Erythrocytes adherent to activated ECs align in the flow direction.

<p>(A) Ionomycin- (1 μM) treated (1 hour) erythrocytes perfused (10 minutes) without (left panel) or with histamine (100 μM) (right panel) over HUVECs. (B) Ionomycin-treated (1 hour) erythrocytes perfused without (left panel) or with (right panel) thrombin (1 U/ml) over HUVECs. The dashed boxes correspond to the zoomed regions. The arrow indicates the direction of the flow. (C) Ionomycin-treated erythrocytes perfused with histamine over HUVECs. Adherent erythrocytes are marked by (*). (D) Platelets perfused (10 minutes) with histamine (100 μM) over HUVECs. Adherent platelets are marked by arrowheads. (E) Ionomycin-treated erythrocytes mixed with monoclonal anti-VWF CLB-RAg20-coupled fluoresbrite YG microspheres (∅ 3 μm) perfused (10 minutes) with histamine over HUVECs. Scale bars represent 50 μm.</p

VWF mediates erythrocyte adhesion to ECs.

<p>(A) Ionomycin- (1 μM) treated (1 hour) erythrocytes labeled with PKH67 (green), mixed with Alexa Fluor^®568-labelled anti-VWF antibody (red) perfused (10 minutes) with histamine (100 μM) over HUVECs. The dashed box corresponds to the zoomed region. Scale bar represents 50 μm. (B) Quantification of adhesion of ionomycin-treated erythrocytes perfused (10 minutes) with histamine over HUVECs (Ctrl), followed by 10 minutes perfusion with (i) control buffer (Buffer), (ii) 5% autologous plasma (Plasma), or (iii) 5 μg/ml ADAMTS13 (ADAMTS13). (C) Quantification of adhesion of ionomycin-treated erythrocytes perfused with histamine over HUVECs transduced with lentivirus encoding a control short hairpin (shCtrl) or a short hairpin against VWF (shVWF). Adherent erythrocytes were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.g001" target="_blank">Fig 1</a>. (D) Western blot and densitometric quantification of VWF levels in cell lysates of the lentivirally transduced HUVECs. Values are means ± SD (n = 3; Each n represents single independent experiments using cells from different donors). Statistical analysis was performed using a 2-tailed Student's <i>t</i> test. * <i>P</i> < 0.05; ** <i>P</i> < 0.01.</p

Intracellular calcium accumulation potentiates erythrocyte adhesion to ECs.

<p>(A) Schematic representation of the different experimental conditions used to analyze the role of intra- and extracellular calcium for erythrocyte adhesion to ECs. (i) Ionomycin- (1 μM) treated (1 hour) erythrocytes perfused (10 minutes) using a calcium-rich buffer. (ii) Ionomycin plus EGTA- (5 mM) treated erythrocytes perfused using a calcium-rich buffer. (iii) Ionomycin-treated erythrocytes perfused using an EGTA-supplemented buffer. (I = Ionomycin, E = EGTA) (B) Quantification of adhesion of erythrocytes treated as described in panel A perfused (10 minutes) with histamine (100 μM) over HUVECs. (C) Quantification of adhesion of erythrocytes loaded with increasing concentrations of calcium perfused (10 minutes) with histamine (100 μM) over HUVECs. Adherent erythrocytes were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.g001" target="_blank">Fig 1</a>. Values are means ± SD (n = 4; Each n represents single independent experiments using cells from different donors). Statistical analysis was performed using a 1-way ANOVA (B) or a 1-way ANOVA in which all means were compared with 0 μM calcium (C) each followed by Bonferroni's post-hoc test. * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001; **** <i>P</i> < 0.0001.</p

Blood cell counts measured from the washed erythrocyte concentrates used in Fig 1C and 1D.

<p>Blood cell counts measured from the washed erythrocyte concentrates used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.g001" target="_blank">Fig 1C and 1D</a>.</p

Erythrocytes bind VWF through a platelet-independent mechanism.

<p>(A) Ionomycin- (1 μM) treated (1 hour) erythrocytes, mixed with platelets and perfused (10 minutes) with histamine (100 μM) over HUVECs. An example of an adherent platelet is marked by (*) and an adherent erythrocyte is marked by an arrowhead. (B) Ionomycin-treated erythrocytes labeled with anti-CD235a-FITC (green), mixed with anti-CD42b-APC-labeled platelets (magenta) and Alexa Fluor^®568-labelled anti-VWF antibody (red) were perfused with histamine over HUVECs. Adherent erythrocytes were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.g001" target="_blank">Fig 1</a>. Values are means ± SD (n = 3; Each n represents single independent experiments using cells from different donors). Statistical analysis was performed using a 1-way ANOVA in which all means were compared with 0 platelets ml^-1 followed by Bonferroni's post-hoc test. (C) Erythrocytes were purified by FACS. The erythrocyte population based on cell size (i) was positive sorted for CD235a-FITC and negative sorted for CD42b-APC (ii). Purified erythrocytes (10⁷ cells/ml) treated (1 hour) with ionomycin (1 μM) (green), mixed with Alexa Fluor^®568-labelled anti-VWF antibody (red) were perfused with histamine (100 μM) over HUVECs. (iii) The dashed box corresponds to the zoomed region. Scale bars represent 50 μm.</p

Erythrocyte binding to ECs.

<p>(A) HUVECs cultured in perfusion chambers and stained for VE-Cadherin (red) and nuclei (blue). The dashed box corresponds to the zoomed region. Scale bar represents 50 μm. (B) Erythrocytes treated for 1 hour with ionomycin (1 μM) were perfused over histamine- (100 μM) activated HUVECs (37°C and 5% CO₂). Adhesion was recorded for 10 minutes. Images are stills from the video in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.s011" target="_blank">S1 Video</a> at the indicated times in minutes. The arrow indicates the direction of the flow. The dashed box corresponds to the zoomed region. Scale bar represents 50 μm. (C) Quantification of adhesion of ionomycin- (1 μM) or control-treated (1 hour) erythrocytes perfused (10 minutes) with or without histamine (100 μM) over HUVECs. A schematic outline of the quantification method is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.s001" target="_blank">S1 Fig</a>. (D) Ionomycin- or control-treated erythrocytes perfused with or without thrombin (1 U/mL) over HUVECs. (E) Ionomycin- or control-treated erythrocytes perfused over TNF-α- (10 ng/ml) or control-treated (24 hours) HUVECs. (F) Kinetics of ionomycin- or control-treated erythrocyte accumulation after perfusion with or without histamine (100 μM) over HUVECs (Control-Control: ●; Histamine-Control: ○; Control-Ionomycin: ■; Histamine-Ionomcyin □). Adhesion of erythrocytes from 3 independent donors was quantified at a 1 minute time interval for 10 minutes. For C-E the average number of adherent erythrocytes within 15 fields of view (FOV; 1 FOV = 419.23x319.41 μm) was used. For F the absolute number of adherent erythrocytes within 1 field of view was used. Values are means ± SD (n = 3; Each n represents single independent experiments using cells from different donors). Statistical analysis was performed using a 2-way ANOVA in which all means were compared with the double-blank control followed by Bonferroni's post-hoc test. ** <i>P</i> < 0.01; **** <i>P</i> < 0.0001.</p

VWF levels determine binding of calcium-loaded erythrocytes to ECs.

<p>(A) Quantification of adhesion of ionomycin- (1 μM) or control-treated (1 hour) erythrocytes perfused (10 minutes) with or without histamine (100 μM) over HUVECs, (B) BOECs, (C) HAECs, or (D) the cell line HMEC-1. Adherent erythrocytes were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.g001" target="_blank">Fig 1</a>. (E) Quantification of VWF-positive area per cell based on VE-Cadherin and VWF stained HUVECs, BOECs, HAECs, and the cell line HMEC-1. (F) Intracellular VWF levels and (G) secreted VWF levels after 10 minutes induction by histamine (100 μM) from HUVECs, BOECs, HAECs, and the cell line HMEC-1 measured by ELISA. (H) The absolute number of ionomycin-treated erythrocytes adherent to histamine-treated ECs (obtained from A-D) are plotted against secreted VWF levels (pmol) (obtained from G). Different EC types are marked: ● HUVEC; ▲ BOEC; ■ HAEC; ▼ HMEC1. Values are means ± SD (n = 3; Each n represents single independent experiments using cells from different donors). Statistical analysis was performed using a 2-way ANOVA in which all means were compared with the double-blank control (A-D) or a 1-way ANOVA in which all means were compared with HUVECs (E-G) each followed by Bonferroni's post-hoc test. * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001; **** <i>P</i> < 0.0001.</p

A double hit model describes erythrocyte adhesion to vascular ECs.

<p>(A) A single hit model for the adhesion of circulating cells to activated endothelium is shown. Endothelial activation by histamine [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.ref022" target="_blank">22</a>] or thrombin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.ref021" target="_blank">21</a>] leads to the release of VWF from Weibel-Palade bodies. Platelets adhere to VWF under flow and thereby contribute to primary hemostasis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173077#pone.0173077.ref027" target="_blank">27</a>]. Normal erythrocytes do not adhere to VWF and remain in circulation. (B) Double hit model for adhesion of circulating cells to ECs. ECs, activated by histamine or thrombin, release VWF from Weibel-Palade bodies. Platelets adhere to the VWF strings. In addition, calcium-loaded erythrocytes can adhere independently of platelets to VWF strings. Micro-thrombi-like structures are formed.</p