Computational compartmentalization of tumor uptake of anxA5 and M1234.

<p>The experimental data of Fig. 6B were fit according to the model of Fig. 8. Panels A–C show representative fits for anxA5 uptake by untreated (A) and cyclophosphamide treated tumor (B) and for M1234 uptake by cyclophosphamide treated tumor (C.) The experimental data (red circles) were used to calculate concentrations in whole tumor (black line), tumor interstitium (Comp. 2, red line) and bound to the PS-target (Comp. 3, blue line). Panel D shows the concentrations of anxA5 and M1234 in compartment 3 (PS-target) of tumor that was either untreated or treated with cyclophosphamide (+CYP) 6 hours post-injection. *p<0.05, **p<0.01.</p

Red blood Cell (RBC) binding of anxA5 variants at low membrane density (1*10⁴ molecules/cell) as determined by calcium titration.

<p>(A) Calcium titration curves of anxA5 (open circles) and anxA5-NP (open squares). Each point is the average of three experiments with bars indicating SEM. (B) Hill coefficient (black bars) and EC50 (grey bars) of anxA5 and anxA5-NP as determined from calcium titration curves.</p

Determination of apoptosis (A–C) and microvessel density (D) in tumors of NMRI nude mice: effects of cyclophosphamide (CYP) treatment.

<p>Untreated (–CYP) and treated tumors (+CYP) were excised from tumor-bearing mice, frozen and sectioned. Sections were stained with TUNEL-assay and hematoxylin. % TUNEL-positive nuclei was determined as a measure of apoptosis (A–C). Panel A shows TUNEL-staining of a section of an untreated tumor and panel B of a CYP-treated tumor. Sections were stained with the endothelial specific antibody anti-CD31 and hematoxylin. Microvessel density was determined as % of tumor area that is CD31-positive (D). **p<0.01.</p

3-compartment model and equations that were employed to fit the experimental data. k is constant, C is concentration (nM) and V is volume (L).

<p>3-compartment model and equations that were employed to fit the experimental data. k is constant, C is concentration (nM) and V is volume (L).</p

Binding of annexin-variants to phosphatidylserine containing membranes.

<p>Panel A shows calcium-dependent binding of anxA5 (closed circles), M1234 (open circle), anxA5-NP (closed squares) and M1234-NP (open squares) to a synthetic phospholipid surface comprising 20 mole% phosphatidylserine and 80 mole% phosphatidylcholine. Binding was measured by ellipsometry and is expressed as change in degree of the analyser (Δ°) as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096749#pone.0096749-Andree1" target="_blank">[24]</a>. Panel B shows the % of apoptotic cells of a population of anti-Fas stimulated Jurkat cells that can be detected using fluorescently labeled annexin-variants and flow cytometry.</p

Analyses of purity of anxA5 and M1234, and stoichiometry of anxA5-biotin and anxA5-AF680.

<p>(A) 50 ng purified anxA5 and M1234 were run on an SDS-PAGE. The proteins were visualized by silver staining. (B) Maldi TOF/TOF spectrum of purified anxA5 showing a peak of the monomer at 35772 Da, the dimer at 71589 Da and the double protonated peak at 17826 Da. (C) Maldi TOF/TOF spectrum of anxA5 labeled with maleimide-biotin showing a peak at 36309 Da. Maleimide-biotin labeling resulted in a Mw increase of ±540 Da indicating 1∶1 stoichiometry. (D) Maldi TOF/TOF spectrum of anxA5 labeled with maleimide-AF680 showing a peak at 36672 Da. Maleimide-AF680 labeling resulted in a Mw increase of ±900 Da indicating 1∶1 stoichiometry. Comparable results were obtained with M1234-biotin and M1234-AF680 (not shown).</p

Blood clearance and biodistribution of annexin-variants in NMRI nude mice.

<p>2-labeled annexin-variant were injected intravenously. At various time-points 25 µl blood samples were collected to measure fluorescence. Blood concentration of annexin-variant was calculated using the constructed reference curves. Panel A shows the blood clearance of anxA5 (grey squares), M1234 (black circles), anxA5-NP (black triangles) and M1234-NP (grey triangles). Half-lives were calculated by mono-exponential fit (B). *p<0.05, **p<0.01, ***p<0.001. 2 nmoles of fluorescently labeled anxA5 (black bars, panel C), M1234 (grey bars, panel C), anxA5-NP (black bars, panel D) and M1234-NP (grey bars, panel D) were injected intravenously into tumor-bearing NMRI nude mice. 1 hour (panel C) and 24 hours (panel D) post-injection organs were collected, weighed and measured for fluorescence. Concentration of annexin-variant were calculated using the constructed reference curves.</p

Non-invasive optical imaging of kinetics of tumor-uptake of fluorescently labeled annexin-variants in tumor bearing NMRI nude mice untreated (–CYP) or treated with cyclophosphamide (+CYP).

<p>Panels A and C show representative pictures of tumors of mice injected with anxA5 and M1234 (A), and anxA5-NP and M1234-NP (C). Panel B illustrates the time courses of tumor levels of anxA5 (closed circles, –CYP; open circles, +CYP) and M1234 (closed squares, –CYP; open squares, +CYP). The inset shows the time-courses during the first 10 hours. Panel D represents the time-courses of tumor levels of anxA5-NP (closed circles, –CYP; open circles, +CYP) and M1234 (closed squares, –CYP; open squares, +CYP).</p

Duration of VKA treatment significantly increases the amount of coronary calcification and number of calcified stenotic plaques.

<p>A non-enhanced scan was performed to measure the Coronary artery calcification (Agatston score). Panel A shows a significant increase in Agatston score in VKA users (p = 0.029), as patients use VKA for a longer time (T = tertile of VKA use). This increase is not visible in individually matched patients not on VKA (panel B; p = 0.965). Next, CT-angiography was performed. All coronary segments were assessed for plaque presence and plaque morphology. In patients using VKA, a significant trend (p = 0.009) was seen towards a higher percentage of calcified plaques in those patients treated longest with VKA (panel C). In contrast, this was not the case for the non-VKA users (panel D). Coronary plaques were identified and categorized as calcified (closed circles), mixed calcified (diamonds) and non-calcified (open circles). T1, T2 and T3 indicate 1^st, 2^nd and 3^rd tertile respectively.</p

Warfarin treatment of apoE deficient mice on Western Type Diet does not influence plaque size but increases vascular calcification.

<p>ApoE^−/− mice developed atherosclerotic lesions in the aortic arch and carotid arteries when maintained on Western type diet (WTD) for 12 weeks (A). From baseline mice were fed with WTD plus vitamin K (VK₁) or WTD plus vitamin K and warfarin (VK₁&W) for the duration of one week or four weeks. Growth of intimal area was not significantly affected by warfarin. Vascular calcium was determined by AAS and revealed significant increase in calcium at 1 and 4 weeks warfarin treatment. Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05.</p