Confirmation with light transmission aggregometry.

<p>Maximal light transmission, i.e. platelet aggregation in platelet-rich plasma from healthy control subjects (A), CAD patients treated with ASA alone (B), and clopidogrel responders (C) and low-responders (D) among CAD patients treated with ASA and clopidogrel. The following agonist/antagonist concentrations were used: 32 µM TRAP, 6.5 µM ADP, 50 µM serotonin, 10 µM ketanserin. * p<0.05 and ** p<0.01 in ANOVA followed by Bonferroni's multiple comparison test.</p

Serotonin enhances ADP-induced aggregation in clopidogrel responders and ketanserin reverts high on-treatment reactivity in clopidogrel low-responders.

<p>Multiple electrode aggregometry of whole blood from clopidogrel responders (A, mean AUC ±standard deviation after incubation with ADP: 280±86 AU/min) and low-responders (B, AUC with ADP 512±127 AU/min) normalized to the response induced by TRAP. The following agonist/antagonist concentrations were used: 32 µM TRAP, 6.5 µM ADP, 1, 50, or 100 µM serotonin, 10 µM ketanserin. ** p<0.01 and *** p<0.001 in ANOVA followed by Bonferroni's multiple comparison test.</p

Serotonin increases platelet reactivity in whole blood impedance aggregometry.

<p>Whole blood from healthy control subjects (A, n = 10), coronary artery disease (CAD) patients treated with ASA alone (B, n = 9), and the total study cohort of CAD patients treated with ASA and clopidogrel (C, n = 42) after incubation with the depicted substances. The following agonist/antagonist concentrations were used: 32 µM TRAP, 0.5 mM AA, 50 µM serotonin, 10 µM ketanserin. * p<0.05 in ANOVA followed by Bonferroni's multiple comparison test.</p

Serotonin release.

<p>Agonist-induced serotonin release was measured by high-sensitivity ELISA in a subset of 13 CAD patients treated with ASA and clopidogrel, among whom 9 were identified as clopidogrel responders (A) and 4 as low-responders (B). The following agonist/antagonist concentrations were used: 32 µM TRAP, 6.5 µM ADP, 10 µM ketanserin. * p<0.05 in ANOVA followed by Bonferroni's multiple comparison test.</p

Baseline clinical characteristics of CAD patients treated with ASA only and CAD patients treated with ASA and clopidogrel.

<p>Healthy control subjects (n = 10) were 34±4 years old, 5 were male, 1 smoked and none suffered from the mentioned diseases. <i>P</i> values for the comparison of CAD patients with ASA only vs. all CAD patients with ASA+clopidogrel and for the comparison of clopidogrel low-responders vs. responders. Number of patients (%), unless otherwise indicated.</p

E-selectin expression is upregulated in the presence of peripheral serotonin.

<p>Immunofluorescence for E-selectin (red) and nuclei (blue). Mesenteric venules at baseline conditions (A), after fluoxetine treatment (B), after serotonin challenge (C) and after lipopolysaccharide stimulation (D) of WT mice. Mesenteric venules of Tph1−/− mice at baseline (E), after fluoxetine treatment (F), after serotonin challenge (G) and after lipopolysaccharide stimulation (H). Semi quantification of fluorescence-levels for E-selectin (I). Scale bar = 50 µm; Flx = fluoxetine, LPS = lipopolysaccharide. ** = p<0.001.</p

Leukocyte-endothelial interactions were amplified by acute fluoxetine treatment without further stimulation.

<p>Rhodamine-stained leukocyte rolling (A), velocity (B) and adhesion (C) in resting mesenteric veins and representative intravitalmicroscopy images of vehicle- (D) and fluoxetine-treated (E) WT mice (n = 10). Leukocyte rolling (F), velocity (G) and adhesion (H) in resting mesenteric veins and representative intravitalmicroscopy images of vehicle- (I) and fluoxetine-treated (J) Tph1−/− mice (n = 10). Rhodamine-stained leukocyte rolling (K), velocity (L) and adhesion (M) in lipopolysaccharide stimulated mesenteric veins 4 hours prior to investigation and representative intravitalmicroscopy images of vehicle- (N) and fluoxetine-treated (O) WT mice (n = 10). n.s. = not significant. Flx = fluoxetine.</p

Serotonin levels in serum and plasma.

<p>Serotonin concentrations in serum (A), plasma (B) and supernatant of PRP after fluoxetine challenge (C) measured by ELISA (n = 14). n.s. = not significant, * = p<0.01.</p

Neutrophil extravasation is unaltered after acute fluoxetine treatment in vivo.

<p>Gating strategies for Gr-1 neutrophil count with flow cytometry (A). Number of Gr-1 positive neutrophils in abdominal lavages of vehicle- and fluoxetine-treated WT mice 4 hours after intraperitoneal injection of 4% thioglycollate or vehicle (B, n = 9–10). n.s. = not significant.</p

<i>Tph1</i> (−/−) mice have a normal response to social novelty.

<p><i>Tph1</i> (−/−) mice demonstrate similar responses to a stranger mouse (A) and to a novel mouse (B). The percentage of time that the test mouse spent exploring stranger 1 was comparable to age-matched WT animals (A) and the time that the test mouse spent exploring stranger 2 was comparable to age-matched WT mice (B).</p