Interaction of collagelin with collagen.

<p>A: B-collagelin was immobilized on a streptavidin-coated sensorchip (∼20 RU). Collagen (10 µg/ml) was injected over the sensorchip. A representative sensorgram (dark line) and interaction fit (gray line) are shown after subtracting the non-specific background signal from a control flow cell coated with an irrelevant peptide. B: B-collagelin (250, 500 µg.mL⁻¹) was injected over a collagen-coated sensorchip. Sensorgrams (black) and interaction fits (gray) are shown. Representative sensorgrams are shown after subtracting the non-specific response from the irrelevant peptide. C: B-collagelin or control peptide (50 µg. mL⁻¹) were incubated with immobilized, fibrillar, type-I collagen in microtitration plates, and detected using HRP-coupled extravidin. In competition experiments, collagelin was mixed with GPVI-Fc (50 µg.mL⁻¹), 9012.2 IgGs (50 µg.mL⁻¹) or 3J24.2 IgGs (50 µg.mL⁻¹) before being added to collagen-coated wells. Means±SD (n = 3) are presented; *** p<0.01. D: B-collagelin (50 µg.mL⁻¹, black) or B-Pc (gray) were incubated with immobilized collagen I or III, CRP, fibrinogen, fibronectin, vitronectin and laminin in microtitration plates, and detected as above. Means±SD (n = 3) are shown.</p

<i>In- vivo</i> scintigraphy, <i>ex vivo</i> myocardial autoradiography and histology using ^99mTc-streptavidin-B-collagelin.

<p>A: Planar thoracic scintigraphy of a control rat (sham). B: Planar and tomographic (frontal and sagittal views) thoracic images of a rat with a fibrotic myocardial infarction: a hot-spot (arrows) can be seen in the left ventricular myocardial area. C: Corresponding myocardial autoradiography and histology (collagen-specific picrosirius red staining,), confirming tracer uptake in the thin, fibrotic (red) myocardial scar (arrows). D: Control experiment: no activity can be seen in the myocardial scar of a rat injected with irrelevant ^99mTc-streptavidin-B-Pc.</p

<i>In vivo</i> scintigraphy, <i>ex vivo</i> myocardial autoradiography and histology using 99mTc-collagelin.

<p>A: Planar thoracic scintigraphy of a rat with fibrotic myocardial infarction: a clear hot-spot (arrows) xcan be seen in the left ventricular myocardial area. B: From left to right, corresponding myocardial histology (Masson's trichrome, picrosirius red) and autoradiography, confirming tracer uptake in the thin, fibrotic (red) myocardial scar (arrow heads). C: Control experiment: very low activity is observed in the myocardial infarction in a rat injected with irrelevant 99mTc-Pc.</p

Identification of collagelin.

<p>A: <i>Identification of 9O12.2 binding bacterial clones</i>. Proteins from bacterial clones were separated by electrophoresis under non-reducing conditions, and analysed by immunoblot using the 9O12.2 IgG. The band at ∼63 kDa corresponds to the FliTrx fusion protein containing a peptide recognized by 9O12.2. Results are from six selected clones (10, 12, 14, 15, 16, 18), and from one clone selected from the same library but using an irrelevant antibody (−). The sequence of clone 14 was retained for peptide synthesis. B–D <i>Surface plasmon resonance (SPR) analysis of collagelin binding to 9O12.2</i>. In B, increasing concentrations of the 9O12.2 IgGs were passed over the sensorchip (4, 6, 8, 10 µg/ml from bottom to top). In C: 9O12.2 IgG (8 µg/ml) was injected over immobilized B-collagelin that was either non-reduced (black) or reduced by DTT (gray) on the sensorchip. In D, 9O12.2 IgG (5 µg/ml) was injected over immobilized B-collagelin in the absence (black) or presence of recombinant soluble GPVI (25 µg/ml) (gray). Representative sensorgrams are shown after subtracting the non-specific response from a control flow cell coated with an irrelevant peptide.</p

Histochemical analysis of peptide binding to tissue collagen.

<p>A: Frozen sections of rat aorta were incubated with B-collagelin or control peptide (200 µg/mL) and detected using HRP-coupled streptavidin. Sections were counter-stained with hematoxylin. Contiguous serial sections were stained with picrosirius red. In a competition experiment, the peptide was mixed with anti-GPVI IgG 9O121.2 (300 µg/mL). B: Paraffin embedded sections of rat tail tendon were treated as above.</p

(A): Amino-acid alignment (Fasta format) of the 20 clones sequenced after screening of the FliTrx random peptide display library against immobilized 9O12 IgG.

<p>The sequence of the peptide that has been selected for synthesis is underlined.</p