Background
Aspirin (acetylsalicylic acid) is one of the most widely used non-steroidal anti-inflammatory drugs (NSAIDs). In addition to possessing anti-inflammatory properties at high doses (1 g/day) [
1] aspirin also demonstrates effective cardioprotective properties at lower doses (75-150 mg/day) [
2]. The proposed mechanism by which aspirin acts is by irreversible inhibition of cyclooxygenase 1 (COX-1) [
3]. Enzyme acetylation effectively blocks formation of the prostaglandin intermediates, PGG
2 and PGH
2 and their resultant downstream bioactive products [
4]. The cardioprotective anti-platelet property of low-dose aspirin is attributed to irreversible inhibition of synthesis of a pro-thrombotic COX-1-derived eicosanoid, thromboxane A
2 (TxA
2) [
5], which is derived from the polyunsaturated fatty acid substrate, arachidonic acid (AA).
Platelet specificity of aspirin action is believed to stem from the fact that platelets lack nuclear DNA, rendering them incapable of
de novo biosynthesis of COX-1, ensuring that inhibition of COX-1 prevails for the lifespan of the platelet. There is an increasing awareness of clinical variability in anti-thrombotic response to aspirin, with a significant proportion of patients failing to respond, a phenomenon referred to as `aspirin resistance' [
6]. Estimates of aspirin resistance vary widely (5-45%) [
7], but resistant patients are known to display an increased risk of cardiovascular morbidity [
8]. There is, however, a lack of association between aspirin-mediated inhibition of platelet function and inhibition of TxA
2 synthesis. This apparent anomaly has been attributed to a perceived lack of reliability of platelet function assays, on the assumption that reduced inhibition of TxA
2 is the main contributory factor in aspirin resistance [
9].
Whilst TxA
2 is undoubtedly an important mediator in the platelet activation process, to date little attention has been paid to other eicosanoids generated by platelets. 12-hydroxyeicosatetraenoic acid (12-HETE), formed by the enzyme 12-lipoxygenase (12-LOX), is another major platelet-derived eicosanoid, although its role in platelet function remains unclear. Reports have suggested 12-HETE to have both anti- and pro-thrombotic properties [
10]-[
12]. It has also been suggested to play a role in promoting expression of platelet CD62 (P-selectin) [
13] and is elevated in patients with essential hypertension [
14]. The increasing recognition of the potential importance of 12-HETE in platelet function has led to a recent resurgence of research in the area, which has resulted in the development of novel 12-LOX inhibitors to help elucidate the function of platelet 12-HETE and its potential as a novel anti-platelet target [
15],[
16]. The impact of aspirin on 12-HETE is not yet fully understood. In this study we have investigated the association of TxA
2 (measured as its degradation product TxB
2) and 12-HETE levels, with low-dose aspirin-mediated inhibition of platelet aggregation in healthy volunteers.
Methods
Subjects
Healthy volunteers (n = 19) were recruited through the Highland Clinical Research Facility. Subjects were free from all non-steroidal anti-inflammatory drugs (NSAIDs) two weeks prior to blood collection. A 5 ml blood sample was drawn into trisodium citrate tubes (Monovette®, Sarstedt, Leicester, UK) from the antecubital fossa (21G) before and 1.5 h after oral dosing with aspirin (Actavis, Barnstaple, UK; 75 mg). For inhibitor studies, a further three healthy volunteers were recruited and blood drawn as above. The study was approved by the North of Scotland Research Ethics Committee and the University of the Highlands and Islands Ethics Committee, and was in accordance with the Declaration of Helsinki and its amendments, with all volunteers providing written informed consent.
Measurement of platelet aggregation and inhibitor studies
Platelet aggregation was measured in response to collagen in platelet-rich plasma (PRP) derived from the freshly drawn blood samples immediately before and 1.5 h after aspirin administration. Briefly, fresh blood was centrifuged (120 g for 10 min at room temperature), PRP aspirated and the platelet count obtained on an AcT Diff 8 Blood Counter (Beckman Coulter, High Wycombe, UK). The remaining blood sample was centrifuged (1000 g for 10 min at room temperature) and the platelet poor plasma (PPP) aspirated for use as a reference and to dilute the PRP to a standard platelet count (150 × 106/ml). Standardized PRP samples were aliquoted (500 μl) into glass cuvettes with a micro-stirrer and pre-warmed to 37°C prior to addition of type I collagen (Labmedics, Manchester, UK; 2.5 μg/ml. Aggregation was recorded for 6 min using a Chrono-Log 700 platelet aggregometer (Chrono-Log, Haverford, PA, USA), at which point the activated platelets were immediately stored at -80°C for subsequent eicosanoid analysis. Aggregation was quantified as the area under the curve (AUC) over the 6 min period post-activation. To determine the effect of 12-LOX inhibition on platelet aggregation, platelets were pre-incubated for 10 min with either 100 μM baicalein (Tocris Bioscience, Bristol, UK) or DMSO as a vehicle control at a final concentration of 0.5% (v/v) prior to addition of 1.25 μg/ml collagen; aggregation was measured as described above and samples retained at the end of the aggregation assay for subsequent eicosanoid measurements.
LC-MS/MS analysis of platelet eicosanoids
Eicosanoids were isolated from the PRP following collagen activation using solid-phase extraction (SPE) chromatography (C18 500 mg, 6 ml, Waters, Manchester, UK). 1 ml ice-cold methanol containing 1 ng of the deuterated internal standards, PGE2-d4 and15-HETE-d8 (Cayman Chemical, Ann Arbor, MI, USA), was added to 0.5 ml PRP to precipitate proteins, and following centrifugation (600 g for 10 min at 4°C), the supernatant was removed, diluted to <10% methanol content by the addition of 9 ml ddH2O and acidified to pH 3.5 with 1 M HCl. Acidified samples were immediately added to SPE cartridges, which had been conditioned with 2 × 6 ml methanol, followed by 2 × 6 ml ddH2O. Samples were then washed with a further 6 ml ddH2O and 2 × 5 ml hexane before elution of the eicosanoids with 2 × 3 ml ethyl acetate. The ethyl acetate fraction was dried under vacuum and then resuspended in 100 μl 50:50 (v/v) solvent A:solvent B where solvent A consisted of H2O:methanol 90:10 (v/v) containing 0.1% (v/v) acetic acid and solvent B consisted of methanol containing 0.1% (v/v) acetic acid. All solvents were LC-MS grade (Fisher Scientific, Loughborough, UK).
Eicosanoids were separated on a Kinetex 1.7 μM XB-C18 column (100 × 2.1 Mm) (Phenomenex, Macclesfield, UK) column using a Thermo Accela 1250 UHPLC system (Thermo Scientific, Hemel Hempstead, UK). The gradient was 45-100% solvent B over 18 min at a flow rate of 400 μl/min. The LC effluent was directed into the source of a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer. The instrument was operated in negative ion mode and data were acquired using the selected reaction monitoring (SRM) mode. Eicosanoids were identified on the basis of their characteristic ion pairs (TxB2, 369/169; PGE2-d4, 355/193; PGE2, 351/189; 12-HETE, 319/179; 15-HETE-d8, 327/226) and matching retention time with authentic standards. Data were acquired and analysed using Thermo LCquan v2.6 software. Concentrations of TxB2 and 12-HETE were determined by comparison to a calibration curve run in parallel for each compound and adjusted for recovery by reference to amounts of the PGE2-d4 and 15-HETE-d8 internal standards respectively.
Statistical analysis
Data were compared with two-tailed, paired Student's t-test or analysed using regression analysis (Pearson's). A P-value of <0.05 was considered statistically significant in all cases with data expressed as mean ± SEM.. The statistical analysis was performed using GraphPad Prism v5.0 software.
Discussion
These findings demonstrate that, as well as inhibiting TxA
2 generation through COX-1, aspirin additionally impacts upon 12-LOX-mediated synthesis of 12-HETE. Whilst the effect of aspirin on TxA
2 was universal and comprehensive, indicating effective aspirin-mediated COX-1 inhibition, the variation in efficacy on 12-HETE inhibition was also closely associated with the extent of inhibition of aggregation. Indeed, the subject with the highest levels of 12-HETE was found to have the lowest aspirin-induced reduction in platelet aggregation. This suggests that aspirin-mediated inhibition of 12-HETE generation may be of use in predicting the efficacy of aspirin with regard to platelet function. Whilst generally considered to be a classic COX-1 inhibitor, aspirin has previously been shown to impact upon the platelet 12-LOX pathway, and can reversibly inhibit 12-HETE generation in a dose-dependent manner [
17],[
18]. This effect appears to be unique to aspirin rather than being due to secondary effects caused by COX inhibition as treatment with indomethacin (COX-1 inhibitor) has no impact upon 12-HETE levels [
19].
Considerable heterogeneity of platelet response to aspirin was observed between the healthy volunteers in this study, despite effective TxB
2 inhibition. This observation parallels a recent study which showed that aspirin reduced TxB
2 levels in all subjects tested [
20] further supporting the notion that an alternative mechanism to TxB
2 may be involved in variable sensitivity to aspirin. Our study examined the acute (1.5 hours) effect of aspirin treatment on eicosanoid levels and platelet function. Despite aspirin being an irreversible inhibitor of platelet COX-1, it is recognised that cumulative dosing of aspirin may be required to achieve maximal inhibition of TxB
2 generation, although how cumulative dosing effects 12-HETE inhibition are currently unknown [
21].
12-HETE has been suggested to play a role in platelet function and is thought to contribute to both platelet adhesion and aggregation [
19],[
22] although its exact role is unclear. Addition of 12-HpETE, the hydroperoxy precursor of 12-HETE has been demonstrated to play a `priming' role and increase aggregation of washed platelets [
23],[
24]. However, other studies have shown that addition of exogenous 12-HpETE inhibits both collagen and thrombin-induced platelet aggregation [
25],[
26] suggestive of anti-aggregatory properties.
Pharmacological inhibition of 12-LOX diminishes aggregation of washed platelets, further suggesting a role for 12-HETE in promoting platelet aggregation [
11],[
27],[
28]. Other evidence for a pro-aggregatory role of 12-HETE comes from a recent study that showed a significant decrease in aggregation of washed platelets in response to collagen with a newly developed inhibitor, NCTT-956 that is highly specific for 12-LOX [
29]. These results are consistent with our experimental findings using baicalein, although it should be noted that this inhibitor may have off-target effects on other eicosanoid pathways. The functional importance of platelet 12-LOX is also emphasized by the fact that 12-LOX-deficient mice display elevated mortality in an ADP-induced model of thromboembolism [
30] and inhibition of 12-LOX inhibit coronary thrombosis in a canine model [
31]. Furthermore, in patients receiving heparin treatment, platelet-derived 12-HETE has been shown to be elevated, whilst TxB
2 levels were unchanged [
32]. This was suggested as a possible mechanism to explain the phenomenon of transient heparin-induced platelet activation.
The signalling pathways involved in eicosanoid-dependent platelet activation are complex and not yet fully understood. Collagen is known to stimulate platelet 12-LOX activity via the glycoprotein VI receptor, in a PECAM-1 and PKC-dependent pathway [
33]. Release of AA from membrane phospholipids by PLA
2 activation is a critical step in eicosanoid biosynthesis and a recent study has shown that COX-1 and 12-LOX are linked to different PLA
2 subsets, suggesting that TxA
2 and 12-HETE are generated from different intracellular pools of AA [
34]. 12-HpETE is known to modulate platelet COX activity, suggesting a degree of `cross-talk' between the LOX/COX pathways although the exact mechanism is unclear.
Some studies have reported a stimulatory effect of low concentrations of 12-HpETE on COX-1 activity [
30], whilst others have shown that 12-HpETE inhibits COX-1 by altering calcium homeostasis via activation of soluble guanylate cyclase [
26]. The exact nature of the inhibitory mechanism of aspirin on the 12-LOX pathway is unclear at present. However, it has been suggested that aspirin exerts its impact by inhibiting the glutathione peroxidase-mediated conversion of 12-HpETE to 12-HETE, preventing 12-HETE formation [
35]. COX-1 has a bifunctional activity producing the labile peroxide, PGG
2, which is reduced to the alcohol PGH
2 via an endoperoxidase mechanism. PGH
2 is then further metabolised by various prostanoid-generating enzymes. This bears a striking similarity to the mechanism of 12-HETE generation, whereby 12-LOX catalyses the regio- and stereo-specific incorporation of molecular oxygen to form the hydroperoxide (12-HpETE), which is then reduced to the corresponding alcohol (12-HETE). Therefore, it is possible that aspirin may be acting in a similar manner, and preventing the reduction of 12-HpETE.
Aspirin remains, and is likely to remain for some time, the mainstay of anti-platelet therapy in vascular patients and so a full understanding of its mechanisms of action is essential. Here we have shown for the first time a correlation between aspirin-mediated platelet aggregation and 12-HETE generation. This finding highlights the importance of the platelet 12-LOX pathway as an additional mechanism by which aspirin can modulate the complex network of AA metabolism, with potential effects on thrombotic events. This study also underlines the importance of monitoring interconnecting pathways when investigating the inhibitory mechanism of pharmacological agents. In future work the investigation of the mechanism and nature of aspirin inhibition of 12-LOX could be extended by using platelet markers of activation ex vivo e.g. P-selectin, larger sample sets and disease cohorts such as patients with type 2 diabetes or cardiovascular disease.
Authors' contributions
BHM performed the mass spectrometric analysis of eicosanoids. BHM, MHL and ATT carried out the platelet aggregation studies. BHM, GFR, JW, SJL, ILM and PDW conceived the study, participated in its design, contributed to the interpretation of the results and helped to draft the manuscript. All authors read and approved the final manuscript.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.