DioxolaneA3-phosphatidylethanolamines are generated by human platelets and stimulate neutrophil integrin expression

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1 ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.


Introduction
Platelets generate eicosanoids including thromboxane A 2 (TXA 2 ), 12-hydroxyeicosatetraenoic acid , and small amounts of prostaglandins (PGs), PGE 2 , and D 2 . We recently described a new platelet eicosanoid, proposed to be 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA 3 ) [1]. Full structural characterization of this lipid remains to be completed once we have sufficient quantities purified from platelets or COX reactions. Importantly, we found that DXA 3 both primes and activates human neutrophils at nM-μM concentrations [1].
Platelets also form enzymatically-oxidized phospholipids (eoxPL) that contain PGs or HETEs at the sn2 position [2,3]. These generally comprise phosphatidylethanolamines (PE) with 16:0p, 18:1p, 18:0p or 18:0a predominating at sn1. We recently showed that thrombin-activated platelets generate over 100 diverse eoxPL on acute activation of platelets, including n3 and n6 fatty acids, and both single and multiply oxygenated forms at sn2 [4]. This indicated that eoxPL formation is a rapid and wide-ranging process of importance to innate immunity. In support, HETE-PEs can modulate a number of relevant events in vitro including enhancing clotting factor activities, modulating monocyte cytokine generation and enhancing neutrophil antibacterial actions [3,5,6]. Furthermore, two recent studies have shown a critical role for these lipids in mediating ferroptotic cell death [7,8].
Herein, we describe the detailed cellular and enzymatic biosynthesis pathways for four PE-esterified forms of DXA 3 . A co-ordinated series of enzymes and signaling mediators are required, including the fast esterification of newly formed DXA 3 free acid. A quantitative assay showed the majority of platelet DXA 3 generated on thrombin activation is PE-esterified. DXA 3 -PEs remain cell associated, are detected in human clots and activate neutrophil integrin expression, independently of their hydrolysis to the free acid analog. In summary, DXA 3 -PEs are platelet-derived lipids that can activate neutrophils adding to the growing evidence for enzymatic phospholipid oxidation as a physiological process of importance during early innate immunity.

Isolation of human platelets
Human blood donations were approved by the Cardiff University School of Medicine Ethics Committee and were with informed consent (SMREC 12/37, SMREC 12/10), and according to the Declaration of Helsinki. Exclusion criteria was a known sensitivity to aspirin. For studies on isolated platelets, whole blood was collected from healthy volunteers free from non-steroidal anti-inflammatory drugs for at least 14 days into acid-citrate-dextrose (ACD; 85 mmol/L trisodium citrate, 65 mmol/L citric acid, 100 mmol/L glucose) (blood:ACD, 8.1:1.9, v/v) and centrifuged at 250g for 10 min at room temperature. Platelet-rich plasma was collected and centrifuged at 900g for 10 min, and the pellet resuspended in Tyrode's buffer (134 mmol/L NaCl, 12 mmol/L NaHCO 3 , 2.9 mmol/L KCl, 0.34 mmol/L Na 2 HPO 4 , 1.0 mmol/L MgCl 2 ,10 mmol/L Hepes, 5 mmol/L glucose, pH 7.4) containing ACD (9:1, v/v). Platelets were centrifuged at 800g for 10 min then resuspended in Tyrode's buffer at 2×108 ml −1 . Platelets were activated at 37°C in the presence of 1 mmol/L CaCl 2 for varying times, with 0.2 U ml −1 thrombin, 10 μg/ml collagen, 10 μmol/L A23187, 20 μmol/ L TFLLR-NH 2 , or 150 μmol/L AY-NH 2 before lipid extraction as below. Experiments involving signaling inhibitors included a 10 min preincubation at room temperature. In some experiments, calcium was omitted from buffers. For separation of cells from microparticles, platelets were centrifuged at 970g for 5 min, then supernatants respun at 16,060g for 5 min. For aspirin supplementation, blood samples were first obtained following a 14-day NSAID-free period for baseline determinations of eicosanoids. Subjects were administered 75 mg/day aspirin for 7 days, then provided a second blood sample. Platelets were isolated and activated in vitro using 0.2 U/ml thrombin, as described above, then lipids extracted as described below.

Clot isolation
Blood was allowed to clot for 1 h at 24°C, and spun at 1730g for 10 min. Clot samples were placed in a pre-frozen mortar and pestle on dry ice and further cooled with liquid nitrogen. 250 mg clot was ground up in 3 ml PBS pH 7.4 containing 10 mM DTPA. Lipids were extracted from 1 ml samples using the method below.

Lipid extraction
Lipids were extracted by adding a solvent mixture (1 mol/L acetic acid, isopropyl alcohol, hexane (2:20:30, v/v/v)) to the sample at a ratio of 2.5-1 ml sample, vortexing, and then adding 2.5 ml of hexane [11]. Where quantitation was required, 5-10 ng PGE 2 -d4, and di-14:0phosphatidylethanolamine (DMPE) were added to samples before extraction, as internal standards. After vortexing and centrifugation, lipids were recovered in the upper hexane layer. The samples were then re-extracted by addition of an equal volume of hexane. The combined hexane layers were dried and analyzed for free or esterified PGs using LC-MS/MS as below.

Reversed phase LC-MS/MS and LC/MS 3 of esterified DXA 3
For MRM analysis, lipids were separated on a C 18 Luna, 3 µm, 150 mm×2 mm column (Phenomenex), using a gradient of 50-100% B over 10 min followed by 30 min at 100% B (Solvent A: methanol:acetonitrile:water, 1 mM ammonium acetate, 60:20:20. Solvent B: methanol, 1 mM ammonium acetate) with a flow rate of 200 μl/min. Esterified DXA 3 was monitored on a 4000 Q-Trap (Sciex) as precursors of m/z 770.6, 796.6, 798.6 and 814.7 fragmenting to product ions at m/z 351.2. For high resolution analysis, a reversed-phase UPLC Fourier Transform MS method was used (Thermo Scientific Orbitrap Elite). Analysis was performed using heated electrospray ionization (h-ESI) in negative ion mode at sheet, auxiliary and sweep gas flows of 70, 20, and 0, and capillary and source heater temperatures at 300 and 350°C, respectively. Data dependent MS 3 of m/z 351 from esterified DXA 3 was carried out in negative FTMS mode with a resolving power of 15,000. Lipid extracts were separated on a C18 Hypersil Gold, 1.9 µm, 100×2.1 mm column using a gradient (A, methanol/acetonitrile/water containing 1 mM ammonium acetate, at a ratio 60:20:20; B, methanol, 1 mM ammonium acetate) with flow rate 200 μl min -1 , starting at 50% B and maintaining for 10 min. The gradient increased to 100% B over 15 min and returning to initial conditions for 5 min. Orbitrap analysis was performed using heated electrospray ionization (h-ESI) in negative ion mode at sheath, auxiliary, and sweep gas flows of 30, 10, and 0, respectively. Capillary and source heater temperatures were 275 and 250°C, respectively, at 30,000 resolution, in full scan mode. LC/MS of precursor ions were monitored using accurate mass in FTMS mode. Negative MS/MS spectra were acquired using higher energy collisioninduced-dissociation (HCD). Data dependent MS 3 of m/z 351 was carried out in ITMS mode on the LTQ Ion Trap. Where fold changes are presented, data are shown as A/IS: analyte divided by internal standard.
2.5.2. Phospholipase A 2 Hydrolysis of esterified DXA 3 and quantitation of free acid form PE was purified from thrombin-activated platelet lipid extracts using normal phase HPLC as follows: extracts resuspended in normal phase solvents (50:50 of solvents A:B (A, hexane: propan-2-ol, 3:2; B, solvent A:water, 94.5:5.5)) were separated on a Spherisorb S5W 4.6×150-mm column (Waters Ltd., Estree, Hertfordshire, UK) using a gradient of 50-100% B over 25 min at a flow rate of 1.5 ml min −1 [12]. Absorbance was monitored at 205 nm and products identified by retention time comparison using a mixture of standard phospholipids (bovine brain PC and PE, 25 mg/ml). Fractions were collected at 30 s intervals and analyzed by direct flow injection, for subsequent analysis by ESI/MS/MS. Flow injection analysis of esterified DXA 3 s was performed by injecting 20 μl of each fraction under flow (1 ml min −1 ) in methanol into the electrospray source, with specific MRM transitions monitored using m/z 351 as the product ion, on a 4000 Q-trap. PE fractions were dried using N 2 , then resuspended in 1 ml buffer (150 mmol/L NaCl, 5 mmol/L CaCl 2 , 10 mmol/L Tris (Trizma base), pH 8.9). 200 μg snake venom phospholipase A 2 (PLA 2 ) from Sigma-Aldrich was added, and incubated for 60 min at 37°C. Lipids were reextracted as above, using hexane:isopropanol:acetic acid. As a purified standard is not yet available, we synthesized and purified a biogenic standard using COX-1 (Hinz et al., in review). Quantitation was achieved using PGE-d4 as internal standard. For analysis of free DXA 3 , the following HPLC conditions were used: C18 Spherisorb ODS2, 5 µm particle size, 150×4.6 mm (Waters Ltd., Elstree, Hertfordshire, UK) on a Sciex 4000 Q-Trap. The solvent system was 75% water, 25% acetonitrile, 1% glacial acetic acid Solvent A, 60% methanol, 40% acetonitrile, 1% glacial acetic acid at 1 ml min −1 Solvent B. Solvent B was increased from 50-90% over 20 min and at 25 min returned to 50% [13].

Isolation and activation of human neutrophils
Human neutrophils were isolated from 20 ml citrate anticoagulated whole blood, and resuspended in Krebs buffer. Briefly, blood was mixed 1:3 with 2% trisodium citrate (wt/vol) and HetaSep (Stemcell technologies) and allowed to sediment for 45 min at 20°C. The upper plasma layer was recovered and under laid with ice-cold Lymphoprep (2:1 for plasma/Lymphoprep) and centrifuged at 800g for 20 min at 4°C. The pellet was resuspended in ice-cold PBS and 0.4% sodium tricitrate (wt/ vol) and centrifuged at 400g for 5 min at 4°C. Contaminating erythrocytes were removed using up to three cycles of hypotonic lysis. Finally, cells were resuspended in a small volume of Krebs buffer (100 mmol/L NaCl, 50 mmol/L HEPES, 5 mmol/L KCl, 1 mmol/L MgCl 2 , 1 mmol/L NaH 2 PO 4 , 1 mmol/L CaCl 2 , and 2 mmol/L D-glucose, pH 7.4), counted and kept on ice. Neutrophils were diluted to 2×106 cells/ml and incubated with or without HPLC purified DXA 3 -PE (equivalent to lipids from 2×108 platelets/ml), vehicle (methanol or DMSO), 1 μM 1-stearoyl-2-arachidonyl-PE (SAPE), or 1 μM fMLP for 20 min at 37°C. In some experiments, neutrophils were pre-treated with 1.60 µM U-75302 (Cayman Chemicals) or 1 µM LY255283 (Sigma-Aldrich) for 10 min at room temperature. Cells were blocked using 5% mouse serum in PBS (containing 0.5% BSA, 5 mmol/L EDTA and 2 mmol/L sodium azide) for 1 h on ice and centrifuged at 320g for 5 min at 4°C. Anti-human CD11b-Alexa Fluor 647 (0.0625 μg, BioLegend) or isotype control were added and incubated for 30 min on ice. Neutrophils were washed twice with ice-cold PBS (containing 0.5% BSA, 5 mmol/L EDTA and 2 mmol/L sodium azide) and analyzed on a Cyan ADP flow cytometer (Beckman) and identified by forward and side scatter and Alexa Fluor 647.

Statistics
Data on platelets are representative of at least three separate donors, with samples run in triplicate for each experiment. Data are expressed as mean ± SEM, of three separate determinations. Statistical significance was assessed using an unpaired, two-tailed Students t-test. Where the difference between more than two sets of data was analyzed, one-way ANOVA was used followed by Bonferroni multiple comparisons test, as indicated on legends. p < 0.05 was considered statistically significant.

Human platelets generate phospholipid-esterified DXA 3 on acute activation
Previously we used precursor LC-MS/MS to screen for parents of m/z 351.2 in lipid extracts from thrombin-activated platelets, in order to find esterified prostaglandins. This demonstrated several series of ions consistent with four phosphatidylethanolamine (PE) species, namely 16:0p, 18:1p, 18:0p and 18:0a-PE, where p refers to plasmalogen and a to acyl, for the sn1 fatty acid [2]. Early eluting peaks were identified as esterified PGE 2 /D 2 , but others remained uncharacterised. Herein, a later eluting series are identified as DXA 3 -PEs using MS/MS and MS 3 both for the esterified forms and following cPLA 2 hydrolysis of purified platelet PE fractions (Figs. 1,2).
Using precursor to product m/z 351.2 in multiple reaction monitoring (MRM) mode, two lipids were detected for each PE parent ion ( Fig. 1A-D). However, when analyzed using high resolution Orbitrap MS, monitoring exact m/z, three lipids were seen (Fig. 1E-H). The first in each MS chromatogram represents co-eluting PGE 2 /D 2 -PEs  Fig. 2A-D) [1]. This showed characteristic product ions, including prominent ones at m/z 163, 165, and also 207, 225 and 271. To further prove DXA 3 was attached to PE, the lipids were purified using HPLC, then saponified using snake venom PLA 2 , as described in Materials and Methods. Analysis of saponified PE using the m/z 351.2 → 271.2 showed some PGE 2 and D 2 at 32.6 and 34.7 min respectively, however DXA 3 was seen at 51.5 min, with MS/MS confirming its structure (Fig. 2E,F). To quantify DXA 3 attached to PE, free DXA 3 in lipid extracts derived from thrombin-activated platelets was quantified before and after hydrolysis using snake venom PLA 2 (Fig. 3A). Total esterified DXA 3 in platelets from four donors was determined to be 24.3 ± 6.1 out of 31.6 ± 8.1 ng /2×108 platelets/ml (mean ± SEM). This indicates that the majority of DXA 3 is esterified, with structural data confirming the four lipids as 16:0p/DXA 3 -PE, 18:1p/DXA 3 -PE, 18:0p/ DXA 3 -PE and 18:0a/DXA 3 -PE.

Characterization of platelet signaling pathways involved in DXA 3 -PE generation
On platelet activation, DXA 3 -PE was generated early, in particular in response to thrombin where it plateaued after 10 min (Fig. 3B-D). DXA 3 -PE was retained by the cells, in contrast to free DXA 3 , which is primarily secreted, indicating that the two forms partition differently (Fig. 3E,F). DXA 3 -PE formation required PLC, but not PI3 kinase, while inhibition of PKC significantly increased DXA 3 -PE generation (Fig. 4A,B). The negative control (U-73373) for the PLC inhibitor (U-73112) showed approximately 50% inhibition, but far less than seen using U-73112 (Fig. 4A). This suggests some off target effects of U-73112, but supports PLC involvement in DXA 3 -PE generation. Formation of DXA 3 -PEs was dependent on intracellular but not extracellular calcium (Fig. 4C). An essential role for COX-1 was revealed through either in vivo (7 days of 75 mg/day aspirin) or in vitro inhibition (SC-560, aspirin, indomethacin) inhibition (Fig. 4D-F), while cPLA 2 but not iPLA 2 or sPLA 2 was also required (Fig. 5A,B). Last, formation of DXA 3 -PE was triggered via PAR-1 and −4 receptors (Fig. 5C). Notably, the timescale for formation of esterified DXA 3 is very similar to that of the free acid form with a fast generation within the first 10 min of activation [1].

Enzymatic mechanism of DXA 3 -PE formation by COX-1
DXA 3 -PE could form via direct PE oxidation or via oxidation of AA released by cPLA 2 followed by esterification into PE. The latter is more likely since COX-1 is unable to directly oxidize complex lipids, and DXA 3 -PE formation was sensitive to cPLA 2 inhibition. In support, thimerosal, an inhibitor of lysophospholipid acyl transferases (LPAT) elevated free DXA 3 , while suppressing DXA 3 -PE formation (Fig. 5D,E). This supports a mechanism whereby AA is hydrolyzed by cPLA 2 , oxidized by COX-1 to DXA 3 , then re-esterified into PE via LPAT enzymes. Triascin C, an inhibitor of fatty acyl Co-A ligase (FACL), inhibited formation of DXA 3 -PE, but also partially blocked free DXA 3 formation (Fig. 5F,G). COX-1 is generally considered unable to oxidize complex substrates. To confirm this, we incubated 18:0a/20:4-PE with COX-1. Hematin controls showed a small oxidation to form DXA 3 -PE, but this was not increased by inclusion of COX-1 (Fig. 6A,B). However, when AA was added, allowing COX-1 turnover, additional DXA 3 -PE was formed. As this was not sensitive to metal chelation, it likely results from secondary oxidation directly mediated by lipid radicals that escape the active site of COX-1 during turnover (Fig. 6A,B). We note that thimerosal blocked approx. 50% of the generation of DXA 3 -PEs (Fig. 5D). Thus, at least half of these lipids form in platelets via esterification, rather than radical-mediated oxidation.

DXA 3 -PE from human platelets activates human neutrophil integrin expression
DXA 3 -PE was isolated from activated platelet lipid extracts using HPLC. When added at concentrations approximating a physiological platelet concentration (equivalent to 2×108 cells/ml), neutrophil Mac-1 expression increased significantly (Fig. 6D,E). In contrast, the unoxidized analog, 1-stearoyl-2-arachidonyl-PE (SAPE) did not activate Mac-1 (Fig. 6D). When incubated for 30 min with neutrophils, no loss of DXA 3 -PE was seen, nor was free DXA 3 generated (Fig. 6F). This indicates that hydrolysis of DXA 3 -PE by neutrophil phospholipases did not occur and indicating that Mac-1 activation is stimulated directly by the esterified form. Instead, levels of DXA 3 -PE appeared to slightly increase, likely a matrix effect on extraction from the presence of neutrophil lipids and proteins. Last, to explore the signaling mechanisms, antagonists of leukotriene B4 receptors, BLT1 and BLT2 were included. However, neither prevented the expression of Mac-1 indicat-ing that DXA 3 -PE activates neutrophils by BLT receptor-independent mechanisms (Fig. 6G).

Discussion
Recent studies identified a platelet lipid, DXA 3 that forms via enzymatic oxidation of AA by COX-1 in response to physiological agonist activation. This lipid primes and activates human neutrophils at nM-μM concentrations in vitro suggesting its likely importance in innate immune responses [1]. In that study, only the free acid lipid was   Fig. 3. Quantification of DXA 3 released after hydrolysis of platelet PE and acute generation of DXA 3 -PE by human platelets, which is retained by the cells. Panel A. Quantification of DXA 3 released after hydrolysis shows higher levels in PE than free. Lipid extract of thrombin activated platelets was, hydrolyzed using snake venom PLA 2 and analyzed for free DXA 3 using LC-MS/MS before and after hydrolysis. Experiment was repeated on four separate donors, a representative donor is shown (n=3, mean ± SEM). Panels B-D. Generation of PEesterified DXA 3 by human platelets. Washed platelets were activated for varying times, using 0.2 unit.ml −1 thrombin, 10 μg/ml collagen, or 10 μmol/L A23187, then lipids extracted and analyzed using reverse-phase LC-MS/MS, monitoring precursor [M-H] -→ m/z 351.2 as described in Methods. Panels E,F. Esterified DXA 3 is retained by the cells while the free acid isomer is released. Thrombin-activated platelets were pelleted, then supernatant centrifuged at 16,060×g for 10 min to pellet microparticles before lipid extraction. DXA 3 -PEs and free DXA 3 were analyzed using reverse-phase LC-MS/MS, as described in Methods. A/IS: analyte:internal standard.

F E
described. Herein, we demonstrate that the majority of DXA 3 is initially formed esterified to four PEs, remains cell-associated and is generated in human clots in vitro. Like the free acid analog, DXA 3 -PE also activates neutrophils at physiologically-relevant concentrations. The study adds to the growing evidence that enzymatic phospholipid oxidation by platelets is a physiological process that occurs during blood clotting and hemostasis and modulates innate immunity [3]. DXA 3 -PE is formed on a similar timescale, within 2-10 min post agonist activation, as free DXA 3 (Fig. 3 B-D) and entirely from endogenous substrate mobilized during physiological platelet activa- Lipids were extracted and analyzed as described in Methods. Levels are expressed as ratio analyte to internal standard. Data are representative of experiments repeated at least three times on different donors (n=3, mean ± SEM). Panel F. In vivo aspirin supplementation blocks generation of DXA 3 -PE. Lipids were analyzed following thrombin activation of washed platelets, before or after supplementation with 75 mg/day aspirin for 7 days. Data are representative of five independent donors (n=5, mean ± SEM); ***p < 0.001 versus thrombin alone, using ANOVA and Bonferroni Post Hoc Test. Levels of DXA 3 -PEs are expressed as ratio analyte to internal standard. A/IS: analyte:internal standard. tion [1]. This indicates that a controlled enzymatic formation, similar to the generation of HETE-PEs by platelet 12-lipoxygenase (LOX), macrophage 12/15-LOX and neutrophil 5-LOX. Pharmacological studies indicated involvement of PLC, intracellular calcium, cPLA 2 and COX-1 [3,5,11]. These signaling mediators are also required for free DXA 3 formation demonstrating that PE-esterified forms require first the generation of the free acid form [1]. This was further supported by observations that an inhibitor of LPAT significantly blocked their formation (Fig. 5D,E). Activation by PAR agonists suggests the involvement of these receptors in thrombin activation, however thrombin can also activate additional receptors, including integrins. Platelets express several PKC isoforms, of which some promote and Washed platelets were activated with a PAR-1 agonist, TFLLR-NH 2 (20 μM), and/or a PAR-4 agonist, AY-NH 2 (150 μM), for 30 min at 37°C then analyzed, as described in Methods. Panels D,E. Generation of DXA 3 -PE is inhibited by thimerosal, while free DXA 3 is enhanced. Washed platelets were incubated with 75 µM of thimerosal for 30 min at 37°C prior to thrombin activation, before lipid extraction and analysis. Panels F,G. Generation of free and esterified DXA 3 -PE is inhibited by triascin C. Washed platelets were incubated with 7 µM of triascin C for 30 min at 37°C prior to thrombin activation, before lipid extraction and analysis. Levels are expressed as analyte:internal standard. Data are representative of experiments repeated at least three times on different donors (n=3, mean ± SEM). ***p < 0.001 versus thrombin, using ANOVA and Bonferroni Post Hoc Test. A/IS: analyte:internal standard. others inhibit platelet activation. For example, PKCα can be pro-while PKCδ can be anti-aggregatory [14]. Go6850 (at 100 nM) will inhibit a number of PKC isoforms, including α,β,δ and ε [15]. We found that this enhanced generation of DXA 3 -PE suggesting that overall, PKC isoforms are suppressing this pathway. Using purified COX-1, we showed that COX-1 does not directly oxidize 18:0a/20:4-PE (Fig. 6A). However, a small amount of co-oxidation could be mediated during AA oxidation. This suggests an additional route to DXA 3 -PE formation and could account for the thimerosal-insensitive generation seen in platelets (Fig. 5D), however it is likely that non-enzymatic routes to synthesis   would be tightly controlled and minimized in platelets via the action of glutathione peroxidases. The majority of DXA 3 is detected as esterified to PE in platelets (Fig. 3A). This contrasts with HETE-PEs of which only 30% is attached to phospholipids, and TXB 2 which has not been found to occur in esterified forms, and suggests that esterification pathways may favor certain oxidized lipids, but this remains to be fully investigated [3]. Recently, we used lipidomics to profile oxidized PL generated by human platelets and found over 100 species all generated on acute activation with thrombin [4]. One family, HETE-PEs, can translocate to the outside of the plasma membrane and is prothrombotic in vitro, raising the possibility that membrane PL oxidation is a general phenomenon that regulates innate immune events during acute injury [3].
Herein, DXA 3 -PE was found to activate neutrophil Mac-1 expression, independently of hydrolysis to free DXA 3 (Fig. 6D-H). This indicates that DXA 3 could potentially act either as a soluble mediator when free, or alternatively when remaining cell-associated, if facing out of the membrane surface and attached to PE (e.g. during plateletneutrophil interactions). This would be a new concept for eicosanoid signaling, since traditionally these were only thought of as soluble signaling agents. Previous, eicosanoids were not considered to be reesterified into phospholipids in significant amounts. This study showing DXA 3 esterification strengthens previous reports of formation of PGE 2 -and HETE-PE formation as a regulated biological process [2,3,5,6,11,16].
We suspected that DXA 3 -PE might activate LTB 4 receptors, either BLT1 or BLT2, since both are present in neutrophils and known to upregulate Mac-1 expression [17,18]. However, both were excluded using receptor antagonists (Fig. 6H). Neutrophils express many cell surface receptors, including other G protein coupled receptors, Tolllike receptors and integrins [17]. Thus DXA 3 -PE likely activates other pathways to upregulate Mac-1 expression and further studies are required to delineate this. Indeed whether the free and esterified forms of this lipid can also stimulate additional neutrophil activities including calcium mobilization, superoxide generation and degranulation, also will be determined.
Neutrophil phospholipids that have incorporated exogenous eicosanoids can be subsequently hydrolyzed by secondary cell stimulation, for example releasing free acid 15-HETE [19]. Thus, during blood clotting, phospholipases such as sPLA 2 could metabolise DXA 3 -PEs from activated platelets generating the free acid analog and thus further supporting innate immune responses to acute injury via soluble diffusion, with this lipid able to signal either in free or esterified forms. In summary, DXA 3 -PEs represent new platelet lipids generated through receptor-regulated cell signaling pathways. The full structural characterization of DXA 3 and generation of synthetic standards will enable further study of its functions in innate immunity.

Conflicts of interest
The authors declare no competing financial interests.