Journal: Scientific Reports

Article Title: Discovery of an uncovered region in fibrin clots and its clinical significance

doi: 10.1038/srep02604

Figure Lengend Snippet: (a) The 102–10 mAb was reactive to fibrin clots only ( n = 6). The results are presented as the means ± s.d., ** P < 0.01. (b) Comparing CBB staining with western blot, the 102–10 mAb reacted exclusively with the Bβ chain of denatured whole fibrinogen (Fng). (c) Western blot was conducted for a sample of the digested Bβ chain, and all positive bands detected with the 102–10 mAb are shown. The amino acid sequence of the 10-kDa peptide detected by the 102–10 mAb was analysed in the next step. (d) Only the No. 5 peptide inhibited the binding of 102–10 mAb to fibrin clots ( n = 3; P < 0.01, No. 4 vs. No. 5). (e) The epitope of 102–10 (Bβ201–216; red) interacted with the γ chain (blue), and Bβ203–205 (red ball) interacted with the region around γ216–218 (blue ball) in a hydrophobic manner. The whole structure of fibrinogen is shown (lower panel).

Article Snippet: To produce new antibodies that recognise epitope regions of the fibrin clot Bβ chain (CNIPVVSGKECEEIIR) or γ chain (KNWIQYKEGFGHLSP), recombinant epitope protein was produced from pET21b (Novagen, Darmstadt, Germany) and fibrinogen Bβ chain DNA or fibrinogen γ chain DNA (Origene Technology, Rockville, MD).

Techniques: Staining, Western Blot, Sequencing, Binding Assay

Journal: Scientific Reports

Article Title: Discovery of an uncovered region in fibrin clots and its clinical significance

doi: 10.1038/srep02604

Figure Lengend Snippet: Hydrophobic, ionic, and hydrogen bonds were detected between the Bβ chain and γ chain in the unique region. (a) The anti-Bβ mAb and (b) anti-γ mAb were reactive only to fibrin clots ( n = 8). The results are presented as the means ± s.d., ** P < 0.01. (c) Image of the structural change that occurred as fibrinogen transformed into a fibrin clot; the unique region was formed only when fibrinogen transformed into a fibrin clot (blue arrow). The epitope of the 102–10 mAb (red) on the Bβ chain (silver) and residues 206–220 (yellow) of the γ chain (light blue) were the components of the unique region. (d) The unique region within the fibrin clot was proposed to be large enough for both mAbs to bind to the region simultaneously. (e) Electrostatic surface representations (−5 kT/e, red, to +5 kT/e, blue) of the Bβ chain and γ chain in the unique region. Yellow characters indicate the epitope of the 102–10 mAb and the counterpart on the γ chain. (f) Hydrophobic interaction between the Bβ chain (yellow dots) and γ chain (black dots) of the unique region. (g) Amino acids (blue characters) on the Bβ chain (yellow) and amino acids (black characters) on the γ chain (blue) form hydrogen bonds in the epitope region. (f, g) Red indicates the epitope of 102–10.

Article Snippet: To produce new antibodies that recognise epitope regions of the fibrin clot Bβ chain (CNIPVVSGKECEEIIR) or γ chain (KNWIQYKEGFGHLSP), recombinant epitope protein was produced from pET21b (Novagen, Darmstadt, Germany) and fibrinogen Bβ chain DNA or fibrinogen γ chain DNA (Origene Technology, Rockville, MD).

Techniques: Transformation Assay

Journal: Nature

Article Title: Fibrin drives thromboinflammation and neuropathology in COVID-19

doi: 10.1038/s41586-024-07873-4

Figure Lengend Snippet: a , Binding ELISA of Spike S1(N501Y) to fibrin. Dissociation constants (K d ). Representative curvefits from two independent biological experiments in duplicates. b , Spike overlap with perivascular fibrin(ogen) deposition in lung of Beta-infected WT mice at 3 d.p.i. The 51% of the calculated proportion of fibrin that colocalizes with Spike protein is significantly higher than the 23% predicted if the correlation were random. Fisher’s exact test (two-tailed); n = 78 images from 5 mice (Methods). Representative confocal images are shown. Scale bar, 200 μm. c , Scatter plot of positive correlation of fibrinogen and Spike immunoreactivity in n = 78 images from 5 mice, Pearson correlation two-tailed (Methods). d . 3D reconstruction of light sheet acquisitions of whole lung tissue from an Alexa546-fibrinogen and Alexa647-Spike S1(N501Y)-injected WT mouse following 3DISCO tissue clearing. Two representative focal fibrinogen deposits from n = 3 mice were selected for 3D visualization. Volumetric rendering reveals close interactions between fibrinogen deposits (green) and trimeric spike (magenta), confirming colocalization. Scale bars, 100 μm (top), 300 μm (bottom). e , Fibrinogen crystal structure (PDB: 3GHG) with mapped peptides (red). Proximity of peptides γ 163-181 and γ 364-395 (inset). f , Peptide array mapping with immobilized peptides of SARS-CoV-2 Spike blotted with fibrinogen and fibrinogen γ chain. Heatmap of signal intensity showing binding sites (white-orange) within the S1-NT Spike domain. Key indicates fluorescence intensities signal values from low (white) to high (orange). Schematic indicating Spike domains and amino acid sequence.

Article Snippet: For the spike peptide array, 1, 0.1 or 0.01 μg ml −1 His-tagged recombinant human fibrinogen γ chain (Novus Bio) was hybridized with the SARS-CoV-2 spike Glycoprotein Variant Collection Peptide Microarray (JPT).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test, Injection, Peptide Microarray, Fluorescence, Sequencing