composition of 5-hour-old thrombi (fibrin, red blood cell [RBC], and leukocytes; black arrows, scale bar = 100 µm). The pink/yellow color represents fibrin and nuclei are darkly stained. This panel shows a magnified image of the area in the black box highlighted in panel C. (E-F) Martius scarlet blue-stained representative images of IVC in α2AP−/− mice 5 hours after IVC ligation. The black arrows indicate areas of minimum thrombosis.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Labeling, Formalin-fixed Paraffin-Embedded, Staining, Ligation

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on acute thrombus formation. (A-B) Representative IVCs in α2AP+/+ and α2AP−/− mice (A) 24 hours after IVC ligation and (B) their thrombus weights. Each symbol in the bar graph represents an animal used in each group (N = 12); ****P < .0001, Student t test. (C) Martius scarlet blue (scale bar = 500 µm) staining shows 24-hour thrombi rich in fibrin (pink color). (D) Magnified image (original magnification ×20; scale bar = 100 µm) of panel C showing a fibrin-rich (pink) thrombus area. (E) Minimum thrombus formation (black arrows) in α2AP−/− mice 24 hours after IVC ligation.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Ligation, Staining

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on chronic thrombus formation. (A) Martius scarlet blue-stained 7-day-old thrombi (scale bar = 500 µm). (B) Magnified images (original magnification ×20) of IVC thrombus showing lamellate fibrin-rich (pink) lines of Zahn. (C) Thrombi were not formed in α2AP−/− mice after 7 days of IVC ligation. Arrow indicates the minimum thrombus formation. (D) Martius scarlet blue stained 14 days old thrombi (scale bar = 500 µm) in α2AP+/+ mice. (E) Magnified image (original magnification ×20) of panel D showing a fibrin-rich (pink), porous thrombus. (F) Martius scarlet blue-stained IVC showing minimum thrombus in α2AP−/− mice. (G) Representative images of IVC thrombus formation in different groups as labeled including sham mice (7 days). (H) Vein thrombus weight milligram/gram of body weight. Each symbol represents an animal used in each group (N = 38). One-way ANOVA; ****P < .0001, **P < .01.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Staining, Ligation, Labeling

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP supplementation/inhibition. (A) Representative images of IVC thrombus formation in α2AP−/− mice after 5 hours’ ligation after administration of α2AP or α2AP + α2AP-inactivating antibody (α2AP-I) as labeled. (B) Vein thrombus weight in these groups in milligram/gram of body weight as labeled. Each symbol represents a single experiment, N = 14. The horizontal red and blue lines in the graph show, for reference, the mean thrombus weight in α2AP+/+ and α2AP−/− mice, respectively. ****P < .0001; one-way ANOVA (among 4 groups).

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Inhibition, Ligation, Labeling

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP on fibrinolysis. (A) Plasma D-dimer levels in α2AP+/+ and α2AP−/− mice after 5 hours of IVC ligation. The mice were supplemented with 10 mg/kg human fibrinogen. *P < .05 (Student t test). (B) Vein thrombus weight milligram/gram of body weight in α2AP+/+ and α2AP−/− mice after 5 hours’ IVC ligation used for D-dimer measurements ***P < .001 (Student t test). (C) Representative images of IVC thrombus formation in EACA-treated α2AP−/− mice 5 hours after ligation as labeled. (D) Vein thrombus weight (milligram/gram of body weight) in α2AP−/− mice with or without EACA treatment. The horizontal red line in the graph shows the mean vein thrombus weight in α2AP+/+ mice for reference. *P < .05, one-way ANOVA (among 3 groups). (E) Vein thrombus weight (milligram/gram of body weight) in Pg−/− mice in comparison with α2AP+/+ mice. (N = 21) ***P < .001, Mann-Whitney test.

Article Snippet: Proteins and reagents Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Ligation, Labeling, MANN-WHITNEY

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Expression of fibrinolytic system components in 24 h and 7 days old venous thrombi. Panels A-F show the expression (red) of (A-B) tPA, (C-D) uPA, (E-F) PAI-1, (G-H) plasminogen (Pg), (I-J) α2AP, and (K-L) their quantification in 8-µm cryosections of 24 hours and 7 days thrombi as labeled. Red color (Alexa Fluor 555) represents each component as labeled and blue color shows 4′,6-diamidino-2-phenylindole–stained nuclei. For comparison, IVC from sham mice were immunostained and quantified for each antibody under the same color range in the histogram mode of Image Pro-Plus software. The total immunostained area (arbitrary units; AU/mm2) for each protein was measured in 500 μm (original magnification ×4) images of an IVC sample. (N = 4-5 per group, mean ± SEM). *P < .05, **P < .01, ***P < .001.

Article Snippet: Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Expressing, Labeling, Staining, Comparison, Software

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on early thrombus formation. (A) Representative IVCs in different groups as labeled. (B) Vein thrombus weight milligram/gram of body weight. Each symbol represents an animal used in each group (N = 33). The bar graph shows the statistical significance between different groups by one-way ANOVA and Newman-Keuls post hoc test. ****P < .0001; ***P < .001; ns, nonsignificant. (C-F) Formalin-fixed paraffin-embedded (5 µm) sections of IVC thrombi were stained with Martius scarlet blue kit (scale bar = 500 µm). Representative image of N = 5 for each group. (C) Thrombus formation in the IVC of α2AP+/+ mice 5 hours after ligation (500 µm). (D) The composition of 5-hour-old thrombi (fibrin, red blood cell [RBC], and leukocytes; black arrows, scale bar = 100 µm). The pink/yellow color represents fibrin and nuclei are darkly stained. This panel shows a magnified image of the area in the black box highlighted in panel C. (E-F) Martius scarlet blue-stained representative images of IVC in α2AP−/− mice 5 hours after IVC ligation. The black arrows indicate areas of minimum thrombosis.

Article Snippet: Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Labeling, Formalin-fixed Paraffin-Embedded, Staining, Ligation

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on acute thrombus formation. (A-B) Representative IVCs in α2AP+/+ and α2AP−/− mice (A) 24 hours after IVC ligation and (B) their thrombus weights. Each symbol in the bar graph represents an animal used in each group (N = 12); ****P < .0001, Student t test. (C) Martius scarlet blue (scale bar = 500 µm) staining shows 24-hour thrombi rich in fibrin (pink color). (D) Magnified image (original magnification ×20; scale bar = 100 µm) of panel C showing a fibrin-rich (pink) thrombus area. (E) Minimum thrombus formation (black arrows) in α2AP−/− mice 24 hours after IVC ligation.

Article Snippet: Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Ligation, Staining

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP deficiency on chronic thrombus formation. (A) Martius scarlet blue-stained 7-day-old thrombi (scale bar = 500 µm). (B) Magnified images (original magnification ×20) of IVC thrombus showing lamellate fibrin-rich (pink) lines of Zahn. (C) Thrombi were not formed in α2AP−/− mice after 7 days of IVC ligation. Arrow indicates the minimum thrombus formation. (D) Martius scarlet blue stained 14 days old thrombi (scale bar = 500 µm) in α2AP+/+ mice. (E) Magnified image (original magnification ×20) of panel D showing a fibrin-rich (pink), porous thrombus. (F) Martius scarlet blue-stained IVC showing minimum thrombus in α2AP−/− mice. (G) Representative images of IVC thrombus formation in different groups as labeled including sham mice (7 days). (H) Vein thrombus weight milligram/gram of body weight. Each symbol represents an animal used in each group (N = 38). One-way ANOVA; ****P < .0001, **P < .01.

Article Snippet: Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Staining, Ligation, Labeling

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP supplementation/inhibition. (A) Representative images of IVC thrombus formation in α2AP−/− mice after 5 hours’ ligation after administration of α2AP or α2AP + α2AP-inactivating antibody (α2AP-I) as labeled. (B) Vein thrombus weight in these groups in milligram/gram of body weight as labeled. Each symbol represents a single experiment, N = 14. The horizontal red and blue lines in the graph show, for reference, the mean thrombus weight in α2AP+/+ and α2AP−/− mice, respectively. ****P < .0001; one-way ANOVA (among 4 groups).

Article Snippet: Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Inhibition, Ligation, Labeling

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP on fibrinolysis. (A) Plasma D-dimer levels in α2AP+/+ and α2AP−/− mice after 5 hours of IVC ligation. The mice were supplemented with 10 mg/kg human fibrinogen. *P < .05 (Student t test). (B) Vein thrombus weight milligram/gram of body weight in α2AP+/+ and α2AP−/− mice after 5 hours’ IVC ligation used for D-dimer measurements ***P < .001 (Student t test). (C) Representative images of IVC thrombus formation in EACA-treated α2AP−/− mice 5 hours after ligation as labeled. (D) Vein thrombus weight (milligram/gram of body weight) in α2AP−/− mice with or without EACA treatment. The horizontal red line in the graph shows the mean vein thrombus weight in α2AP+/+ mice for reference. *P < .05, one-way ANOVA (among 3 groups). (E) Vein thrombus weight (milligram/gram of body weight) in Pg−/− mice in comparison with α2AP+/+ mice. (N = 21) ***P < .001, Mann-Whitney test.

Article Snippet: Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Clinical Proteomics, Ligation, Labeling, Comparison, MANN-WHITNEY

Journal: Blood

Article Title: Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin

doi: 10.1182/blood.2019000049

Figure Lengend Snippet: Effects of α2AP on fibrinolysis. (A) Plasma D-dimer levels in α2AP+/+ and α2AP−/− mice after 5 hours of IVC ligation. The mice were supplemented with 10 mg/kg human fibrinogen. *P < .05 (Student t test). (B) Vein thrombus weight milligram/gram of body weight in α2AP+/+ and α2AP−/− mice after 5 hours’ IVC ligation used for D-dimer measurements ***P < .001 (Student t test). (C) Representative images of IVC thrombus formation in EACA-treated α2AP−/− mice 5 hours after ligation as labeled. (D) Vein thrombus weight (milligram/gram of body weight) in α2AP−/− mice with or without EACA treatment. The horizontal red line in the graph shows the mean vein thrombus weight in α2AP+/+ mice for reference. *P < .05, one-way ANOVA (among 3 groups). (E) Vein thrombus weight (milligram/gram of body weight) in Pg−/− mice in comparison with α2AP+/+ mice. (N = 21) ***P < .001, Mann-Whitney test.

Article Snippet: Reagents were obtained from the following sources: human α2AP (Athens Research and Technology, Athens, GA); ε-aminocaproic acid (EACA) (Sigma, St. Louis, MO); inhibitory mouse monoclonal antibody against mouse α2AP (#MAP4H9, Molecular Innovations, Novi, MI); and inhibitory monoclonal antibody against human α2AP (TS23, Translational Sciences, Memphis, TN); all the other reagents if not specified (Sigma).

Techniques: Ligation, Labeling, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Requirement of Plasminogen Binding to Its Cell-Surface Receptor α-Enolase for Efficient Regeneration of Normal and Dystrophic Skeletal Muscle

doi: 10.1371/journal.pone.0050477

Figure Lengend Snippet: A . Immunocytochemical staining for eMHC of MPCs in DM in the presence of MAb11G1, EACA and α2-antiplasmin. B . Quantification of the Differentiation ratio (n° eMHC positive cells/total cells). C . Quantification of the Fusion ratio (n° eMHC positive cells fused in a myotube >4 nuclei/total cells) or Myogenic Index. are mean +/− SEM (error bars). ** P <0.005. Experiments were performed in triplicates.

Article Snippet: Monoclonal antibodies MAb11G1 and MAb7H8 against α-enolase, produced in our laboratory ; ε-aminocaproic acid (EACA), Sigma; α2-antiplasmin, Loxo GmbH.

Techniques: Staining