Comparison of iron deficiency classifications.

<p>For patients presenting between 2006 and 2010, iron deficiency was assigned as absent (No ID) if ferritin, iron and T<i>f</i>SI were all clearly in the normal range (ferritin>20 µg/L, serum iron>11 µmol/L and T<i>f</i>SI>20%, N = 93). The groupings to examine iron deficiency were ferritin <15 µg/L (ferr<15, N = 20); ferritin <20 µg/L (ferr<20, N = 26); and serum iron <11 µmol/L (iron <11, N = 50). BMI, body mass index. MCV, mean corpuscular volume. MCH, mean corpuscular haemoglobin. MCHC, mean corpuscular haemoglobin concentration. T<i>f</i>SI, transferrin saturation index.</p

Stroke incidence.

<p><b>A</b>) Cumulative survival until first stroke: The solid line is modelled from the 129 patients with serum iron <8 µmol/L; dotted line from the 161 patients with serum iron >19 µmol/L. Shaded areas indicate 95% confidence intervals. <b>B</b>) Comparison of the stroke risk ROC models from myocardial infarction and serum iron (base model, black line/symbols), and strongest model generated from captured physiological variables, excluding the outcome measure of myocardial infarction (red line/symbols). The two models provide equivalent areas under the curve of 0.65 and 0·66 (p = 0.88). In the physiological ROC model, stroke risk was higher not only with lower serum iron (OR 0.95 [95% CI 0.90, 1.01]), but also with lower PAP(mean) (OR 0.94 [0.86, 1.03]); higher fibrinogen (OR 1.50 [0.95, 2.33]), lower SaO₂ (OR 0.98 [95% CI 0.93, 1·03]), and in women (OR 1.57 [0.71, 3.47]).</p

Right-to-left shunt and hypoxaemia evaluations.

<p><b>A</b>: Cartoon of the circulations indicating site of the pulmonary capillary filter, the dual pulmonary and bronchial/systemic arterial supply to lung tissue, and a pulmonary arteriovenous malformations (PAVM, red arrow). <b>B</b>: Relationship between quantified right-to-left shunt (measured using with ^99mTc-labelled albumin macroaggregates (10–80 µm) or microspheres (7–25 µm)), with same-day oxygen saturation (SaO₂), representing 309 paired values in 198 individuals since 1999. The linear regression coefficient of −1.22 (95% CI −1.31, −1.14; p<0.0001) indicates a strong relationship that explains 73% of the total variance in erect SaO₂ (adjusted r² 0.73). The shunt explained a smaller proportion of the total variance in supine SaO₂ (adjusted r² 0.54, data not shown). <b>C–F</b>: Representative right lateral brain images following injection of ^99mTc-labelled albumin macroaggregates for shunt diagnosis and quantification: <b>C</b>) R-L shunt 48.8% of the cardiac output, associated with a resting SaO₂ of 59%. <b>D</b>) R-L shunt 25%; SaO₂ 83%. <b>E</b>) R-L shunt 7.7%; SaO₂ 93.7%. Note the intense activity in the lung apices as expected. <b>F</b>) R-L shunt 3.3%; SaO₂ 96%. Note that the gain has been turned up but no cerebral activity is visible. This is the same individual as in <b>D</b>), with the images taken 6 months before (<b>D</b>) and 3 months after (<b>F</b>) embolisation which obliterated the causative PAVMs.</p

Details of clinical ischaemic strokes.

<p>*Note that silent lacunar infarcts, or silent infarcts at other sites, were excluded by the study methodology. Data exclude four iatrogenic strokes of known aetiology (one following thrombus injection through giving set; one at time of cerebral angiography; one at time of pulmonary angiography; and one progressive following stereotactic radiotherapy), but otherwise include all first clinical strokes of ischaemic aetiology.</p

Representative platelet dose response curves.

<p><b>A</b>) Typical control and iron deficient responses to ADP at 5 (blue), 10 (green), 20 (red) and 50 (black)µM. <b>B</b>) Typical control dose response curves to 5HT at 20 (blue), 200 (green), 2,000 (red) and 20,000 (black) µM–note the absence of the secondary wave of aggregation. <b>C</b>) Representative 5HT dose response curves displaying delayed secondary wave of aggregation observed in all severely iron deficient patients (hemoglobins 5.0–7.5 g/dl). The traces illustrated (5HT at 20 (blue), 200 (green), 2,000 (red) and 20,000 (black)) µM were from an individual with ferritin 4 µg/L, iron 3 µmol/l, hemoglobin 7.5 g/dl. <b>D</b>) Traces from the same individual as in (<b>C</b>), following a 6 month course of iron that resulted in improved iron indices (ferritin 31 µg/L, iron 7 µmol/l, hemoglobin 10.5 g/dl). Note that despite further treatments, iron deficiency persisted due to ongoing hemorrhagic losses. Aggregation curves are displayed for 5HT at 20 (black), 200 (green), 2,000 (red) and 20,000 (blue)µM.</p

SaO₂ measurement reproducibility.

<p>SaO₂ values measured by pulse oximetry following 7, 8, 9 and10 minutes standing. The variability within these measurements has not been presented previously. 522 consecutive datasets for the 165 PAVM patients first presenting between 2006 and 2010 were analysed and represent their datasets at presentation and in follow up. To illustrate reproducibility across all severities of hypoxaemia, datasets were divided into quintiles based on SaO₂, each with over 100 datasets. SD, standard deviation.</p

Comparison of platelet dose response curves in response to agonists in iron deficient patients and controls.

<p>Solid lines indicate controls; dotted lines represent the iron deficient group. Error bars represent standard error of the mean. <b>A</b>) Total aggregation in response to ADP at 5–50 µmol/L. <b>B</b>) Rate of aggregation in response to ADP. Since circulating blood should spend less than two seconds between pulmonary transits, the rate of aggregation may be particularly relevant. <b>C</b>) Total aggregation in response to 5HT. <b>D</b>) Rate of aggregation in response to 5HT.</p

Adjusted odds ratios for ischaemic stroke risk.

<p>Full list of age/gender, and serum iron-adjusted odds ratios for ischaemic stroke risk for the specified variable, where an inverse association with stroke risk is indicated by an odds ratio <1. N: number of datasets available. CI, confidence intervals, AF, atrial fibrillation. Pseudo r² indicates the proportion of stroke variance explained by age, gender, and the specified variable. P values were calculated by the non parametric Wald test which does not assume independence of variables. † Quadratic regression plots for stroke risk versus serum iron or SaO₂ presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088812#pone.0088812.s001" target="_blank">Figure S1</a>. Note <i>p</i> = 0.047 significant at FDR = 0.05 level.</p

Immunofluorescence images of FVIII expression by EC.

<p><b>a</b>: Sequential confocal fluorescence microscopy images in primary human EC. <b>a</b> and <b>b</b>: Representative HPMEC and HPAEC control images using To-Pro-3 nuclear counterstain (first panel, monochrome), murine control IgG₁ (second panel monochrome) and merged images (third panel: To-Pro-3 nuclear counterstain white, control IgG₁ red) using maximum gain used for imaging. <b>c</b>) Comparison of proportion of <b>i</b>) <b>HPMEC</b> and <b>ii</b>) <b>HPAEC</b> expressing vWF and FVIII protein (expression levels defined in methods). <b>d, e, f, g</b>) Sequential confocal fluorescence microscopy images comparing vWF (monochrome in first panel); FVIII (monochrome in second panel, specific mAb as denoted), and merged images (third panel; vWF green, anti-FVIII reacting protein red) in <b>d</b>): HPMEC, and <b>e, f, g</b>): HPAEC. FVIII mAb images displayed here are representative of all FVIII mAbs examined, and all cell lots. Note yellow merged images suggesting degree of FVIII/vWF colocalisation in <b>e, f, g</b>, with white colouring denoting the nuclei (TO-PRO3 nuclear counterstain). Scale bars indicate 5 µm.</p

FVIII splice isoforms.

<p><b>a: Variants identified by ExonMine</b>: Variants 1 and 2 correspond to major and minor RefSeq isoforms; variants 3–5 to expressed sequence tag (EST) sequences deposited in Genbank. Note none of the alternate variants encode the FVIII mAb epitopes. <b>b: Variants identified in EC</b>. Simultaneous expression of variants 1–4 in HPAEC (PA), HPMEC (PM), and HUVEC (H). Gels: φx, HaeIII-digested φx marker, C^A negative water control for HPAEC/HUVEC, C^M negative water control for HPMEC. The apparent difference in size of variant 4 is an artefact due to gel running (note differential site of 194 marker band in first and last lanes). Cartoons: Thin and thick arrows indicate sites of PCR and sequencing oligonucleotide primers respectively. Sequence chromatograms were obtained using nested reverse internal primers in exon 23 (variants 1–3) or exon 3 (variant 4; low concentration first round product sequenced). Note V5 sequences (exons U1-1-2-3) were not amplified from EC in any reaction.</p