Roisin D. Moriarty

The application of water soluble, mega-Stokes-shifted BODIPY fluorophores to cell and tissue imaging.

BODIPY (4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene) fluorophores are widely used in bioimaging to label proteins, lipids and nucleotides, but in spite of their attractive optical properties they tend to be prone to self‐quenching because of their notably small Stokes shift. Herein, we compare two BODIPY compounds from a recently developed family of naphthyridine substituted BODIPY derivatives, one a visible emitting derivative (BODIPY‐VIS) and one a near‐infrared emitting fluorophore with a Stokes shift of approximately 165 nm as contrast reagents for live mammalian cells and murine brain tissue. The compounds were rendered water soluble by their conjugation to polyethylene glycol (PEG). Both PEGylated compounds exhibited good cell uptake compared with their parent compounds and confocal fluorescence microscopy revealed all dyes explored to be nuclear excluding, localizing predominantly within the lipophilic organelles; the endoplasmic reticulum and mitochondria. Cytotoxicity studies revealed that these BODIPY derivatives are modestly cytotoxic at concentrations exceeding 10 μM where they induce apoptosis and necrosis. Although the quantum yield of emission of the visible emitting fluorophore was over an order of magnitude greater than the Mega‐Stokes shifted probe, the latter showed considerably reduced tendency to self quench and less interference from autofluorescence. The near‐infrared probe also showed good penetrability and staining in live tissue samples. In the latter case similar tendency to exclude the nucleus and to localize in the mitochondria and endoplasmic reticulum was observed as in live cells. This to our knowledge is the first demonstration of such a Mega‐Stokes BODIPY probe applied to cell and tissue imaging.

C-reactive protein binds to αIIbβ3

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A novel role for the fibrinogen Asn-Gly-Arg (NGR) motif in platelet function

The integrin αIIbβ3 on resting platelets can bind to immobilised fibrinogen resulting in platelet spreading and activation but requires activation to bind to soluble fibrinogen. αIIbβ3 is known to interact with the general integrin-recognition motif RGD (arginine-glycine-aspartate) as well as the fibrinogen-specific γ-chain dodecapeptide; however, it is not known how fibrinogen binding triggers platelet activation. NGR (asparagine-glycine-arginine) is another integrin-recognition sequence present in fibrinogen and this study aims to determine if it plays a role in the interaction between fibrinogen and αIIbβ3. NGR-containing peptides inhibited resting platelet adhesion to fibrinogen with an IC50 of 175 µM but failed to inhibit the adhesion of activated platelets to fibrinogen (IC50> 500 µM). Resting platelet adhesion to mutant fibrinogens lacking the NGR sequences was reduced compared to normal fibrinogen under both static and shear conditions (200 s⁻¹). However, pre-activated platelets were able to fully spread on all types of fibrinogen. Thus, the NGR motif in fibrinogen is the site that is primarily responsible for the interaction with resting αIIbβ3 and is responsible for triggering platelet activation.