antibody against α2ap  (Santa Cruz Biotechnology)

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: Journal of Thrombosis and Haemostasis

Article Title: On the localization of the cleavage site in human alpha‐2‐antiplasmin, involved in the generation of the non‐plasminogen binding form

doi: 10.1111/jth.14761

Figure Lengend Snippet: In‐house purified α2AP visualized by SDS‐PAGE and Western blot. (A) SDS‐gel of purified α2AP. (B) Immunoblot obtained with anti‐Asn‐α2AP. (C) Immunoblot obtained with TC 3AP. (D) Merged anti‐Asn‐α2AP and TC 3AP blots. The relative migration distances of molecular weight marker proteins are indicated on the left. PB‐α2AP is indicated by arrowhead. NPB‐α2AP is indicated by an asterisk, as well as by a red box in (A)

Article Snippet: The mouse monoclonal antibody against the C‐terminus of α2AP (TC 3AP) was purchased from Technoclone.

Techniques: Purification, SDS Page, Western Blot, SDS-Gel, Migration, Molecular Weight, Marker

Journal: Journal of Thrombosis and Haemostasis

Article Title: On the localization of the cleavage site in human alpha‐2‐antiplasmin, involved in the generation of the non‐plasminogen binding form

doi: 10.1111/jth.14761

Figure Lengend Snippet: MS/MS fragmentation spectra of the (A) nondeamidated and (B) deamidated Asn408‐Gln421 peptide in an overnight Arg‐C digest of NPB‐α2AP as determined by Mascot and MaxQuant analysis, respectively. Best spectrum data are shown. The overlapping labels of the peaks between m/z 730 and 750 in (A) should be read as the following: b(13)++ m/z 731.3759; y0(13)++,y*(13)++, z + 1(13)**, m/z 738.3837, 738.8757, 739.3796; y(13)++ m/z 747.3890

Article Snippet: The mouse monoclonal antibody against the C‐terminus of α2AP (TC 3AP) was purchased from Technoclone.

Techniques: Tandem Mass Spectroscopy

Journal: Journal of Thrombosis and Haemostasis

Article Title: On the localization of the cleavage site in human alpha‐2‐antiplasmin, involved in the generation of the non‐plasminogen binding form

doi: 10.1111/jth.14761

Figure Lengend Snippet: Three‐dimensional structure of human α2AP as predicted by I‐TASSER. Red, β‐sheet A; green, β‐sheet B; yellow, β‐sheet C; magenta, reactive center loop; cyan, C‐terminal cleavage area Leu417‐Asp422 (LKEQQD); blue, C‐terminus; orange, epitope of TC 3AP. Left panel: front view. Right panel: side view, 90°C clockwise rotation

Article Snippet: The mouse monoclonal antibody against the C‐terminus of α2AP (TC 3AP) was purchased from Technoclone.

Techniques:

Journal: Journal of Thrombosis and Haemostasis

Article Title: On the localization of the cleavage site in human alpha‐2‐antiplasmin, involved in the generation of the non‐plasminogen binding form

doi: 10.1111/jth.14761

Figure Lengend Snippet: Schematic representation of the α2AP C‐terminus from Asn408 to Lys464. Arrows indicate the in vivo C‐terminal cleavage sites (preferred site between Gln421 and Asp422), the bold underlined sequence represents the area that includes the TC 3AP epitope, and the brace indicates the area that includes the in vitro C‐terminal cleavage site as described by Sasaki et al.

Article Snippet: The mouse monoclonal antibody against the C‐terminus of α2AP (TC 3AP) was purchased from Technoclone.

Techniques: In Vivo, Sequencing, In Vitro

Journal: Journal of Thrombosis and Haemostasis

Article Title: On the localization of the cleavage site in human alpha‐2‐antiplasmin, involved in the generation of the non‐plasminogen binding form

doi: 10.1111/jth.14761

Figure Lengend Snippet: NPB‐α2AP peptides identified by Mascot and MaxQuant analyses after Arg‐C digest (Figures S1‐S6 )

Article Snippet: The mouse monoclonal antibody against the C‐terminus of α2AP (TC 3AP) was purchased from Technoclone.

Techniques: Sequencing