Optimized plasma preparation is essential to monitor platelet-stored molecules in humans

<div><p>Platelets store a plethora of different molecules within their granules, modulating numerous pathways, not only in coagulation, but also in angiogenesis, wound healing, and inflammatory diseases. These molecules get rapidly released upon activation and therefore represent an easily accessible indirect marker for platelet activation. Accurate analysis of platelet-derived molecules in the plasma requires appropriate anticoagulation to avoid <i>in vitro</i> activation and subsequent degranulation of platelets, potentially causing artificially high levels and masking biologically relevant differences within translational research studies. However, there is still enormous heterogeneity among anticoagulants used to prevent unwanted platelet activation, so that plasma levels reported for platelet granule contents range over several orders of magnitude. To address this problem and to define the most robust method of plasma preparation to avoid <i>in vitro</i> platelet activation during processing, we compared plasma concentrations of the three platelet-stored factors thrombospondin (TSP-1), platelet factor 4 (PF4), and soluble P-selectin (sCD62P) between human blood samples anticoagulated with either citrate-theophylline-adenosine-dipyridamole (CTAD), acid-citrate-dextrose (ACD), citrate, ethylenediaminetetraacetic acid (EDTA) or heparin. Additionally, we assessed the effect of storage temperature and time between blood drawing and sample processing within the differentially anticoagulated samples. Our data strongly support the use of CTAD as anticoagulant for determining plasma concentrations of platelet-stored molecules, as anticoagulation with heparin or EDTA led to a 12.4- or 8.3-fold increase in plasma levels of PF4, respectively. Whereas ACD was similar effective as CTAD, citrate only showed comparable PF4 plasma levels when plasma was kept at 4°C. Moreover, blood sampling with CTAD as anticoagulant resulted in the most reproducible values, even when samples were processed at ambient temperature or after storage over 6 hours. In the latter case, anticoagulation with heparin or EDTA led to artificially high plasma levels indicative of <i>in vitro</i> platelet activation. Therefore, we want to raise scientific awareness for choosing CTAD as optimal anticoagulant for the detection of platelet-stored molecules in plasma.</p></div

Plasma levels of platelet-stored factors vary among different anticoagulants.

<p>Plasma levels of PF4 (A, C) and TSP-1 (B, D) were determined in 8 healthy individuals. Plasma was prepared from CTAD (black bar), ACD (dark grey bar), citrate (grey bar), heparin (white bar) or EDTA (patterned bar) blood at 4°C (A-B) or at room temperature (C-D) within 30 min after blood draw. Plasma concentration was measured using ELISA. Significant differences were analyzed using one-way ANOVA with Dunnett correction and were depicted as *p<0.05, **p<0.01, ***p<0.001 (in comparison to CTAD).</p

Fold-increase of platelet-stored factors due to suboptimal plasma generation.

<p>Plasma concentrations of PF4 (A, C) and TSP-1 (B, D) were calculated as fold of plasma levels relative to CTAD plasma 30 min 4°C (8 healthy donors). Fold-increase is depicted for ACD (dark grey bar), citrate (grey bar), heparin (white bar) or EDTA (patterned bar) plasma at 0.5 h, 2 h, 6 h, and 24 h. Samples were either processed at 4°C (A-B) or at ambient temperature (C-D). Significant differences were analyzed using one-way ANOVA with Dunnett correction and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (in comparison to CTAD 30 min 4°C).</p

Effect of <i>in vitro</i> platelet activation on concentration of plasma factors.

<p>Blood of 8 healthy donors was anticoagulated with CTAD, ACD, citrate, heparin or EDTA (grey bar) and stimulated with the platelet activator thrombin (dark grey bar) at a submaximal dose for 0.5 h and 6 h at room temperature. Subsequently, plasma was prepared and analyzed for PF4 (A, C) and TSP-1 (B, D) levels. Significant differences were analyzed using two-way ANOVA with Bonferroni correction and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (in comparison to untreated).</p

Time-dependent differences in plasma levels of platelet-stored factors.

<p>Blood from 8 healthy donors was anticoagulated with CTAD (black bar), ACD (dark grey bar), citrate (grey bar), heparin (white bar) or EDTA (patterned bar) and stored at 4°C (A-B) or at room temperature (C-D) for 0.5 h, 2 h, 6 h or 24 h until plasma preparation. Concentrations of PF4 (A, C) and TSP-1 (B, D) were determined for each time point. Significant differences were analyzed using two-way ANOVA with Dunnett correction (with CTAD as reference) and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.</p

Optimized plasma preparation is essential to monitor platelet-stored molecules in humans - Table 1

<p>Optimized plasma preparation is essential to monitor platelet-stored molecules in humans</p> - Table

Experimental set-up to compare the effect of different anticoagulants on plasma levels of platelet-stored factors.

<p>(A) Plasma preparation. Anticoagulated blood of 8 healthy volunteers was centrifuged at 1,000 x g and 4°C for 10 minutes. Supernatants were transferred to new tubes and centrifuged at 10,000 x g and 4°C for 10 minutes to generate platelet-free plasma. Supernatants of the second centrifugation step were aliquoted and stored at –80°C until further use. (B) Sampling. Blood of each donor was collected using CTAD (blue), ACD (pink), citrate (green), EDTA (red) and heparin (yellow) as anticoagulant. Half of the samples were kept either at 4°C or at room temperature. Plasma was generated at 0.5 h, 2 h, 6 h, and 24 h. To analyze the effect of <i>in vitro</i> platelet activation a subgroup of samples was stimulated with thrombin at a submaximal concentration and plasma was generated after 0.5 h and 6 h.</p

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Image_1_Activation of the Regulatory T-Cell/Indoleamine 2,3-Dioxygenase Axis Reduces Vascular Inflammation and Atherosclerosis in Hyperlipidemic Mice.PDF

<p>T-cell activation is characteristic during the development of atherosclerosis. While overall T-cell responses have been implicated in disease acceleration, regulatory T cells (Tregs) exhibit atheroprotective effects. The expression of the enzyme indoleamine 2,3-dioxygenase-1 (IDO1), which catalyzes the degradation of tryptophan (Trp) along the kynurenine pathway, has been implicated in the induction and expansion of Treg populations. Hence, Tregs can reciprocally promote IDO1 expression in dendritic cells (DCs) via reverse signaling mechanisms during antigen presentation. In this study, we hypothesize that triggering the “Treg/IDO axis” in the artery wall is atheroprotective. We show that apolipoprotein B100-pulsed tumor growth factor beta 2-treated tolerogenic DCs promote de novo FoxP3⁺ Treg expansion in vivo. This local increase in Treg numbers is associated with increased vascular IDO1 expression and a robust reduction in the atherosclerotic burden. Using human primary cell cultures, we show for the first time that IDO1 expression and activity can be regulated by cytotoxic T-lymphocyte associated protein-4, which is a constitutive molecule expressed and secreted by Tregs, in smooth muscle cells, endothelial cells, and macrophages. Altogether, our data suggest that Tregs and IDO1-mediated Trp metabolism can mutually regulate one another in the vessel wall to promote vascular tolerance mechanisms that limit inflammation and atherosclerosis.</p

Image_3_Activation of the Regulatory T-Cell/Indoleamine 2,3-Dioxygenase Axis Reduces Vascular Inflammation and Atherosclerosis in Hyperlipidemic Mice.PDF

<p>T-cell activation is characteristic during the development of atherosclerosis. While overall T-cell responses have been implicated in disease acceleration, regulatory T cells (Tregs) exhibit atheroprotective effects. The expression of the enzyme indoleamine 2,3-dioxygenase-1 (IDO1), which catalyzes the degradation of tryptophan (Trp) along the kynurenine pathway, has been implicated in the induction and expansion of Treg populations. Hence, Tregs can reciprocally promote IDO1 expression in dendritic cells (DCs) via reverse signaling mechanisms during antigen presentation. In this study, we hypothesize that triggering the “Treg/IDO axis” in the artery wall is atheroprotective. We show that apolipoprotein B100-pulsed tumor growth factor beta 2-treated tolerogenic DCs promote de novo FoxP3⁺ Treg expansion in vivo. This local increase in Treg numbers is associated with increased vascular IDO1 expression and a robust reduction in the atherosclerotic burden. Using human primary cell cultures, we show for the first time that IDO1 expression and activity can be regulated by cytotoxic T-lymphocyte associated protein-4, which is a constitutive molecule expressed and secreted by Tregs, in smooth muscle cells, endothelial cells, and macrophages. Altogether, our data suggest that Tregs and IDO1-mediated Trp metabolism can mutually regulate one another in the vessel wall to promote vascular tolerance mechanisms that limit inflammation and atherosclerosis.</p