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: bioRxiv

Article Title: Harnessing micrometer-scale tPA beads for high plasmin generation and accelerated fibrinolysis

doi: 10.1101/2024.11.06.621942

Figure Lengend Snippet: Fibrin gels were formed with ( A ) normal pooled plasma, tissue factor (1.25 pM) and CaCl 2 (8.3 mM), ( B ) fibrinogen (2 mg/mL), glu-plasminogen (10 μM), thrombin (2 U/mL), ( C ) fibrinogen (2 mg/mL), glu-plasminogen (10 μM), thrombin (2 U/mL), α2-antiplasmin (1 μM) with free tPA or tPA-beads at 37°C. Fibrinolysis was measured by absorbance (405 nm) for tPA-beads (2.25 mU/mL) with diameters of 0.1 μm (3.55×10 10 beads/mL) and 1.0 μm (1.69×10 8 beads/mL) or free tPA (2.25 mU/mL). Each line and shaded region represent the average and standard deviation of n=3.

Article Snippet: Human fibrinogen depleted of plasminogen, von Willebrand Factor and fibronectin (FIB3), human glu-plasminogen (HPG 2001) and α2-antiplasmin (α2AP) was obtained from Enzyme Research Laboratories (South Bend, IN).

Techniques: Standard Deviation