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. 2010 Oct;30(10):2005-13.
doi: 10.1161/ATVBAHA.110.209908. Epub 2010 Aug 11.

Resolvin E1 regulates adenosine diphosphate activation of human platelets

Gabrielle Fredman  1 , Thomas E Van Dyke, Charles N Serhan
Affiliations

Resolvin E1 regulates adenosine diphosphate activation of human platelets

Gabrielle Fredman et al. Arterioscler Thromb Vasc Biol. 2010 Oct.

Abstract

Objective: To investigate the ability of resolvin E1 (RvE1) to regulate adenosine diphosphate (ADP) activation of platelets via specific receptors because RvE1 reduces platelet aggregation with certain agonists, including ADP.

Methods and results: RvE1 is an eicosapentaenoic acid-derived specialized proresolving mediator generated during the resolution of acute inflammation. RvE1 exhibits potent organ-protective actions in vivo and acts on specific cell types, including platelets. RvE1, 0.1 to 100 nmol/L, incubated with platelets gave reduced ADP-stimulated P-selectin mobilization (IC(50), approximately 1.6×10(-12) mol/L) and polymerized actin content compared with control platelets. RvE1, 1 to 100 nmol/L, did not stimulate or block intracellular Ca(2+) mobilization. By using a new P2Y(12)-β-arrestin-coupled cell system, ADP-activated P2Y(12) with an EC(50) of 5×10(-6) mol/L and RvE1 did not directly stimulate P2Y(12) or block the ADP-P2Y(12) signals. In this system, another eicosanoid, leukotriene E(4) (LTE(4)) (EC(50), 1.3×10(-11) mol/L), dose dependently activated P2Y(12). When recombinant P2Y(12)-expressing cells were transiently transfected with an RvE1 receptor, human ChemR23 (present on human platelets), with the addition of RvE1 (0.1-10.0 nmol/L), blocked ADP signals (IC(50), approximately 1.6×10(-11) mol/L) in P2Y(12)-ChemR23-expressing cells compared with mock transfections.

Conclusions: RvE1's regulatory actions (ie, reducing ADP-stimulated P-selectin mobilization and actin polymerization) are human (h)ChemR23-dependent. Moreover, specific platelet actions of RvE1 selectively engaged with ADP-activated platelets that illuminate a new cellular mechanism and affect ω-3 eicosapentaenoic acid, which may contribute to both resolution of vascular inflammation and ADP-dependent platelet activation relevant in pathological cardiovascular events.

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Figures

Figure 1
Figure 1. RvE1 reduces ADP-stimulated P-selectin surface mobilization
PRP was incubated with either vehicle, (A) RvE1 (0.01–100 nM, blue), EPA (0.01–100 nM, red) (B) 19-para-flurorophenoxy-RvE1 (0.01–100 nM) was incubated for 15 minutes, 37°C, and ADP (10 μM) was added for 3 minutes, 37°C. Platelets were stained with PE-anti-human CD62P (P-selectin) and subjected to flow cytometry. (C) Representative histograms of PE anti-human CD62P surface expression. Vehicle (shaded), ADP (10μM) alone, solid line, RvE1 or 19-para-fluorophenoxy-RvE1 (dashed lines). (Ci) and (Ciii) indicate SPM alone while (Cii) and (Civ) display SPM + ADP. Histograms are representative of n=5–6 separate donors. (Cv) Representative histogram of ChemR23+ platelets. (Cvi) 19-para-fluourophenoxy-RvE1 (10 nM) reduction of ADP-stimulated platelet aggregation, representative of n=3. (D) Direct Comparison between PGE1 (0.01 nM-100 nM) and RvE1 at equi-molar concentrations. Results are mean ± SEM n=3, separate donors, *p<0.05, #p<0.03.
Figure 2
Figure 2. RvE1 reduces ADP-stimulated actin polymerization: Ca2+ independent
(A,B) PRP was incubated with either vehicle or RvE1 (100 nM) for 15 minutes, 37°C with gentle mixing then stimulated with ADP (10 μM) for 0, 15, 30, or 60 seconds. Incubation was stopped by the addition of ice-cold formalin (6%) and platelets were permeabilized and stained with FITC-phalloidin (1:100) for 1 hour, 4°C. Changes in platelet morphology were quantified via flow cytometry and Cell Quest software. (B) RvE1 (100nM) percent inhibition of ADP-stimulated actin polymerization. Results are mean±SEM n=3, *p≤ 0.03. (C,D) Fluorescent intracellular calcium indicator, Fura-2. (C) RvE1 (1–100nM) as compared to vehicle and ADP. (D) RvE1 (1–100nM) plus ADP as compared to vehicle. Fluorescence was monitored by an EnVision plate reader and accompanying software. Results are representative of n=3.
Figure 3
Figure 3. RvE1 reduces ADP-stimulated actin polymerization in MEG-01
(A–C) MEG-01 cells (1×105/treatment) were cultured and stimulated with vehicle, ADP (10 μM), RvE1 (100 nM) and ADP plus RvE1 for 0, 15, 30, 60 or 180 seconds, 37°C. Actin polymerization was visualized via fluorescent microscopy (Nikon Eclipse E600 and imported into Spot Diagnostic analysis software) (C) or quantified via flow cytometry (D,E). (A) Images are representative of n=3 and (B,C) values are mean ± SEM of n=3. (D) Representative gel of P2Y12, P2Y1, ChemR23 and BLT1 expression. Note that all products were 0.3kb with the exception of full-length human ChemR23, which was 1kb. (E) MEG-01 surface expression of ChemR23 (left, solid line) or BLT1 are shown as compared to their respective isotype matched controls (shaded). (F) Mock pcDNA3 or hChemR23 was transiently transfected into CHO cells using Lipofectamine 2000 for 48 hours, 37°C. CHO-Mock (grey bar) or CHO-hChemR23 (black bar) cells (1×105) were stimulated with RvE1 (10 nM) 15 minutes prior to ADP (10 μM) stimulation (60 minutes, 37°C). FITC-phalloidin fluorescence was assessed via an EnVision plate reader and software. Values are mean ± SEM of n=3, *p<0.01.
Figure 4
Figure 4. RvE1 counter regulates P2Y12 signaling
ADP (10−10 M – 10−4 M) added to CHO-P2Y12 (1×105 cells/well), RvE1 (10−10 M – 10−6 M), LTE4 (10−12 M – 10−8 M) or LTD4 (10−12 M – 10−8 M) were added (60 minutes at 37°C). All incubations were stopped with lysis buffer containing emerald substrate reagent for (60 minutes, 21°C). EC50 values were determined by curve fit analysis using GraphPad software (see methods). (A) ADP (squares) and RvE1 (triangles) dose response. Values are mean ± SEM of n=5. (B) RvE1 (10−10 M – 10−7 M), when added simultaneously with ADP. (C) LTE4 (circles) and LTD4 (triangles) dose response. Values are mean ± SEM of n=6. (D) LTE4 (10−9 M – 10−8 M) followed by ADP (10−5 M). Values are mean ± SEM , n=3, *p<0.05.
Figure 5
Figure 5. RvE1 regulates P2Y12 signaling in a ChemR23-dependant manner
(A,B) CHO-hP2Y12β-arrestin cells were transiently transected with mock-pcDNA3 or hChemR23 using FuGene transfection reagent for 48 hours, 37°C. Cells were plated (1×105 cells/well), and ADP (10−5 M) or ADP plus RvE1 (10−10 M – 10−8 M) were incubated for 60 minutes, 37°C. (A) Human ChemR23 transient transfection was monitored using flow cytometry. Histograms are representative of n=4. (B) Inhibition of P2Y12 by RvE1 was monitored in mock-pcDNA3 (grey bars) or hChemR23 (black bars) Values are mean ± SEM of n=4, * p<0.03, 2-way ANOVA with Bonferroni post testing. (C) Proposed Scheme for RvE1 actions on human platelets (see discussion for details).
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