Lipids in Platelets
Lipids are low molecular weight, typically hydrophobic and
amphipathic molecules found in all cell types. They are either
generated endogenously or incorporated into cells from di-
etary sources. Their formation, trafficking, and metabolism are
tightly controlled by cellular proteins, which include members
of large families of phospholipases, lipid synthetases, ligases,
oxidases/reductases, and transporters. Lipids fulfill 3 primary
roles: structural, energy storage, and signaling. There are sev-
eral distinct families of lipids in platelets, characterized by
(1) common functional groups and structural motifs, such as
phospholipids, sphingolipids, steroids, and prenol lipids and
© 2014 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.114.301597
Abstract: Lipids are diverse families of biomolecules that perform essential structural and signaling roles in
platelets. Their formation and metabolism are tightly controlled by enzymes and signal transduction pathways,
and their dysregulation leads to significant defects in platelet function and disease. Platelet activation is associated
with significant changes to membrane lipids, and formation of diverse bioactive lipids plays essential roles in
hemostasis. In recent years, new generation mass spectrometry analysis of lipids (termed lipidomics) has begun to
alter our understanding of how these molecules participate in key cellular processes. Although the application of
lipidomics to platelet biology is still in its infancy, seminal earlier studies have shaped our knowledge of how lipids
regulate key aspects of platelet biology, including aggregation, shape change, coagulation, and degranulation,
as well as how lipids generated by platelets influence other cells, such as leukocytes and the vascular wall, and
thus how they regulate hemostasis, vascular integrity, and inflammation, as well as contribute to pathologies,
including arterial/deep vein thrombosis and atherosclerosis. This review will provide a brief historical perspective
on the characterization of lipids in platelets, then an overview of the new generation lipidomic approaches,
their recent application to platelet biology, and future perspectives for research in this area. The major platelet-
regulatory lipid families, their formation, metabolism, and their role in health and disease, will be summarized.
(Circ Res. 2014;114:1185-1203.)
Key Words: blood platelets ■ mass spectrometry
Modern Day Perspective on Lipid Discovery and
Characterization in Platelets
Valerie B. O’Donnell, Robert C. Murphy, Steve P. Watson
This Review is in a thematic series on Sticky Notes on Blood Platelets, which includes the following articles:
Testing Cardiovascular Drug Safety and Efficacy in Randomized Trials [Circ Res. 2014;114:1156–1161]
Thiol Isomerases in Thrombus Formation [Circ Res. 2014;114:1162–1173]
Platelet Immunoreceptor Tyrosine-Based Activation Motif (ITAM) Signaling and Vascular Integrity [Circ Res. 2014;114:1174–1184]
Platelet Lipidomics: Modern Day Perspective on Lipid Discovery and Characterization in Platelets
What Can Proteomics Tell Us About Platelets?
Assessment of Platelet Function and the Arguable Need for More Antiplatelet Drugs
Genomics of Megakaryocytes and Platelets
Garret FitzGerald, Guest Editor
Original received August 30, 2013; revision received December 11, 2013; accepted December 12, 2013. In January 2014, the average time from
submission to first decision for all original research papers submitted to Circulation Research was 14.35 days.
From the Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom (V.B.O’D.); Department of Pharmacology,
University of Colorado at Denver, Aurora (R.C.M.); and Birmingham Platelet Group, Centre for Cardiovascular Sciences, Institute of Biomedical Research,
College of Medical and Dental Sciences, Birmingham, United Kingdom (S.P.W.).
Correspondence to Valerie B. O’Donnell, PhD, Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, CF14 4XN, United
Kingdom. E-mail firstname.lastname@example.org
1186 Circulation Research March 28, 2014
(2) minor structural differences, including positional isomers,
fatty acid (FA) chain length, and hydrocarbon saturation,
which render a complex mixture of molecular species. In com-
mon with all mammalian cells, the major structural lipids in
platelets are phospholipid, which arrange themselves in mem-
branes with hydrophobic FAs orientated to the core and polar
headgroups facing the aqueous phase (Figure 1). Phospholipid
membranes include both the plasma membrane and also the
numerous intracellular organelle membranes in platelets.
During activation, they provide substrates that are converted
enzymatically to bioactive species, including 1,2-diacyglyc-
erol, FAs, eicosanoids/prostaglandins, phosphatidylinositides
(PI), lysophospholipids, and lysophosphatidic acid (LPA).
They are also indirectly oxidized to form phospholipid-es-
terified eicosanoids and prostaglandins by lipoxygenases and
cyclooxygenases. Major remodeling of lipids occurs during
platelet activation and is associated with significant structural
alterations to platelet membranes, including shape change,
spreading, microvesicle formation, and degranulation, as well
as generation of bioactive prothrombotic species. Platelet
membranes contain sphingomyelins and free cholesterol that
are enriched in specialized signaling areas termed lipid rafts.
Platelets also contain appreciable amounts of neutral lipids,
including 1,2-diacylglycerol, triglycerides, and cholesteryl es-
ters (CE). Some additional lipids present in smaller amounts,
but with important signaling roles include members of the
sphingolipids and glycolipids/ceramide families.
In this review, we will summarize what is currently known
about each lipid class in platelets, how they are metabolized,
and their major functions. Their roles in platelet-dependent
pathologies will also be described.
Historical Perspective, Early Studies
on Platelet Lipids
Before the advent of lipidomics, cellular lipids were studied
using traditional techniques that included thin layer chro-
matography, gas chromatography/mass spectrometry (MS),
and high-pressure liquid chromatography coupled with ra-
diochemical, ultraviolet, or fluorescence detection. Research
using these approaches still informs most of what we know
about platelet lipids today.
One of the earliest comprehensive analyses of platelet phos-
pholipids was performed in 1962 by Marcus et al.1–4 After
this, studies were performed in the 1970s to 1980s comparing
platelet lipid composition in a diverse and somewhat unusual
array of population groups and species.5–15 Also, with interest
in the role of lipids causing vascular disease increasing around
that time, the effects of dietary supplementation with lipids on
Nonstandard Abbreviations and Acronyms
liquid chromatography tandem mass spectrometry
liquid chromatography/mass spectrometry
Figure 1. Phospholipids in
the membrane. A, Typical
structure of a phospholipid:
phosphatidylcholine. B, Struc-
ture of the plasma membrane
lipid compartment and its
relationship to formation of
lipid mediators. Note, for
simplicity, proteins have not
been shown in this figure.
DAG indicates diacylglyceride;
PA, phosphatidic acid; PC,
prostaglandin; PL, phospholipid;
PLD, phospholipase D; PS,
inositol triphosphate; SM,
sphingomyelin; and TX,
O’Donnell et al Platelet Lipidomics 1187
platelet lipid FA composition (omega-3, fish oil, corn oil, or
varying dietary fat) were characterized.16–18
Platelet eicosanoids and related species were first identi-
fied in the early 1970s by Hamberg and Samuelsson19 and
Hamberg et al20 in the Karolinska Institute, Stockholm, in par-
allel with Sir John Vane, in London. In 1974, the major plate-
let products 12S-HETE and 12-hydroxyheptadecatrienoic
acid were demonstrated; then in 1975, thromboxane A2
(TXA2) was discovered as a platelet-derived lipid that causes
irreversible aggregation. This seminal work contributed to the
awarding of a Nobel Prize to Samuelsson and Vane for discov-
eries on prostaglandins, TXs, and related biologically active
substances. In the early 1980s, thrombin was demonstrated
to cause significant alterations to membrane lipids, with PI
showing losses of ≤45%. Arachidonic acid (AA) specifically
decreased in PI and phosphatidylcholine pools, suggesting
these to be likely sources of substrate for eicosanoid genera-
tion.21,22 Release of AA from PI was proposed to involve the
sequential action of phospholipase C generating 1,2-diacylg-
lycerol followed by diacylglycerol lipase acting on 1,2-dia-
cylglycerol, representing an alternative to phospholipase A2
(PLA2).22 These studies established the overall lipid composi-
tion of resting and activated platelets, in particular about the
more abundant species such as phospholipids and FAs. In con-
trast, 1980s to 2000s saw intense interest in characterizing the
role of specific platelet signaling lipids in regulating platelet
function and contributing to both physiological hemostasis
and human disease. Examples that will be discussed herein
and that deserve special mention include (1) the central role
of TXA2 in regulating hemostasis and contributing to patho-
logical clot formation, (2) the role of 1,2-diacylglycerol, PI,
and inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) in promoting
calcium mobilization after receptor-dependent activation of
platelets, and (3) the role of anionic phospholipids in promot-
Lipidomics: Current State-of-the-Art
Since the mid-2000s, the advent of ’omics, largely driven
by the development of sensitive benchtop mass liquid chro-
matography–MS (LC/MS) instruments, for example, elec-
trospray ionization coupled to tandem (triple quadrupole or
MS/MS) or time-of-flight instruments, has revolutionized our
ability to study small amounts of complex mixtures of di-
verse lipids in biological samples, hence the term lipidomics.
These days, the term tends to be synonymous with the MS
of lipids. The major disadvantages of older approaches in-
stead of LC/MS were (1) low sensitivity and selectivity (thin
layer chromatography and high-pressure liquid chromatogra-
phy), (2) the need for time-consuming derivatization methods
(eg, for gas chromatography/MS), and (3) the requirement
for radioisotopes with their inherent health issues, notably
in regard to 32P-orthophosphate. When compared with this,
LC/MS combines high sensitivity with the ability to detect,
characterize, and quantify individual molecular species di-
rectly without derivatization or purification. Older methods
were generally unable to analyze specific lipids directly (eg,
Figure 2. Summary of traditional and new lipidomic technologies used to discover and characterize cellular lipids. GC/MS
indicates gas chromatography/mass spectrometry; HPLC-UV, high-pressure liquid chromatography/ultraviolet; and LC/MS/MS, liquid
chromatography/tandem mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; and TLC, thin layer chromatography.
1188 Circulation Research March 28, 2014
1-stearoyl-2-arachidonyl-phosphatidylcholine would not have
been measurable as a single species in a complex mixture).
Traditional and newer lipidomic methodologies are summa-
rized in Figure 2.
Lipidomics is broadly divided into 2 separate approaches:
(1) liquid chromatography-tandem MS (LC/MS/MS), which
is targeted, highly sensitive, and quantitative and (2) shotgun
lipidomics, which is high throughput but can only detect the
most abundant species.23,24 In LC/MS/MS, several molecu-
lar species are analyzed in the same sample after separation,
by their characteristic parent m/z (mass:charge ratio, where
mass is divided by the total net charge of the molecule) and
daughter ions that form after collision-induced-fragmentation
(where molecules are collisionally activated using an in-
ert gas then break apart into small daughter ions that can be
separately analyzed). These methods typically use a tandem
MS instrument (eg, tandem in space as a triple quadrupole
or tandem in time, as an ion trap). These comprise 3 sepa-
rate chambers: the first and third of which are mass analyz-
ers that house 4 parallel gold-plated rods (quadrupoles). The
second is a collision cell where nitrogen or argon fragments
the molecule for MS/MS analysis (Figure 3A). Thus, LC/MS/
MS methods are best suited to studies where researchers are
interested in specific lipids and require accurate quantitation.
However, separations can be long (eg, ≥1 hour for phospho-
lipids). However, shotgun lipidomics involves direct infusion
of complex mixtures, without separation, into a mass spec-
trometer, followed by MS scanning of a defined mass window,
often using high-resolution instruments (where the MS can
distinguish molecules that are extremely close in mass, eg,
differing in mass by ≤1–5 ppm). The advantage is that many
samples can be analyzed in a relatively short time; however, it
is not generally quantitative, and low abundance (often highly
biologically relevant) species are missed. A newer quantitative
approach combining LC separation with high-resolution MS/
MS on rapid scanning Fourier transform or time-of-flight in-
struments, termed multidimensional MS, is now increasing in
popularity in particular for profiling studies.23 In Fourier trans-
form MS, the m/z is determined based on the cyclotron fre-
quency of the ions in a fixed magnetic field. One example of
this is the Orbitrap, a benchtop instrument currently popular
for lipidomics that contains an outer barrel-like electrode and
a coaxial inner spindle-like electrode that together generate
an electrostatic field (Figure 3B). In contrast, time-of-flight
instruments calculate mass based on the length of time it takes
for molecules accelerated by an electric field to reach a detec-
tor at a known distance, with heavier particles having lower
speeds (Figure 3C).
Up to now, platelet lipids have not been extensively studied
using lipidomics. LC/MS/MS has been applied to quantitation
of eicosanoids, such as TX B2 (TXB2) and HETE, and to iden-
tification of sphingolipids molecular species25,26 (Figure 4A).
Shotgun methods have been applied to the characterization of
platelet phospholipids, lysophospholipids, CEs, and ceramides
in a small number of studies.27–29 Using traditional methods,
individual lipid pools first had to be isolated using thin layer
Figure 3. Examples of mass spectrometry (MS) instrument configurations. A, Triple quadrupole instrument. Ions are selected in
Q1, fragmented in Q2, and daughter ions scanned out in Q3. B, Fourier transform Orbitrap MS. Ion trajectories in an Orbitrap mass
spectrometer. C, Time-of-flight (ToF) MS. In the example shown, matrix-assisted laser desorption ionization is used to generate ions that
are selected in an electric field and m/z determined based on time taken to reach the detector.
O’Donnell et al Platelet Lipidomics 1189
chromatography, then hydrolyzed to release FAs for determi-
nation as free acid species, a considerably slower approach
that requires more material.1–4 Recently, the composition of
stored platelets and extracellular vesicles was characterized,
showing that vesicles became enriched with LPA, cholesterol,
and other lipids.28,29
In the past 2 to 3 years, MS technology has undergone sig-
nificant increases in both sensitivity and scanning speed, with
new benchtop instrument configurations becoming available,
and only now are researchers beginning to apply these to the
study of cells and tissues. At this time, we are only learning
what these new technologies are capable of, and what ques-
tions they might answer. One approach, ion mobility (Waters
Synapt; AB Sciex SelexION), allows separate detection of
lipids that have the same accurate mass (eg, elemental compo-
sition) and retention time on high-pressure liquid chromatog-
raphy, giving an extra layer of molecular differentiation that
was not possible before. This is based on the differing mobil-
ity of the ions in a carrier gas. For example, 2 triacylglycerides
each containing 2 stearic acids and 1 palmitic acid, but with
the palmitic acid at different positions on the glyceride back-
bone can be distinguished. A second approach, using Fourier
transform instruments, such as the Orbitrap (ThermoFisher),
allows rapid high-resolution scanning that differentiates be-
tween lipids based on mass differences down to 1 to 5 ppm (eg,
can distinguish coeluting lipids with masses of m/z 516.280
and 516.289; Figure 4B). Coupled with high-pressure liquid
chromatography, these instruments are particularly suited to
structural characterization and global lipidomic screening ap-
proaches. Another configuration of relevance to lipidomics is
the combination of matrix-assisted laser desorption/ionization
with high-resolution MS (eg, the matrix-assisted laser desorp-
tion/ionization; Synapt G2, Waters), which is currently being
applied to lipid imaging in tissue samples to determine spatial
localization of molecular species (eg, in lung30; Figure 4C)
The resolution of lipid imaging, however, is not yet at the
cellular or subcellular level. Potential applications and new
frontiers for lipidomic MS will be described in the General
Summary of this article.
Methodological Issues: Working With Platelet
Lipids In Vitro
Washed human platelets are isolated from whole blood using
centrifugation. Care needs to be taken during bloodletting, thus
it is important to use a needle that allows the blood to flow
freely to ensure that platelets are not exposed to shear. This can
itself activate the cells to generate lipid mediators even before
isolation. Several protocols are listed in detail by Watson and
Authi31 and generally involve a low-speed centrifugation of
whole blood anticoagulated with acid citrate-dextrose (lowers
pH and chelates Ca2+) to generate platelet-rich plasma followed
by a faster centrifugation of the recovered plasma to pellet the
platelets themselves. This is followed by a single wash step in
Tyrode buffer with acid citrate-dextrose (9:1). Anticoagulants,
Figure 4. Illustrations of data from 3 of the newer mass spectrometry (MS) technologies. A, Liquid chromatography tandem mass
spectrometry (LC/MS/MS) of eicosanoid standards.34 B, Illustration of resolving power of Fourier transform MS on an Orbitrap Elite, at
different resolution settings, as shown. C, Left to right, H&E stain of a distal human lung slice (15-μm thick) showing both airways and
blood vessels. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric image of the positive ions (m/z 788.6 blue color)
derived from 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC) a phospholipid found in the cells of the airways. MALDI mass spectrometric
image of the positive ions (m/z 703.6, red) derived from the sphingomyelin molecular species SM(d18:1/16:0) an abundant phospholipid
present in the pulmonary blood vessels. MALDI mass spectrometric image of the positive ions (m/z 810.6, green) derived from 1-oleoyl-
2-arachidonoyl-phosphatidylcholine (SAPC) localized to both airways and blood vessels. Merged MALDI mass spectrometric images
indicating colocalization of phospholipids in cells of the lung parenchyma.
1190 Circulation Research March 28, 2014
such as prostacyclin and indomethacin, are included during
washing by many investigators but should be avoided in stud-
ies of lipids, to avoid interference to lipid-sensitive signaling
pathways. In this case, care needs to be taken because the
platelets may activate after the last spin when anticoagulant is
removed. The plasma/cells need to be maintained at ≈20° and
gently pipetted at all times. On final resuspension in Tyrode
buffer, they may appear slightly clumpy but after ≈15 to 20
minutes will have dispersed to a single cell suspension, with-
out requiring manipulation. It is advisable to avoid repeated
pipetting, or shaking/inverting of the cells during isolation and
handling. Once isolated, platelets should be kept at room tem-
perature and used within 2 to 3 hours.
Platelet lipids can be isolated using several different meth-
ods, including liquid:liquid extraction methods such as Bligh
and Dyer using chloroform/methanol, or as in our laboratory,
hexane/isopropanol based solvent mixtures that broadly ex-
tract most molecular species.32,33 The advantage of the latter is
lipid extraction into the upper rather than lower organic phase.
Specific lipids, such as eicosanoids, can also be more selective-
ly isolated using solid phase extraction methods, such as C18
columns.34 Once isolated, we typically resuspend in a small
volume of methanol or chloroform/methanol when highly con-
centrated and stored at −80°C before analysis, under inert gas.
Unsaturated lipids can undergo facile oxidation ex vivo,
so they need careful handling. Lipid extracts should be kept
on ice when in use but stored under inert gas at −80°C.
Although some investigators routinely include metal chela-
tors and antioxidants when studying oxidized species, this is
not generally necessary with platelet lipid extracts, if they are
handled appropriately, and analyzed within a few weeks of
generation. As confirmation, we only detect expected enan-
tiomeric species of eicosanoids specific to platelet enzymes
(eg, prostaglandin [PGE]2, 12-HETE, but little 8-iso-PGE2 or
other HETE isomers) in our assays.26,35 Although antibody-
based ELISA kits for eicosanoids are commercially available
and may seem attractive where MS lipidomics is not locally
available, these should be avoided for measurement of lipids
in complex samples because of lack of antibody specificity.
However, they may be acceptable for washed platelet eico-
sanoid measurements because only a selected low number of
eicosanoids and related species are formed, but careful valida-
tion and comparison should be performed with MS before use.
Specific Lipid Families in Platelets
Like all mammalian cells, the prominent phospholipids in
platelets are phosphatidylcholine and phosphatidylethanol-
amine accounting for ≈40% and 28% of total phospholipids,
respectively. Sphingomyelin and phosphatidylserine are also
relatively abundant (≈18% and 10%) with smaller propor-
tions of PI (3%–5%).1,36,37 The FA composition of platelet
phospholipids is described in Historical Perspective, Early
Studies on Platelet Lipids of this article and varies for each
phospholipid class. Studies in the mid-1970s, using chemi-
cal labeling or exogenous phospholipases, established that
platelet phospholipids are distributed asymmetrically between
plasma membrane bilayers, with the aminophospholipids
phosphatidylethanolamine and phosphatidylserine facing the
cytosol, and phosphatidylcholine and sphingomyelin facing
the outside. On stimulation, a substantial proportion of ami-
nophospholipid is externalized generating a procoagulant or
thrombogenic surface.38 In contrast, lipids required for in-
tracellular signaling are generated from lipids that face the
inside. These include Ins(1,4,5)P3 and 1,2-diacylglycerol
generated from PI 4,5-bisphosphate (PI(4,5)P2), hydrolysis
of AA to act as a substrate for TX generation and formation
of PI-3,4,5-trisphosphate (PI(3,4,5)P3 [PIP3]). Thus, phos-
pholipids act as a major reservoir for substrates for enzymes
that generate key platelet signaling mediators. These will be
described in more detail below.
In 1977, Zwaal et al39 noticed that unactivated platelets were
inert to plasma coagulation because of the absence of phos-
phatidylserine on their surface. Later, Bevers et al40 found that
activation of platelets with thrombin/collagen caused expo-
sure of aminophospholipid and correlated with procoagulant
activity. Around this time, and for many years later, studies
on mechanisms of phospholipid translocation, primarily in
erythrocytes, revealed several regulatory mechanisms that
maintain phospholipid asymmetry. These included a flippase
(proposed as P4 ATPase),41,42 and a floppase that is promoted
by ATP-binding cassette protein transporter C1/multidrug re-
sistance protein multidrug resistance protein 1.43,44 The identi-
ties and mechanisms of action of these proteins are still not
fully known. Opposing enzymes, termed scramblases, are
Ca2+-dependent and required for effective exposure of amino-
phospholipid on the platelet surface to promote coagulation.
The role of the platelet membrane in acting as a critical me-
diator of coagulation, through promoting factor activity, was
first proposed in the 1980s.45 It is now known that at physi-
ological pH, surface exposed phosphatidylserine provides the
negatively charged platform that enables calcium ions to form
bridges with γ-carboxyglutamic acid–containing Gla domains
on coagulation factors. In the case of the prothrombinase
complex, FVa is thought to undergo a conformational change
that forms a high-affinity binding site for FXa.46,47 This brings
thrombin generation to the site of platelet activation, enabling
coagulation and aggregation to be focused together at the site
of hemostatic need. The hunt for the platelet scramblase has
been of clinical importance because its absence accounts for
the rare bleeding disorder, Scott Syndrome, first described in
1979 by Weiss et al.48 In 2010, the involvement of a protein,
TMEM16F, in supporting scramblase activity in platelets was
identified.49 Since then, the 5 patients with Scott Syndrome
to date identified have been reported to have different muta-
tions in the gene encoding TMEM16F, including in intron 6
(G-to-A), disrupting the donor splice site consensus sequence,
and in exon 11 as a single-nucleotide insertion that predicts a
frame shift and premature termination of translation at codon
411.50 At this time, whether TMEM16F is the scramblase it-
self or an accessory protein is the subject of debate.
There seem to be ≥2 independent mechanisms for achiev-
ing aminophospholipid externalization. Recent studies have
established that TMEM16F is required for agonist-triggered
scramblase but not that mediated during platelet ageing/
apoptosis.51,52 Also, only agonist-triggered trafficking of ami-
nophospholipids requires influx of extracellular Ca2+.53 Up to
O’Donnell et al Platelet Lipidomics 1191
now, aminophospholipid externalization has generally mea-
sured using the flow cytometry probe, annexin V-fluorescein
isothiocyanate. However, this gives no information on either
the molecular species of aminophospholipid or amounts.
Using a newly developed lipidomic assay, where amine head-
groups of external facing aminophospholipid are derivatized
using a cell impermeable-reagent, we recently showed that
platelets (agonist activated, apoptotic, or energy depleted)
externalize ≈3% to 5% of the total cellular pool of 5 phos-
phatidylethanolamine and 2 phosphatidylserine distinct mo-
lecular species (Figure 5). Those externalized were the most
abundant platelet aminophospholipid molecular species, with
the same for either apoptosis, agonist activation, or energy
depletion, indicating that the process is not selective for ami-
nophospholipid with particular FA chains.51 We also showed
that these aminophospholipid were not efficiently externalized
in Scott Syndrome platelets, and that for phosphatidylethanol-
amine, the species externalized by platelets were the most
effective in in vitro thrombogenic assays when compared
with those with longer or shorter FA chains.51 Thus, FA side
chains are molecular determinants that can contribute to the
coagulation-regulating activity of phospholipid. The detailed
biophysical mechanisms of this are not known but may relate
to accessibility of aminophospholipid phosphate groups with
Ca2+ and Gla domains. Next, it will be important to compare
how other membrane lipids, for example, CEs, triglycerides,
and sphingolipids with platelet FAs, influence coagulation
factor activities. For this, knowing which FAs predominate in
platelet plasma membrane lipids of these classes would also
be important. Lipidomics will be of central importance in
helping answer these questions through defining the composi-
tion of the platelet plasma membrane during both health and
disease and different activation states. This could be achieved
through either targeted or global lipidomic strategies.
PIs comprise several interrelated species that contain an inosi-
tol headgroup. Two are shown in Figure 6, along with a diagram
describing their inter-relationship. These lipids play important
roles in cell signaling in health and disease, through revers-
ible phosphorylation of the inositol headgroup that is tightly
controlled by enzymes. The phosphoinositide PI(4,5)P2 is me-
tabolized in response to receptor activation through 2 distinct
pathways, namely (1) irreversible cleavage of the phosphodi-
ester bond and (2) reversible phosphorylation of the inositol
headgroup. The latter generates the major product of the PI
3-kinase pathway, PIP3, and the former, the 2 second mes-
sengers, 1,2-diacylglycerol and Ins(1,4,5)P3, which activate
protein kinase C and mobilize intracellular Ca2+, respectively.
1. The phosphodiester cleavage of PI(4,5)P2 is mediated by
the phospholipase C (PLC) family of phosphodiesterases,
which is composed of 10 isoforms, several of which are
regulated by the major G-protein–coupled and tyrosine
kinase–linked receptors in platelets. 1,2-Diacylglycerol
Figure 5. Molecular species of phosphatidylethanolamine (PE) and phosphatidylserine (PS) that are externalized by activated,
aging, or apoptotic human platelets.
1192 Circulation Research March 28, 2014
activates several isoforms of protein kinase C which,
in combination with the release of intracellular Ca2+ by
Ins(1,4,5)P3, leads to powerful platelet activation re-
sponses, such as shape change, aggregation, and secre-
tion. Platelets express variants of PLC-β (β1, β2, β3, β4,
and PLC-γ54–57 PLC-β is stimulated by G-protein–coupled
receptors (including TX and protease activated receptor
1/4), whereas PLC-γ is stimulated by single transmem-
brane glycoprotein receptors (eg, GPIIb–IIIa, GPIbα,
GPVI).58 Ins(1,4,5)P3 activates receptors that function as
Ca2+ channels in the dense tubular system.
2. PI(4,5)P2 is also converted to the lipid second messenger,
PIP3, by the class I family of PI3-kinases. These cata-
lyze the phosphorylation of PI, PI(4)P, or PI(4,5)P2 at
position of 3 of the inositol ring.59 PI3Ks are divided into
class I, II, and III, with class I further subdivided into α,
β, δ, and γ isoforms. Platelets contain all class I isoforms
of PI3K although the level of the δ isoform is lower than
others.60 Stimulation of platelet G-protein–coupled recep-
tors results in phosphorylation of PI(4,5)P2 by the PI3Kγ
isoform to generate PIP3. Deficiency of α, β, and γ iso-
forms of PI3K in mice results in a mild platelet aggrega-
tion defect and impaired thrombosis in vivo.61 In vitro,
PI3Kγ-deficient platelets disaggregate faster after ADP
activation and show mildly impaired ability to mobilize
intracellular Ca2+.62 Several studies show that this isoform
controls a major part of platelet ADP responses.61,63,64 The
protein targets for PIP3 binding and activation down-
stream of PI3Kγ that promote ADP signaling include
Akt isoforms. The PI3Kβ isoform has been proposed to
play several roles in regulating platelet activation, includ-
ing via promoting integrin-dependent Ca2+ flux and Gi-
dependent activation of Rap1b. In recent years, a small
molecule inhibitor of PI3Kβ, TGX-221, has been devel-
oped as a potential antithrombotic therapy.60,65,66
PIP3 binds to a domain of ≈120 amino acids, known as a pleck-
strin homology (PH) domain, which takes its name from the
major protein kinase C substrate in platelets, pleckstrin (which
is one of few proteins to have 2 PH domains). PH domains are
found in a variety of signaling and cytoskeletal proteins, in-
cluding, in platelets, (PLC)γ2, and the tyrosine kinase Btk. PH
domains are usually found with other domains, such as SH2
and SH3 domains, that bind to phosphotyrosine and proline-
rich regions, respectively. PH domains can also bind to other
PIs. In addition, PIP3 is rapidly metabolized in platelets to
PI(3,4)P2 by the Src homology 2 domain-containing inositol
5-phosphatase 1, which itself is regulated by tyrosine phos-
phorylation. This enzyme removes the phosphate at D-5 of the
inositol ring generating PI(3,4)P2 which can then be degraded
Figure 6. Phosphoinositides and their metabolic pathways. A, Structure of PI and PI(3,4,5)P3. B, The known PIs, their kinases, and
phosphatases. DAG indicates diacyglyceride; MTMR, myotubularin-related phosphoinositides phosphatase; OCRL, oculocerebrorenal
syndrome of Lowe; PTEN, phosphatase and tensin homolog; PIKfyve, phosphoinositides kinase with specificity for the 5 position
containing a FYVE finger; R, fatty acid side chains; and SHIP, SH2 domain-containing inositol-5-phosphatase.
O’Donnell et al Platelet Lipidomics 1193
by additional phosphatases. PI(3,4)P2 is generated in large
amounts, accumulating slowly, but independently of aggre-
gation and integrin activity.67,68 It was originally suggested to
regulate aggregation, integrin signaling, and thrombus growth,
but its targets and their overall significance in platelets remain
unknown.69,70 Although Src homology 2 domain-containing
inositol 5-phosphatase 1 is clearly established as a regulator of
PI lipid levels in platelets, the roles of additional phosphatases
also expressed in these cells (notably SHIP2 and phosphatase
and tensin homolog) are currently unclear.
Thus, there is bewildering number of pathways of metabo-
lism of PIs and regulation of PH domain-containing proteins
in platelets such that we still have a relatively poor under-
standing of the role of the PI 3-kinase pathway at the molecu-
lar level. However, the functional significance of PI 3-kinase
in platelets has been widely demonstrated using mutant mice
and both broad spectrum (eg, wortmannin and LY294002) and
PI 3-kinase isoform-specific inhibitors.
Many PIs are generated rapidly and degraded at distinct cel-
lular sites by specific PI-metabolizing enzymes that include
lipid kinases, lipid phosphatases, and phospholipases, some
of which have been described herein (Figure 4).71,72 Several of
these enzymes are involved in platelet function during agonist-
induced activation. For example, PI-4-phosphate-5-kinase
type I phosphorylates PI-4-phosphate (PI(4)P) to gener-
ate PI(4,5)P2 on the plasma membrane.73 Platelets lacking
PIP4KI-γ show impaired generation of PI(4,5)P2 and a sig-
nificant defect in anchoring their cell membranes to the un-
derlying cytoskeleton.74,75 PI(4,5)P2 has also been proposed
to bind talin and to support its role in integrin activation.76–78
PIP3 is generated rapidly during platelet activation and plays a
key role in recruitment and activation of PLCγ2 and other PH
domain containing enzymes.79
Measurement and quantitation of PI molecular species us-
ing MS-based lipidomics approaches has been a major goal
for several years, particularly in the area of cancer research.
However, these lipids are extremely difficult to analyze using
LC/MS primarily because of (1) difficulty in achieving effi-
cient extraction and (2) in-source fragmentation that occurs
on ionization of poly-phosphorylated forms, leading to loss
of phosphate groups. A recent review highlighted available
methods that allow detection and quantitation of some indi-
vidual PI species, but to date, there is no single methodology
that allows robust identification and quantitation of all.80 This
has significantly hampered the study of these species in liv-
ing cells because they form transiently and at extremely low
concentrations during cell signaling. For example, the specific
molecular species of PI regulated in activated platelets includ-
ing in terms of FA chain length and saturation are still un-
clear. The development of sensitive and specific methods for
quantifying PI molecular species is thus a major ongoing goal
in lipidomics that may become possible because newer meth-
odologies become available and is of importance in further-
ing our understanding of the roles of these important platelet
lipids in platelet activation and platelet-driven pathologies.
FAs, Eicosanoids, and Related Species
A major early response of platelets to activation is the switch-
ing on of several phospholipases, including cytosolic PLA2α
isoforms, which cleave FAs from the sn2 bond of phospho-
lipids. Early studies demonstrated that the prominent FAs
released were AA, linoleic acid, palmitic acid, and stearic
acid.81–83 However, the most important in terms of signaling
is AA, the precursor for oxidative transformation to several
eicosanoids via lipoxygenase and cyclooxygenase enzymes
(Figure 7). Platelets express several PLA2 isoforms, includ-
ing Ca2+ sensitive and insensitive forms. In general, the
Ca2+-sensitive cytosolic PLA2α is considered the source of
AA for eicosanoid generation. In support, a recent study dem-
onstrated that genetic deficiency of this isoform leads to sig-
nificantly impaired platelet eicosanoid generation, along with
platelet dysfunction in humans.84
Platelet-generated eicosanoids are best exemplified by the
potent proaggregatory TXA2, generated by coordinated action
of cyclooxygenase-1 and TX synthase. This lipid is extremely
unstable and rapidly rearranges to TXB2, which is released
by platelets at ng amounts per 2×108 cells (Figure 5). TXA2
binds the Gq-coupled receptor TPα with high affinity, trig-
gering activation of PLCβ. A number of receptor agonists to
TP have been developed, including U46 619 and SQ 26 655
that mimic the action of TXA2. Because of its short plasma
half-life, in vivo generation of platelet TX in both humans
and mice is best measured through analysis of urinary me-
tabolites, 11-dehydroTXB2, or 2,3-dinor TXB2.85–91 This has
been achieved using either gas chromatography or LC/MS and
is considered a reliable surrogate for measurement of in vivo
platelet reactivity, which is significantly raised in both cardio-
vascular disease and smokers.92–95
However, 12-HETE, generated
ase, is quantitatively more abundant but does not dis-
play any potent bioactivity toward platelets in vitro. In
contrast, the 12-lipoxygenase–deficient mice display a mild
hyper-responsiveness to ADP; however, their collagen re-
sponses were normal.96 12-Hydroxyheptadecatrienoic acid
is also abundant, accounting for ≤30% of the total AA flux
through cyclooxygenase-1. This is formed through conversion
of prostaglandin H2 (PGH2) either nonenzymatically or via TX
synthase although its function is unknown.97–99 Platelets also
generate smaller amounts of PGs, particularly PGE2 and pros-
taglandin D2 (PGD2), through nonenzymatic rearrangement
of PGH2. They also generate isoprostanes via radical-based
biochemistry, notably 8-epi-PGF2α, but levels are ≈1000-fold
less than corresponding 12-HETE and TX.100,101 PGE2 is either
pro (low dose)– or anti (high dose)– aggregatory, through ac-
tivation of either EP3 or IP receptors, respectively.102 In con-
trast, 8-epi-PGF2, a specific isoprostane isomer, is primarily
antiaggregatory through acting as a TX receptor antagonist
although the physiological relevance of this is unclear.103–105
The importance of eicosanoid signaling in terms of platelet
physiology and vascular disease is underscored by the wide-
spread use of the cyclooxygenase inhibitor aspirin in prevention
of cardiovascular events, as well as an antithrombotic agent in
stroke, antiphospholipid syndrome, and other prothrombotic
conditions.106–108 This was established by seminal work from
several laboratories notably those of Vane, Samuelsson, Roth
and Majerus, Patrono, and FitzGerald20,109–115 and was recent-
ly the subject of the 2013 Grand Prix Scientific (Lefoulon-
Delalande Foundation, Institute of France) awarded to Garret
1194 Circulation Research March 28, 2014
FitzGerald and Carlo Patrono. More recently, a number of
studies found that low-dose (considered platelet-selective) as-
pirin can reduce the spread of existing cancers and the risk of
developing others, in particular adenocarcinoma of the gut,
some lung cancers and breast and prostate cancer. The mecha-
nisms have not been elucidated but have been suggested to
involve an antiplatelet effect, particularly in terms of prevent-
ing spread of existing cancers through the bloodstream.116–121
The biological basis of this is uncertain, in part because plate-
lets are implicated in multiple stages of cancer progression,
but it may involve platelet-derived signaling lipids that are
generated either primarily or secondarily via cyclooxygenase-
1–dependent signaling. Lipidomics could help address this
question in the future. For example, through targeted or un-
targeted analysis of platelet lipids that are generated on ac-
tivation in an aspirin-sensitive manner, candidate molecules
that may play a role in cancer progression could be identified.
Known lipids that could already be considered would include
PGE2, which is known to help cancer cells both evade the im-
mune system and resist drug treatment.
Additional eicosanoids, including leukotrienes and li-
poxins, can be generated through transcellular pathways in-
volving platelet–leukocyte interactions. In 1989, Maclouf
and Murphy122 demonstrated that platelets efficiently con-
verted neutrophil-derived leukotrienes A4 to leukotrienes
C4, in mixed cell incubations. After this, neutrophil–platelet
incubations were shown to generate lipoxin A4 from endog-
enous substrate, but this required activation with ionophore.123
In 1990, receptor-mediated interactions were also shown to
promote this activity.124,125 However, whether transcellular bio-
synthesis itself is a significant source of these lipids in vivo is
still unclear because determining cellular origin of lipids in
complex biological samples is extremely difficult.
Lipidomics has already been extremely powerful in facilitat-
ing studies of platelet eicosanoids, primarily through enabling
rapid and accurate, high-sensitivity quantitation of these spe-
cies using LC/MS/MS approaches in both isolated cells and
in human and murine disease. Newer lipidomics technolo-
gies could further our understanding of the biology of these
species, through a number of ways including imaging their
spatial and temporal generation in tissues (eg, matrix-assisted
laser desorption/ionization-MS), and discovery and structural
characterization of new members of this important class of
biomolecules, using high-resolution scanning methods.
Oxidized phospholipid has long been known as biproducts of
nonenzymatic peroxidation, and several are found at relative-
ly high concentrations in atheroma lesions. Recently, we used
a targeted lipidomic approach to demonstrate that human
platelets generate significant amounts of specific oxidized
phospholipid molecular species via enzymatic mechanisms,
Figure 7. Arachidonate metabolism to form eicosanoids and prostaglandins by cyclooxygenase (COX)-1 in platelets. Arachidonic
acid (AA), liberated by phospholipase A2, is oxidized by COX-1 to form PGG2, then prostaglandin H2 (PGH2). PGH2 is further metabolized
by thromboxane synthase (TXS) to TXA2, which rapidly decomposes to TXB2. PGH2 can also decompose to prostaglandin (PGE2),
prostaglandin D2 (PGD2), and hydroxyheptadecatrienoic acid (HHT). Separately, AA can be oxidized by 12-lipoxygenase (LOX) to 12-HETE.
O’Donnell et al Platelet Lipidomics 1195
during acute activation by thrombin, collagen, or Ca2+ iono-
phore.26,126 This indicates that phospholipid oxidation is not
simply an accidental consequence of inflammatory disease
but a regulated process of likely importance during physi-
ological hemostasis. To date, 3 main families have been
described, 2 from 12-lipoxygenase (10 total lipids) and 4
families of 16 esterified PGs generated via cyclooxygenase-1
(total 26 lipids; Figure 8).
The approach used for their discovery, precursor-LC/MS/
MS, is a tandem MS mode that enables molecules to be de-
tected that contain a specific functional group. In this case,
we used precursor LC/MS/MS to search specifically for es-
terified eicosanoids because on collision-induced fragmen-
tation, they generate a robust carboxylate anion signal in
negative ion mode. Initial studies demonstrated a family of
six 12-HETE–containing phospholipids, comprising 4 phos-
phatidylethanolamines and 2 phosphatidylcholines. The phos-
phatidylethanolamines include several plasmalogens and all
represent oxidized forms of the most abundant phosphatidyl-
ethanolamine and phosphatidylcholine species in platelets.
The positional and enantiomeric specificity of the HETE was
confirmed using several chromatographic approaches, and the
lipids are absent in platelets from 12-lipoxygenase–deficient
mice (M. Aldrovandi and V.B. O’Donnell, unpublished data,
2012). Up to 30% of the total 12-HETE generated by plate-
let 12-lipoxygenase is incorporated into phospholipid, rep-
resenting a significant proportion of the endogenous pool.26
HETE-phospholipid generation is highly coordinated using
intracellular signaling pathways that include Src tyrosine
kinases, Ca2+ mobilization, and phospholipases and they re-
main cell associated after their generation.26 Their formation
requires PLA2-dependent hydrolysis of AA from the plasma
membrane, followed by its oxidation by 12-lipoxygenase,
then re-esterification using Co-A–dependent ligases.26 In vi-
tro, liposomes containing physiological amounts of HETE-
phospholipids significantly enhance tissue factor-dependent
thrombin generation in plasma.26 This indicates that they may
play a key role in hemostasis and is consistent with the obser-
vation that in myeloproliferative disorders, patients with 12-li-
poxygenase deficiency hemorrhage is greater than those with
normal levels of the enzyme.127 Pathways for their formation
and cellular localization are summarized in Figure 9.
Platelet 12-lipoxygenase also generates 4 oxidized
acid, in place of 12-HETE.126 Recently, we scanned for
precursors of m/z 351 and uncovered 4 new families of
Figure 8. Generation of oxidized phospholipids (PLs) by enzymes. PL substrates (phosphatidylethanolamine [PE] is shown here) are
hydrolyzed by phospholipase A2 (PLA2) generating arachidonic acid (AA) that is oxidized by cyclooxygenase (COX)-1 or 12-lipoxygenase
(LOX) to form eicosanoids, which are then re-esterified into PE by fatty acyl Co-A ligases (FACL). PGD2 indicates prostaglandin D2; and
1196 Circulation Research March 28, 2014
phosphatidylethanolamine-esterified PGs, including 4 mo-
lecular species each of PGE2-phosphatidylethanolamine and
PGD2-phosphatidylethanolamine. Similar to the other esteri-
fied eicosanoids, these are generated acutely via coordinated
pathways and remain cell associated.35 Their inhibition by as-
pirin or indomethacin in vitro or in vivo by low-dose aspirin
indicates a requirement for platelet cyclooxygenase-1.35 PGs
are considerably more hydrophobic than HETEs and might not
be expected to remain within the cell membrane itself. Thus,
they may protrude out, anchored by the sn1 FA, in a man-
ner similar to that proposed in the Lipid Whisker Hypothesis,
available to interact with receptors on adjacent cells and medi-
ate paracrine signaling.128 Currently, their physiological func-
tions are unknown.
In summary, human platelets generate a diverse array of
oxidized phospholipid acutely on activation. We have also
found that lipoxygenase isoforms in other innate immune
cells, including neutrophils and monocytes/macrophages,
generate analogous lipids on activation, indicating that this is
a common theme of likely importance in acute response to
injury.33,129–131 All these cell types undergo significant changes
to the plasma membrane compartment on activation, includ-
ing shape change, vesiculation, phagocytosis, and microvilli
generation. Although we know much on how proteins control
these events, far less is known about how lipid oxidation influ-
ences the dynamic behavior of the cell membrane. Chemical
oxidation of model membranes has long been known to cause
changes in headgroup distance, membrane thickness, and wa-
ter permeability, whereas high concentrations (milligram per
milliliter) of purified 15-lipoxygenase can cause pore forma-
tion in purified organelle membranes, through lipid peroxida-
tion, and its overexpression in nonerythroid cells is associated
with mitochondrial membrane collapse.132,133 Thus, the signifi-
cant oxidation that occurs on platelet activation might have
similar consequences. Future work using biophysical methods
will address these questions.
Lysophospholipids and LPA
Lysophospholipids and LPA are generated from phospholip-
ids via the actions of phospholipases: PLA2, phospholipase
Figure 9. Enzymatic pathways that form oxidized phospholipids (PLs) in platelets. A, Thrombin activation of protease activated
receptors (PAR) triggers formation of HETE-phosphatidylethanolamine (PE) and HETE-phosphatidylcholines (PCs), via several intracellular
signaling cascades. Glutathione peroxidase (GPX) is required to reduce the initial hydroperoxide product to 12-HETE-PLs.26 B, Formation
of prostaglandin (PGE2)- and prostaglandin D2 (PGD2)-PE in platelets after thrombin activation requires several intracellular signaling
pathways, and most likely occurs in dense tubular membranes, where cyclooxygenase (COX)-1 has been localized.35 FACL indicates fatty
acyl Co-A ligases; MAPK, mitogen-activated protein kinase; PLA2, phospholipase A2; and LOX, lipoxygenase.
O’Donnell et al Platelet Lipidomics 1197
D (PLD), or through phosphorylation of 1,2-diacylglycerol.
They can also be generated nonenzymatically although, how-
ever, there is no evidence currently that this happens in plate-
lets.134 The structure of lysophosphatidylcholine is shown
(Figure 10). During platelet activation, powerful stimulation
of cytosolic PLA2α occurs, generating AA for eicosanoid
generation and resulting in formation of lysophospholipids.
Using [14C]-stearic acid to prelabel phospholipids, generation
of stearic acid-lysophospholipids of several classes, includ-
ing PI, phosphatidylcholine, phosphatidylethanolamine, and
phosphatidylserine, was reported in response to stimulation
by collagen.135 Lysophosphatidylethanolamine and phospha-
tidylcholine appeared within 30 s, with lyso-PI and phospha-
tidylserine appearing ≈2 to 3 minutes. However, the overall
increase from the baseline was relatively small (2–3-fold).
Although only stearic acid–containing species were studied,
platelets might be expected to generate phosphatidic acid and
plasmalogen species also for phosphatidylcholine and phos-
phatidylethanolamine, reflecting the predominant molecular
species of phospholipid in these cells.135 The temporal gen-
eration of specific lysophospholipid species in platelets is not
currently known. These questions have not yet been addressed
using lipidomics but could easily be through either targeted
quantitative or multidimensional MS approaches.
LPA is generated by thrombin-stimulated platelets and can
itself stimulate aggregation through G-protein–coupled re-
ceptors.136 High concentrations exist in serum (5–10 μmol/L)
and were suggested to originate directly from platelets al-
though this would seem unlikely given their rate of produc-
tion of lysophospholipid precursors.136,137 More recently,
a key role for autotaxin, a plasma lysophospholase D, in
cleaving platelet-derived lysophospholipid to generate high
serum levels of LPA has been demonstrated using heterozy-
gous knockout mice (the homozygous knockout is embry-
onically lethal).138,139 Lysophospholipid substrates themselves
originate at least in part via the action of a platelet-derived
PLA1.140 At this time, the exact physiological significance of
platelet-derived LPA is still not clear although it is thought to
contribute to the development of the vasculature potentially
and possibly in atherosclerosis and in hypertension.141,142
Neutral Lipids: Glycerides, CEs, and Free
Neutral lipids are uncharged species that include glycer-
ides, CEs, and free cholesterol. In platelets, only small
amounts of these are present, with the most abundant being
cholesterol at >90%.3 Free cholesterol is essential for lipid
raft function, described in more detail in Sphingolipids and
Ceramides of this article. Glycerides include triglycerides
and 1,2-diacylglycerols, with triglycerides representing ≈2%
of neutral lipids.3 Although present at only trace amounts in
resting cells, 1,2-diacylglycerol is formed during platelet ac-
tivation by the action of PLC, as described earlier, and acti-
vates classical and novel isoforms of protein kinase C. Thus,
1,2-diacylglycerol is an important second messenger involved
in receptor-dependent activation of platelets. Currently, the de-
tailed composition of platelet 1,2-diacylglycerol is generated
endogenously, in terms of FAs is not known, or whether distinct
1,2-diacylglycerol species are more effective as second mes-
sengers. These questions could potentially be answered in the
future using new generation lipidomic approaches (eg, through
identifying and quantifying platelet-specific 1,2-diacylglycerol
s by LC/MS/MS or multidimensional MS methods).
Sphingolipids and Ceramides
Sphingolipids are a class of lipids that contain a sphingoid
base backbone, the simplest of which is ceramide and consists
of sphingosine and a FA (Figure 10). They are involved in
both dynamic membrane functions (eg, lipid raft–dependent
signaling) and acting as intracellular signaling messengers
although their role in platelets is uncertain.143 Sphingosine
is phosphorylated by sphingosine kinases 1 and 2 to form
sphingosine-1-phosphate (S1P; Figure 10), which binds to
specific G-protein–coupled receptors to influence vascular
development, carcinogenesis, chemotaxis, and proliferation.25
S1P is generated in platelets via the action of sphingosine ki-
nase, then stored and released on degranulation, contributing
to the potent angiogenic activity of serum.144–147 Separately, a
lipidomic study characterized the sphingolipid composition
of murine platelets and demonstrated that resting cells do not
contain S1P, in contrast to human.25 Instead, they contained
large amounts of dihydro-S1P and C24 and C24:1 ceramides.
Thrombin activation of mouse platelets in vitro leads to loss
of dihydro-S1P and ceramide, with major increases in sphin-
gosine and dihydrosphingosine. In vivo, this was observed as
an elevation in plasma dihydro-S1P. In contrast to this, human
plasma contains S1P and also dihydrosphingosine but little
ceramide.25 Separately, stored platelets were found to transfer
phospholipids and sphingolipids to newly released extracel-
lular vesicles, resulting in increased ceramide and decreased
S1P in the cells.28
S1P has long been recognized as a regulator of hemato-
poietic cell trafficking, immune regulation, vascular devel-
opment, and brain inflammation, released from activated
Figure 10 . Structures for lyso-
phosphatidylcholine (PC), ceramide
and sphingosine-1-phosphate (S1P). R
indicates fatty acid group.
1198 Circulation Research March 28, 2014
platelets. More recently, a role for this lipid in thrombopoiesis
was demonstrated using genetically deficient mice.148 S1P sig-
naling via the multifunctional S1P1 receptor is required for 2
specific events involved in platelet formation and release, the
directional migration of proplatelet-containing cytoplasmic
extensions into the circulatory compartment, and the shed-
ding of proplatelets in a Rac-dependent manner.148,149 Because
it is known that S1P1 couples to Rac activation, this sug-
gests that active signaling via S1P is required for key stages
of thrombopoiesis. In support, S1P1-deficient mice display
Currently, relatively little is known about how lipid mo-
lecular dynamics during cell activation can regulate platelet
function; however, one area of intense investigation in re-
cent years relates to the formation and action of cholesterol
and sphingomyelin-rich lipid rafts. These are specialized
membrane microdomains that compartmentalize cellular
processes relevant for signaling, membrane fluidity, and
protein trafficking. Lipid rafts were proposed in the 1970s
using biophysical approaches, but more formally recog-
nized through the work of Simons and Toomre150 in the
early 2000s. Raft microdomains are estimated to account
for ≤10% of the total surface area, with each containing
≈3500 sphingolipids molecules and 10 to 20 protein mol-
ecules. They are thought to enhance signaling functions by
providing platforms that allow clustering of receptors, ki-
nases, and adaptor proteins involved in signaling pathways.
The ability of functions of 2 additional proteins involved
in adhesion, GP Ib-IX-V and GP VI/Fcγ to activate plate-
lets is dependent on their translocation to lipid rafts.151–155
Furthermore, additional platelet proteins also reported to
localize to rafts include the scavenger receptor CD36 and
the tetraspanin CD63.156–158 Lipidomic methods have not
yet been applied to the characterization of platelet lipid
rafts, in terms of profiling individual molecular species. In
other cells (KB oral and KBC pancreatic tumor cell lines),
these domains are enriched in AA-containing plasmalogen
phosphatidylethanolamines although the molecular species
of sphingomyelin and phosphatidylserine were not deter-
mined.159 Whether platelet rafts differ from this is currently
not known. Furthermore, matrix-assisted laser desorption/
ionization imaging methodologies are not yet sufficiently
high resolution to enable subcellular localization of lipids to
be determined directly. If this were possible, then determi-
nation of raft lipids through approaches that do not involve
cellular disruption using detergents would significantly in-
crease our knowledge of the composition of these special-
ized domains and generate new information on how lipids
regulate key signaling events in platelets.
General Summary: Future Perspectives and
New lipidomic methodologies, in particular those afforded
by the latest high-resolution rapid scanning instruments and
imaging methodologies, are allowing us to open an exciting
window into the world of lipid mediators in platelet and other
cells, both in health and in disease. The opportunities pre-
sented are considerable, but their potential is yet to be fully
realized. Understanding the diversity and number of unique
lipid species in cells is of potential importance for elucidating
mechanisms of cell biology and disease and also identifying
biomarkers for stratified/personalized medicine. This is one
area where new generation lipidomic MS has the potential for
transforming our understanding of lipids in health and disease.
Although prelipidomic techniques reported overall composi-
tion of FAs in a particular lipid pool (eg, phospholipids), in-
dividual molecular species could not be detected. Analyzing
all molecular species is important if particular species display
biological actions not shared by others or are key biomarkers
of early disease. Although this field of research is still in its
infancy, it is exemplified by the observation that a particular
molecular species of triglycerides identified using MS can be
used for early diabetes mellitus prediction, and that a specific
phosphatidylcholine integrates hepatic lipogenesis and pe-
ripheral FA use.160,161
Combining this level of analytic ability with the study of
large cohort sample sets, new generation MS technology has
the potential to transform our understanding of disease mecha-
nisms. While cohort collections become larger and more com-
prehensive, it becomes important that omics technologies,
including lipidomics are both analytically robust and used to
investigate relevant questions. Considerable development work
is still required to analyze large data sets from clinical studies
using lipidomic MS screening, but the potential in terms of un-
derstanding disease mechanisms is significant. A recent study
that highlights this approach used MS to demonstrate a series
of phosphatidylcholine metabolites in plasma that predict risk
of cardiovascular disease. These were found to be generated in
the gut through bacterial metabolism of dietary phosphatidyl-
choline, absorbed into the bloodstream, and exerted proinflam-
matory effects of relevance to cardiovascular disease.162
A major challenge lies in the development of computational
and bioinformatic tools for analysis of the large data sets gen-
erated by high-resolution instruments. A number of packages
for processing MS data have been developed both by instru-
mentation companies and by research groups, but postprocess-
ing software is still lacking. Several databases provide spectral
libraries for lipids, including LipidMaps, HMDB, and Metlin;
however, coverage of each alone is incomplete. Recently,
Kind et al163 described the creation of a database including
in silico generated tandem MS spectra using cheminformat-
ics, which is now freely available (LipidBlast: http://fiehnlab.
ucdavis.edu/projects/LipidBlast/). This begins to address the
bioinformatics gap, but much remains to be done.
The application of lipidomics technologies can be either
exploratory or hypothesis driven. Many cohort and large data
set studies use lipidomics to compare sets of tissue or plasma
samples, where large numbers of abundant lipids (eg, phos-
pholipids and CEs) are profiled without a definitive biological
or hypothesis-driven question. This can be accomplished using
either targeted approaches (eg, monitoring several lipids from
well-defined pathways, such as eicosanoids, quantitatively us-
ing a triple quad LC/MS/MS approach with deuterated internal
standards for all species) but increasingly uses multidimen-
sional MS to profile several families of complex lipids simulta-
neously but with less accurate quantitation (a single deuterated
standard for 1 class of lipid is often used). This is a powerful
approach that may lead to discovery of biomarkers that could
O’Donnell et al Platelet Lipidomics 1199
guide individualized treatment strategies or facilitate monitor-
ing of drug responses (eg, which patients should be prescribed
aspirin, statins, or other drugs of relevance to disorders of lipid
metabolism) or open new avenues for understanding disease
mechanisms and could be considered analogous to genomic
approaches, such as genome-wide association study. However,
targeted lipidomic approaches ask specific hypothesis-driven
questions about the behavior of lipid mediators in health and
disease. As examples, in platelets, we have used targeted ap-
proaches to demonstrate that FA side chains regulate the
procoagulant actions of phosphatidylethanolamine and phos-
phatidylserine, and that platelets generate novel families of
procoagulant oxidized phospholipid through enzymatically
controlled pathways.26,51 Both approaches are equally relevant
and powerful, but with the new era of personalized/stratified
medicine and bioinformatics in medicine, it is likely that a new
dawn of discovery for lipid mediators will be driven by explor-
atory approaches that will advance our understanding of their
direct roles in human disease. At this time, we can only specu-
late on the potential applications for lipidomics approaches
in furthering our understanding of disease mechanisms and
in the development of new therapeutic approaches, but these
could involve: (1) predicting risk of disease (eg, cardiovascu-
lar disease or cancer), (2) making decisions on preventative or
treatment strategies, and (3) monitoring treatment efficacy and
guiding ongoing clinical decisions, using plasma, urine, or tis-
sue global lipidome features or specific lipid levels.
We currently have no robust estimates of the total number of
individual lipid molecular species in platelets or in any other
mammalian cell type. To address this, we are currently develop-
ing high-resolution LC/MS/MS and in-house generated bioin-
formatic methodologies to define the total number of unique
platelet lipids (knowns and unknowns) and how these change
on agonist stimulation (eg, thrombin) and cyclooxygenase inhi-
bition (eg, aspirin) in genetically unrelated donors (M. Mondhe
and V.B. O’Donnell, unpublished data). This is a challenging
endeavor because the number of false signals is high and ensur-
ing that accurate identification of real lipids is laborious.
Lipidomic MS can be used for screening, the identifica-
tion of new lipids based on the presence of a characteristic
functional group, or to study the movement of lipids from
inside to the outside of the cell. In our laboratory, we applied
a targeted lipidomic method, termed precursor scanning, to
uncover several families of new platelet lipids comprising
phospholipids with PGs, eicosanoids, or docosanoids at-
tached, that form within 2 to 5 minutes of platelet activation.
This was based on the idea that during collision-induced frag-
mentation, a characteristic eicosanoid or prostaglandin car-
boxylate anion would be generated. This is described in the
Oxidized Phospholipids section of this article.26,126 Separately,
we developed a method that identifies molecular species of
phosphatidylethanolamine and phosphatidylserine that traffic
to the cell surface on platelet activation, apoptosis, or aging.
This uses derivatization of external facing amino-containing
phospholipids using a cell-impermeable reagent and has al-
lowed us to identify not only which lipids are externalized but
also which platelet-specific phosphatidylethanolamine/phos-
phatidylserine are more procoagulant based on side chain FA
composition (see Aminophospholipids of this article).51
An important area where lipidomics can be used to provide
new information is in the characterization of lipid adduction to
nonlipid molecules during cell activation, signaling, and prolif-
eration. This generates novel species that are readily amenable
to MS structural analysis. One example is the reaction of lipid
electrophiles with key transcriptional regulators via Michael
addition, which occurs during prostaglandin-dependent acti-
vation of the transcription factor Nrf2.164 In addition, lipids
can adduct to proteins to form membrane anchors, for exam-
ple, via palmityolation, which is used to localize Src family
kinases and the adapter linker of activated T-cells to lipid rafts.
Lipidomics combined with proteomic MS could then be used
to inform on position of lipidation within the protein critically,
as well as the specific lipid itself.
Although many challenges remain in ultimately defining the
platelet lipidome, the new advances in technology are likely to
make this a reality both in terms of characterization and quan-
titation of all lipid species. However, this on its own is only
the beginning of a new era. The challenge will be to establish
the functional significance of this vast amount of information.
Perhaps the biggest area of effect in relation to platelet regula-
tion will be around the therapeutic manipulation of products
of phospholipases, in particular, phospholipases A2, C, and D
and in the regulation of PI 3-kinases. It remains to be seen to
what extent the variation in lipid backbone influences signal-
ing, and how this varies between donors, health/disease, and
with diet/drug therapy. With an aging population, and the criti-
cal role of platelets in thrombosis and bleeding, the question
will emerge as to whether the knowledge of the lipid com-
position and functional activity of second messengers, such
as 1,2-diacylglycerol, can be targeted therapeutically or in
the diet. However, the challenge of this is perhaps illustrated
by the demonstration of a critical role for PLD in pathologi-
cal thrombus formation and ischemic stroke as shown using
both mutant mice and a small pharmacological inhibitor.165,166
Activation of PLD contributes to GPIb-mediated activation of
platelet integrins, but the molecular basis of this effect, and the
possible involvement of formation of 1,2-diacylglycerol from
phosphatidic acid, remains to be established.
We thank Drs Mondhe, Slatter, and Zemski-Berry for generating pan-
els for Figure 4.
Sources of Funding
This work was supported in part by grants from the British Heart
Foundation (S.P. Watson and V.B. O’Donnell) and Wellcome Trust
(S.P. Watson and V.B. O’Donnell) and grants from the National
Institutes of Health (USA) U54 HL117798 The Personalized NSAID
Therapeutics Consortium and R01 ES022172 (RCM). S.P. Watson is
a British Heart Foundation Chair.
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