affimer highly purified human α2ap  (Athens Research)

Journal: Blood Advances

Article Title: Use of Affimer technology for inhibition of α2-antiplasmin and enhancement of fibrinolysis

doi: 10.1182/bloodadvances.2024014235

Figure Lengend Snippet: The effect of Affimer A11 in plasmin activity and plasmin generation rate and interaction with plasminogen. (A) Fluorometric plasmin activity assay investigating the effects of α2AP on plasmin activity in the absence and presence of Affimer A11. Data are presented as the mean ± SD of 3 independent experiments. One-way ANOVA test was used for statistical analysis to compare A11 with buffer only control. (B) Chromogenic substrate S2251 hydrolysis assay analyzing plasminogen conversion to plasmin in the absence and presence of Affimer A11. Data are presented as the mean ± SD of 3 independent experiments. One-tailed Mann Whitney test was used for statistical analysis to compare A11 with buffer only control. (C) Competitive inhibition of plasminogen binding to α2AP in the presence of increasing concentrations of Affimer. The absorbance units were converted to the percentage of α2AP-bound plasminogen, with results normalized to the binding of plasminogen in the absence of Affimer. Data are presented as the mean ± SD of 3 independent experiments. (D) Predicted binding pose of Affimer A11 (magenta) with α2AP (green). The 2 loops of A11 are highlighted as sticks; the residues of α2AP interacting with the Affimer are shown as coral sticks, and RGD domain is highlighted blue. Figure produced using PyMOL version 2.5.2.

Article Snippet: Highly purified human α2AP (Athens Research & Technology, Athens, GA) was biotinylated, and functional studies were performed to ensure the α2AP protein does not lose activity after biotinylation.

Techniques: Activity Assay, Control, Hydrolysis Assay, One-tailed Test, MANN-WHITNEY, Inhibition, Binding Assay, Produced

Journal: Blood Advances

Article Title: Use of Affimer technology for inhibition of α2-antiplasmin and enhancement of fibrinolysis

doi: 10.1182/bloodadvances.2024014235

Figure Lengend Snippet: The effect of Affimer A11 on fibrin clot formation and lysis tested in turbidimetric assays. (A) Representative turbidity and lysis curves in a purified system. (B-D) The effect of Affimer A11 and control SQT on lag time (B) and maximum absorbance (C) and clot lysis time (D) of clots made from purified fibrinogen in the presence of α2AP and factor XIII. Numbers depict Affimer-to-α2AP molar ratios. (E) Representative turbidity and lysis curves in plasma. (F-H) The effect of Affimer A11 and SQT on lag time (F), maximum absorbance (G), and lysis time (H) of clots made from pooled human plasma. Data are presented as the mean ± standard deviation (SD) of 3 independent experiments performed in duplicate. One-way analysis of variance (ANOVA) test was used for statistical analysis to compare A11 with buffer only control. Max, maximum; OD, optical density.

Article Snippet: Highly purified human α2AP (Athens Research & Technology, Athens, GA) was biotinylated, and functional studies were performed to ensure the α2AP protein does not lose activity after biotinylation.

Techniques: Lysis, Purification, Control, Clinical Proteomics, Standard Deviation

Journal: Blood Advances

Article Title: Use of Affimer technology for inhibition of α2-antiplasmin and enhancement of fibrinolysis

doi: 10.1182/bloodadvances.2024014235

Figure Lengend Snippet: The effect of Affimer A11 in fibrin clot structure. Plasma clots in the presence of Affimer A11 at 10:1, 20:1, and 40:1 Affimer-to-α2AP molar ratio (scaffold-only Affimer and buffer with no Affimer were used as controls) were visualized using LSCM and scanning electron microscopy (SEM). (A) Z stacks (20.30 μm; 30 slices) were compiled to form 3-dimensional (3D) images that are presented here (scale bar, 20 μm). (B) SEM images (scale bar, 1 μm). (C) Fiber density was determined by counting the number of fibers using ImageJ software and presented as the average number of fibers per 100 μm. Two clots were made for each condition, and 3 images were taken in different areas of each clot. (D) Mean fiber thickness of fibers calculated in SEM images using ImageJ software. Two clots were made for each condition, and images were taken in 5 different areas of each clot; 20 fibers were measured in each image. Data presented as mean ± SD. Statistical analysis was performed using 1-way ANOVA; ∗ P value < .05 represents difference from buffer control.

Article Snippet: Highly purified human α2AP (Athens Research & Technology, Athens, GA) was biotinylated, and functional studies were performed to ensure the α2AP protein does not lose activity after biotinylation.

Techniques: Clinical Proteomics, Electron Microscopy, Software, Control

Journal: Blood Advances

Article Title: Use of Affimer technology for inhibition of α2-antiplasmin and enhancement of fibrinolysis

doi: 10.1182/bloodadvances.2024014235

Figure Lengend Snippet: Incorporation of α2AP into the fibrin clot. (A) 3D images of compiled Z stacks of plasma clots made using AlexaFluor 594–labeled fibrinogen (red) and AlexaFluor 488–labeled α2AP (green; scale bar, 20 μm). (B) Histograms of pixel intensity within fibrin fibers demonstrating α2AP incorporation into the clot in the presence of A11, scaffold-only Affimer, or buffer only. (C) α2AP crosslinked to fibrin with or without factor XIII (FXIII) in the presence of Affimer A11, scaffold-only Affimer control protein, or buffer only studied using an enzyme-linked immunosorbent assay. Data presented as the mean ± SD of 3 independent experiments. One-way ANOVA test was used for statistical analysis.

Article Snippet: Highly purified human α2AP (Athens Research & Technology, Athens, GA) was biotinylated, and functional studies were performed to ensure the α2AP protein does not lose activity after biotinylation.

Techniques: Clinical Proteomics, Labeling, Control, Enzyme-linked Immunosorbent Assay

Journal: Blood Advances

Article Title: Use of Affimer technology for inhibition of α2-antiplasmin and enhancement of fibrinolysis

doi: 10.1182/bloodadvances.2024014235

Figure Lengend Snippet: Schematic representation of Affimer A11 dual mechanism. (A) Affimer A11 increases plasmin production by facilitating plasminogen binding to fibrinogen through modulation of α2AP-plasminogen interaction. (B) Affimer A11 interferes with the suppression of plasmin activity by α2AP.

Article Snippet: Highly purified human α2AP (Athens Research & Technology, Athens, GA) was biotinylated, and functional studies were performed to ensure the α2AP protein does not lose activity after biotinylation.

Techniques: Binding Assay, Activity Assay

Journal: Journal of Clinical Medicine

Article Title: Functional and Structural Characterization of Nucleic Acid Ligands That Bind to Activated Coagulation Factor XIII

doi: 10.3390/jcm10040677

Figure Lengend Snippet: Key characteristics of FXIIIa aptamers.

Article Snippet: Peroxidase-conjugated goat anti-human α2-antiplasmin (α2AP) antibody was purchased from Affinity Biologicals (Ancaster, Canada).

Techniques: Activity Assay, Inhibition

Journal: Journal of Clinical Medicine

Article Title: Functional and Structural Characterization of Nucleic Acid Ligands That Bind to Activated Coagulation Factor XIII

doi: 10.3390/jcm10040677

Figure Lengend Snippet: Aptamer binding reduced incorporation of FXIIIa and α2-antiplasmin (α2AP) to fibrinogen. Retention of ( A ) FXIIIa or ( B ) α2AP in fibrinogen coated microtiter plates in the presence of increasing concentrations of aptamers was detected using fluorogenic peptide substrate or a polyclonal antibody against α2AP, respectively. All results are shown as means with standard deviations of triplicates. ● FA1, ■ FA2, ▲ FA3, ▼ FA6, ♦ FA8, ○ FA12, □ negative control sequence.

Article Snippet: Peroxidase-conjugated goat anti-human α2-antiplasmin (α2AP) antibody was purchased from Affinity Biologicals (Ancaster, Canada).

Techniques: Binding Assay, Negative Control, Sequencing

Journal: Journal of Clinical Medicine

Article Title: Functional and Structural Characterization of Nucleic Acid Ligands That Bind to Activated Coagulation Factor XIII

doi: 10.3390/jcm10040677

Figure Lengend Snippet: Aptamer docking on FXIIIa. The highest scoring docking pose for each of the six aptamers on FXIIIa is illustrated. The aptamer models are colored brown and represented in stick format while the FXIIIa backbone is represented in blue colored ribbon format. The fibrinogen and α2AP binding sites on FXIIIa, based on a previous docking study have been colored cyan and red, respectively . The catalytic triad residues on FXIIIa are colored yellow.

Article Snippet: Peroxidase-conjugated goat anti-human α2-antiplasmin (α2AP) antibody was purchased from Affinity Biologicals (Ancaster, Canada).

Techniques: Binding Assay

Journal: Molecular Brain

Article Title: α2-Antiplasmin as a potential regulator of the spatial memory process and age-related cognitive decline

doi: 10.1186/s13041-020-00677-3

Figure Lengend Snippet: The effects of anti-α2AP neutralizing antibodies on adult hippocampal neurogenesis. a Representative images of immunostaining of BrdU and Ki67. Anti-α2AP neutralizing antibodies or control IgG were intracerebroventricularly injected in 12-week-old C57BL/6J mice 2 h after the first intraperitoneal injection of BrdU, and BrdU was administered at 24-h intervals for 7 days. Coronal brain slices were immunostained with antibodies. Arrow heads indicate Ki67 + cells, and arrows indicate examples of BrdU + /Ki67 − cells. Scale bar: 50 μm. b The numbers of BrdU + /Ki67 − cells in the DG were counted in a blinded manner. c Representative images of immunostaining of Dcx. Scale bar: 200 μm. d The numbers of Dcx + cells in the DG were counted in a blinded manner. The values represent the means ± S.E. (control IgG: n = 4, α2AP Ab: n = 5). Statistical significance was evaluated using Student’s t -test. *P < 0.05

Article Snippet: After the first day of MWM training, mice were anesthetized with 1.8–1.9% isoflurane, and then were injected 200 nM of α2AP (Calbiochem, MA, USA) or 0.1 μg/μL of an anti-α2AP neutralizing goat antibody (AF1470, R&D System, MN, USA) as well as saline or normal goat IgG control, respectively (AB-108-C, R&D System) in a total volume of 20 μL into the lateral ventricle (0.5 mm caudally and 1.0 mm laterally to the bregma and 2.0 mm vertically from the skull surface) with a two-step needle (Star needle; Seiseido, Tokyo, Japan) attached to a glass syringe (As one, Osaka, Japan).

Techniques: Immunostaining, Control, Injection

Journal: Molecular Brain

Article Title: α2-Antiplasmin as a potential regulator of the spatial memory process and age-related cognitive decline

doi: 10.1186/s13041-020-00677-3

Figure Lengend Snippet: The Effects of anti-α2AP neutralizing antibodies on spatial memory. a Anti-α2AP neutralizing antibodies or control IgG were intracerebroventricularly injected in 12-week-old C57BL/6J mice after the first day of training in the MWM test. On the second day, mice were repeatedly trained, and then the probe tests were performed 30 min, 1 month and 3 months later. b The results of the training sessions. The latency to the target in each trial was measured. The values represent the mean values of 4 trials in each session. c – e The results of the probe tests at 30 min ( c ), 1 month ( d ) and 3 months ( e ) after training. The time in each quadrant was measured. The values represent the mean ± S.E. (control IgG: n = 10, α2AP Ab: n = 9). Statistical significance was evaluated using an ANOVA with an LSD post-hoc test. **P < 0.01, ns non-significant

Article Snippet: After the first day of MWM training, mice were anesthetized with 1.8–1.9% isoflurane, and then were injected 200 nM of α2AP (Calbiochem, MA, USA) or 0.1 μg/μL of an anti-α2AP neutralizing goat antibody (AF1470, R&D System, MN, USA) as well as saline or normal goat IgG control, respectively (AB-108-C, R&D System) in a total volume of 20 μL into the lateral ventricle (0.5 mm caudally and 1.0 mm laterally to the bregma and 2.0 mm vertically from the skull surface) with a two-step needle (Star needle; Seiseido, Tokyo, Japan) attached to a glass syringe (As one, Osaka, Japan).

Techniques: Control, Injection

Journal: Molecular Brain

Article Title: α2-Antiplasmin as a potential regulator of the spatial memory process and age-related cognitive decline

doi: 10.1186/s13041-020-00677-3

Figure Lengend Snippet: The effects of the injection of α2AP on adult hippocampal neurogenesis. a Representative images of immunostaining of BrdU and Ki67. α2AP or saline was intracerebroventricularly injected in 12-week-old C57BL/6J mice, 2 h after the first intraperitoneal injection of BrdU, and BrdU was administered at 24-h intervals for 7 days. Coronal brain slices were immunostained with antibodies. Arrow heads indicate Ki67 + cells. Scale bar: 50 μm. b The numbers of BrdU + /Ki67 − cells in the DG were counted in a blinded manner. c Representative images of immunostaining of Dcx. Scale bar: 200 μm. d The numbers of Dcx + cells in the DG were counted in a blinded manner. The values represent the mean ± S.E. (saline: n = 5, α2AP: n = 6). Statistical significance was evaluated using Student’s t -test. *P < 0.05

Article Snippet: After the first day of MWM training, mice were anesthetized with 1.8–1.9% isoflurane, and then were injected 200 nM of α2AP (Calbiochem, MA, USA) or 0.1 μg/μL of an anti-α2AP neutralizing goat antibody (AF1470, R&D System, MN, USA) as well as saline or normal goat IgG control, respectively (AB-108-C, R&D System) in a total volume of 20 μL into the lateral ventricle (0.5 mm caudally and 1.0 mm laterally to the bregma and 2.0 mm vertically from the skull surface) with a two-step needle (Star needle; Seiseido, Tokyo, Japan) attached to a glass syringe (As one, Osaka, Japan).

Techniques: Injection, Immunostaining, Saline

Journal: Molecular Brain

Article Title: α2-Antiplasmin as a potential regulator of the spatial memory process and age-related cognitive decline

doi: 10.1186/s13041-020-00677-3

Figure Lengend Snippet: The effects of the injection of α2AP on spatial memory. a α2AP or saline was intracerebroventricularly injected in 12-week-old C57BL/6J mice after the first day of training in the MWM test. On the second day, mice were repeatedly trained, and then the probe tests were performed 30 min and 1 month later. b The results of the training sessions. The latency to the target in each trial was measured, and the values represent the mean values of 4 trials in each session. c and d The results of the probe tests performed 30 min ( c ) and 1 month ( d ) after training. The time in each quadrant was measured. The values represent the mean ± S.E. (saline: n = 8, α2AP: n = 10). Statistical significance was evaluated using an ANOVA with an LSD post-hoc test. **P < 0.01

Article Snippet: After the first day of MWM training, mice were anesthetized with 1.8–1.9% isoflurane, and then were injected 200 nM of α2AP (Calbiochem, MA, USA) or 0.1 μg/μL of an anti-α2AP neutralizing goat antibody (AF1470, R&D System, MN, USA) as well as saline or normal goat IgG control, respectively (AB-108-C, R&D System) in a total volume of 20 μL into the lateral ventricle (0.5 mm caudally and 1.0 mm laterally to the bregma and 2.0 mm vertically from the skull surface) with a two-step needle (Star needle; Seiseido, Tokyo, Japan) attached to a glass syringe (As one, Osaka, Japan).

Techniques: Injection, Saline

Journal: Molecular Brain

Article Title: α2-Antiplasmin as a potential regulator of the spatial memory process and age-related cognitive decline

doi: 10.1186/s13041-020-00677-3

Figure Lengend Snippet: Correlation of α2AP levels in the brain and spatial working memory between young and old mice. a and b The levels of α2AP and plasmin relative to those of GAPDH in the hippocampus and cerebral cortex of young and old C57BL/6J mice were determined by Western blotting (young mice: 12–16 weeks of age, old mice: > 25 months of age). The band intensity was measured using the NIH ImageJ software program and normalized to that of GAPDH. The values were divided by the mean of the normalized intensity of young mice. The bar graphs represent the mean ± SE (arbitrary units: A.U., young mice: n = 8, old mice: n = 9). Statistical significance was evaluated using Student’s t -test. **P < 0.01. c and d The relative intensity of α2AP to GAPDH and the scores of the Y-maze test were analyzed by Pearson’s correlation test. Correlation was evaluated using contribution rate (r 2 ): values of r 2 > 0.16 were considered to indicate a correlation. The values of young mice and old mice were presented with a blank rhombus and filled rhombus, respectively

Article Snippet: After the first day of MWM training, mice were anesthetized with 1.8–1.9% isoflurane, and then were injected 200 nM of α2AP (Calbiochem, MA, USA) or 0.1 μg/μL of an anti-α2AP neutralizing goat antibody (AF1470, R&D System, MN, USA) as well as saline or normal goat IgG control, respectively (AB-108-C, R&D System) in a total volume of 20 μL into the lateral ventricle (0.5 mm caudally and 1.0 mm laterally to the bregma and 2.0 mm vertically from the skull surface) with a two-step needle (Star needle; Seiseido, Tokyo, Japan) attached to a glass syringe (As one, Osaka, Japan).

Techniques: Western Blot, Software

Journal: Molecular Brain

Article Title: α2-Antiplasmin as a potential regulator of the spatial memory process and age-related cognitive decline

doi: 10.1186/s13041-020-00677-3

Figure Lengend Snippet: The effects of α2AP deficiency on age-dependent oxidative stress and neuroinflammation. a and b The levels of 13-HPODE-adducted protein relative to those of GAPDH in the hippocampus and cerebral cortex of young and old WT and α2AP −/− mice were determined by Western blotting (young mice: 11 weeks of age, old mice: 60 weeks of age). The relative intensity was measured using the NIH ImageJ software program and normalized to that of GAPDH. The bar graphs represent the mean ± SE (young WT mice: n = 5, young α2AP −/− mice: n = 4, old WT mice: n = 5, old α2AP −/− mice: n = 4). c The levels of IL-6 mRNA in the hippocampus were shown (young mice: n = 7, old mice: n = 5). Statistical significance was evaluated by an ANOVA with an LSD post-hoc test. *P < 0.05., **P < 0.01

Article Snippet: After the first day of MWM training, mice were anesthetized with 1.8–1.9% isoflurane, and then were injected 200 nM of α2AP (Calbiochem, MA, USA) or 0.1 μg/μL of an anti-α2AP neutralizing goat antibody (AF1470, R&D System, MN, USA) as well as saline or normal goat IgG control, respectively (AB-108-C, R&D System) in a total volume of 20 μL into the lateral ventricle (0.5 mm caudally and 1.0 mm laterally to the bregma and 2.0 mm vertically from the skull surface) with a two-step needle (Star needle; Seiseido, Tokyo, Japan) attached to a glass syringe (As one, Osaka, Japan).

Techniques: Western Blot, Software