Journal: Journal of materials chemistry. B

Article Title: Attenuation of Thrombin-Mediated Fibrin Formation via Changes in Fibrinogen Conformation Induced by Reaction with S- nitroso- N -acetylpenicillamine, but not S- nitrosoglutathione

doi: 10.1039/C8TB02103A

Figure Lengend Snippet: Fibrinogen Structure and Fibrin Formation. (A) Schematic of human fibrinogen showing the three main subunits, alpha, beta and gamma. Also shown is the position of the fibrinopeptides A and B on fibrinogen which are cleaved by thrombin. (B) Schematic of fibrin formation after initiation by thrombin. A working hypothesis is that S-nitrosothiols (RSNO) negatively affect the formation of fibrin through structural changes in fibrinogen and not inhibiting thrombin activity.

Article Snippet: For evaluating the alpha, beta and gamma subunits of fibrinogen, membranes were blocked with Tris-buffered saline containing 0.05% Tween-20 and 3% nonfat dry milk (Bio-Rad, Hercules, CA) for 1 h at room temperature followed by an overnight incubation at 4° C with mouse anti-human alpha fibrinogen, rabbit anti-human beta fibrinogen and goat anti-human gamma fibrinogen antibodies (all diluted 1:200).

Techniques: Activity Assay

Journal: Journal of materials chemistry. B

Article Title: Attenuation of Thrombin-Mediated Fibrin Formation via Changes in Fibrinogen Conformation Induced by Reaction with S- nitroso- N -acetylpenicillamine, but not S- nitrosoglutathione

doi: 10.1039/C8TB02103A

Figure Lengend Snippet: Western immunoblot analysis of fibrinogen subunits, Aa, Bb and γ after 1 h exposure with SNAP, NAP and SNP in absence and presence of thrombin. (A), Representative immunoblots. Aliquots (15 μl) of samples were separated on 12% polyacrylamide gels, protein transferred to poly(vinyl-difluoride) (PVDF) membranes and then immunostained with mouse anti-human alpha fibrinogen, rabbit anti-human beta fibrinogen and goat anti-human gamma fibrinogen antibodies (all diluted 1:200). After membrane washings, the membranes were incubated with the secondary antibodies chicken anti-mouse-AF488, chicken anti-rabbit-AF647 and donkey anti-goat-AF555 (all diluted 1:500) for 1 h. After two 10 min washes the blots were imaged in a fluorescence imager (Bio-Rad VersaDoc MP4000 Hercules, CA). Molecular weight markers are shown on left with positions of the relevant fibrinogen chains and Aαγ dimers indicated on the right. (B) Western analysis of samples in (A), after the 1 h incubation, were quantified by densitometry using Quanity One imaging software (Bio-Rad Hercules, CA). The specific Aαγ dimer formation was calculated as the ratio (density) of the Aαγ dimer band over the sum of dimers plus monomers. The data was collected after 1 h in presence of thrombin. The ratio data are means + SEM (n=3). * = p<0.05, SNAP or NAP vs SNP.

Article Snippet: For evaluating the alpha, beta and gamma subunits of fibrinogen, membranes were blocked with Tris-buffered saline containing 0.05% Tween-20 and 3% nonfat dry milk (Bio-Rad, Hercules, CA) for 1 h at room temperature followed by an overnight incubation at 4° C with mouse anti-human alpha fibrinogen, rabbit anti-human beta fibrinogen and goat anti-human gamma fibrinogen antibodies (all diluted 1:200).

Techniques: Western Blot, Incubation, Fluorescence, Molecular Weight, Imaging, Software