Keywords platelets - acquired - atherogenesis - lipids - adhesion molecules - inflammation
Introduction
We have experienced during the last decades a continuous progress in treating the
complications of atherosclerosis such as myocardial infarction (MI), stroke, and acute
limb ischemia by diagnostic improvements and continued development of new medical
devices and drugs. Still, a large part of patients with acute ischemia does not manage
in time to profit from these medical advances.[1 ] Thus, the quest for novel targets aimed at a further individual reduction of the
risk for a cardiovascular event by preventing atherosclerosis is justified. It has
been argued that the sole causal risk factor for atherosclerosis is simply hypercholesterolemia
and that other epidemiologically associated factors are either exacerbating or only
bystander phenomena.[2 ] It may be that by abolishing circulating low-density lipoprotein (LDL)-cholesterol
atherogenesis might be completely preventable; whether such an approach is realistic
remains questionable. In the meantime, it will be worthwhile to understand the mechanisms
of atherogenesis to identify novel targets. Beyond the metabolic, there is an intricate
chronic inflammatory component to consider. With the CANTOS trial, the hypothesis
that cardiovascular events can be prevented by blocking a potent inflammatory target
such as interleukin-1β has been proven.[3 ] However, a clear cardiovascular benefit was outbalanced by the moderate effect size
and fatal infections warranting further refinement through an individualized approach
or testing new inflammatory targets.[4 ] Especially T cells or monocytes, giving rise to intimal macrophages, but also almost
every circulating blood cell type, have been described in plaques or to contribute
to the inflammatory infiltrate and stability of the plaque.[5 ] Irrespective of their pivotal role in arterial thrombosis, a causative role of platelets
in atherogenesis has been suggested in the 1960s based on the concept that platelets
represent a link between hemodynamic factors, lipids, and the characteristic localization
of plaques.[6 ] The concept of platelets as important inflammatory agents has been refreshed again
in 2005 by Gawaz and colleagues explaining the proinflammatory machinery of platelets
that intimately links thrombosis and atherosclerosis.[7 ] In recent years, rare genetic mutations with atherosclerosis phenotypes have been
discovered and genome-wide association studies as well as Mendelian randomization
studies have emerged as powerful tools generating big data that shed light and spark
discussions about how platelets and blood cells may contribute to human atherosclerosis,
defining novel targets for primary prevention.
Platelets, Initiators, and Amplifiers of Atherogenic Leukocyte Recruitment
Platelets, Initiators, and Amplifiers of Atherogenic Leukocyte Recruitment
Nearly our entire knowledge about the mechanisms of how platelets affect inflammation
and atherosclerosis originates from animal models; preclinical data, however, have
only partially been translated to the human system. The current understanding is that
platelet activation is a requirement for their atherogenic properties. Hyperreactive
platelets are associated with greater atherosclerotic plaque burden and increased
plaque vulnerability, especially in culprit lesions in patients undergoing percutaneous
coronary intervention (PCI) as measured by intravascular ultrasound (IVUS), and patients
with more extensive coronary atherosclerosis have a higher number of hyperreactive
platelets.[8 ]
[9 ] It is thus possible that increased platelet reactivity may potentiate arterial thrombosis
at the time of rupture, thereby driving inflammation and atherosclerotic lesion progression.
A crucial factor after platelet activation is the upregulation and activation of adhesion
receptors that initiate and enhance the contact of platelets with (1) leukocytes to
form aggregates and with (2) endothelial cells or their underlying matrix when exposed
after injury. Key players are αIIbβ3, P-selectin, the von Willebrand factor (VWF)
receptor complex (GPIbα/V/IX), and glycoprotein VI (GPVI).[7 ] These adhesion molecules and their binding partners lead to an increase in rolling
and firm adhesion of platelets on endothelial cells, and tether circulating leukocytes
to the artery as a requirement for subsequent migration into the intima.[10 ] Other adhesion molecules behave in a counterintuitive way: the selective genetic
deficiency of JAM-A in platelets results in hyperreactive platelets and an increase
in the formation of atherosclerotic lesions as JAM-A interacts with and inhibits αIIbβ3
activation, which also results in chemokine release ([Fig. 1 ]).[11 ] However, a cell-type-dependent expression of JAM-A may be decisive whether JAM-A
is atherogenic or atheroprotective. The expression of JAM-A on endothelial cells guides
monocytes into flow-dependent predilection sites of atherosclerosis and JAM-A plasma
levels are increased in coronary artery disease (CAD).[12 ]
[13 ] Therefore, generalized JAM-A inhibition could turn out to be a two-sided sword.
Fig. 1 Atheroprotective role of platelet JAM-A. The integrin αIIbβ3 and JAM-A are coexpressed
on platelets. Either by direct interaction or possibly via contacts through intermediary
proteins lead the presence of JAM-A to reduced outside-in signaling of αIIbβ3 (A ). Genetic depletion of JAM-A from platelets results in increased outside-in signaling
and consequently hyperreactive platelets (B ). These hyperreactive platelets tend to form aggregates and complexes with monocytes
and interact with dysfunctional endothelium to release chemokines such as CXCL4, CCL5,
and CXCL12 which drives the generation of early atherosclerotic lesions.
Several functionally relevant chemokines are expressed and released by platelets.[14 ] Chemokines tend to oligomerize which leads to the formation of mostly homodimers
and heterodimers of either a CC-type (interaction of the N-terminal part) or a CXC-type
(extension of the β-sheet). This is important because atherogenic monocyte recruitment
by CCL5, CXCL4, and their heterodimers depends also on these features. It can be therapeutically
addressed by peptide inhibitors that are derived from amino acid sequences of the
interface and protect from atherosclerosis.[15 ]
[16 ]
GP1bα Interactions with von Willebrand Factor Mediate Platelet Adhesion and Promote
Atherosclerosis
GP1bα Interactions with von Willebrand Factor Mediate Platelet Adhesion and Promote
Atherosclerosis
Platelets have been described as initiators of atherosclerosis because they adhere
to the arterial endothelium of the carotid artery in ApoE
−/− mice before atherosclerotic lesions become visible in a VWF- and GP1b-dependent process.[17 ] VWF is stored and released upon injury or under inflammatory conditions from endothelial
Weibel–Palade bodies or platelet α-granules. It bridges collagen and activates a receptor
complex upon multimerization (GPIbα/V/IX), which is exclusively expressed by megakaryocytes
and platelets leading to platelet adhesion on endothelial cells and driving early
as well as midstage atherosclerosis in mouse models.[17 ] The presence of activated VWF on atherosclerosis-prone endothelium has been confirmed
by molecular imaging detecting GP1b-conjugated microbubbles by ultrasound in vivo.[18 ] Notably, the source of VWF affects its functionality, possibly by altered glycosylation
such as reduced N-terminal sialylation and reduced affinity for GPIbα.[19 ] This might explain why in mouse models only endothelial cell-derived VWF but not
platelet-derived VWF promotes atherosclerosis.[20 ]
After binding and activation of endothelial CD40 by CD40L, ultralarge VWF–platelet
strings arise and facilitate monocyte diapedesis.[21 ] In conjunction with a reduced activity of ADAMTS13, which cleaves ultralarge VWF,
and consequently higher amounts of ultralarge VWF in plasma of patients with CAD,
this mechanism has been proposed to contribute to enhanced monocyte recruitment at
atherosclerotic predilection sites.[21 ] Elevated VWF levels in humans are strongly associated with an increased risk of
ischemic cardiovascular events. Whether this relation is causal or whether increased
VWF levels just reflect disturbances of endothelial function remains to be elucidated.[22 ] It would be very interesting to translate these finding to human genetic disorders
that are comparable to mouse models. Robust large-scale prospective clinical data
that confirm a protection from atherosclerosis are, however, missing so far for both
the rare Bernard–Soulier syndrome (GP1bα deficiency) and the common von Willebrand
disease (VWD) where VWF is dysfunctional and levels are decreased. Lately, a Swedish
registry of 2,790 individuals found that cardiovascular disease (CVD) mortality was
more than halved in patients with VWD compared with controls while hospitalization
due to a cardiovascular event was increased by 30%.[23 ] This might indicate an increase of rupture-prone lesions or lower stability of atherosclerotic
lesions, whereas the lower mortality might be associated with a protection from arterial
thrombosis. In aggregate, VWF appears to be a therapeutic target but experimental
and clinical data are not consistent and require further investigation.
P-selectin
P-selectin is expressed on both endothelial cells and platelets. It is known for quite
some time that the presence of P-selectin on both cellular entities can exacerbate
atherosclerosis as shown by bone marrow transplantation models and adoptive transfer
of P-selectin-deficient platelets.[24 ]
[25 ] More recently, based on these findings and aiming to prevent the anti-inflammatory
and antithrombotic platelet-dependent processes that are instrumental in atherogenesis,
a P-selectin blocking monoclonal antibody (inclacumab) has been developed and tested
in humans with NSTEMI, which resulted in decreased peri-interventional myocardial
damage.[26 ] As a side note, P-selectin expression is enhanced in platelets and endothelial cells
in patients with sickle cell disease contributing to the risk of vaso-occlusive crises.
A humanized monoclonal antibody, crizanlizumab, has been shown in the SUSTAIN trial
to protect patients with sickle cell disease from vaso-occlusive crises and has recently
been approved by the Food and Drug Administration for this indication.[27 ]
Although promising, randomized controlled trials over a longer period in a preventive
setting and monitoring plaque development would be warranted to draw conclusions on
beneficial effects of blocking P-selectin on atherogenesis in humans.
Glycoprotein VI
GPVI is one more target in atherogenesis. GPVI is a platelet-specific membrane protein
that is primarily known for its interaction with fibrillar collagen but other ligands
such as fibronectin or vitronectin are also known.[28 ] These ligands are thought to be the binding partners for GPVI in atherogenesis since
the subendothelial localization of collagen precludes their encounter at intact endothelium.[29 ] Platelets adhere to the endothelium of early atherosclerotic arteries which can
be diminished by antagonists like GPVI-Fc that can be coupled to microbubbles or by
monoclonal antibodies, which goes hand in hand with a lesser extent of atherosclerosis.[29 ]
[30 ] The inhibition of GPVI appears to be especially attractive as side effects are expected
to be low since humans with a genetic deficiency of GPVI and respective knockout mice
have only a mild bleeding diathesis.[31 ] GPVI-Fc is envisioned to be powerful in preventing atherothrombosis without causing
bleeding because collagen is an important component of atherosclerotic plaque activating
platelets via GPVI and GPVI-Fc is most effective under high shear stress, but not
low shear rates.[32 ]
[33 ] GPVI-Fc (revacept) has entered clinical trials. A phase I trial showed that the
drug was well tolerated and currently phase II trials are being performed to test
its effectiveness in preventing periprocedural PCI-associated events.[34 ]
[35 ] The parenteral application of GPVI-Fc however hampers its use in primary prevention.
An alternative option is orally available inhibitors of the Bruton's tyrosine kinase
that interfere with the downstream signaling of GPVI and GPIb.[36 ]
[37 ]
[38 ]
Platelet and NETs in Atherosclerosis: Guilty by Association
Platelet and NETs in Atherosclerosis: Guilty by Association
Activities of platelets and neutrophils are closely intertwined and join forces in
inflammation and atherosclerosis. Formation of neutrophil extracellular traps (NETs;
NETosis) emerges as a potential important link between these cellular entities.[39 ] Upon activation, neutrophils release decondensed chromatin decorated with granule
proteins forming extracellular fibers that bind and kill bacteria.[40 ] Critical for the unfolding of the chromatin structure is the enzyme peptidylarginine
deiminase (PAD4) that catalyzes the citrullination of histones thereby uncoiling chromatin.[41 ]
The presence of NETs has been shown in sections of human atherosclerotic lesions,
both at the luminal aspect and within murine atherosclerotic lesions.[42 ]
[43 ] A role of NETs in propagating atherosclerosis is further supported by the finding
that pharmacological interventions blocking NET formation via PAD4 inhibition can
reduce atherosclerosis and arterial thrombosis in mice.[44 ] Several mechanisms have been proposed to explain the proatherosclerotic role of
NETs: e.g., neutrophil-derived granule proteins (e.g., cathelicidin) stimulate a type
I interferon response and cause endothelial dysfunction[45 ] and cholesterol crystals induce NETs which prime macrophages for atherogenic IL-1β
release.[43 ] Furthermore, smooth muscle cells (SMCs) from atherosclerotic lesions attract neutrophils
and trigger NETosis which in turn causes arterial tissue damage and inflammation.[46 ]
A role of platelets in NETosis was noted by observing that plasma from humans suffering
from severe sepsis induced TLR4-dependent platelet–neutrophil interactions, leading
to the production of NETs and clearing of bacteria.[47 ] Under various conditions of activation, platelets have been demonstrated to trigger
neutrophils to expel their NETs. In addition, platelet inhibitors proved to be protective
by preventing NET formation in neutrophil–platelet-dependent diseases such as acute
lung injury and atherosclerosis.[39 ]
[48 ] Furthermore, in chronic inflammation platelet microparticles contribute to vasculopathy.
This is fostered by their interaction with neutrophils and depends on the nuclear
danger molecule HMGB1, which triggers neutrophils to cast their NETs.[49 ]
[50 ]
Platelets store and release chemokines that can form heteromers such as the chemokine
CXCL4 that binds to CCL5 leading to synergistic effects on leukocyte recruitment and
which can elicit NETosis in combination but not alone.[51 ] Blocking this heterodimerization reduces atherosclerosis, lung injury, and the formation
of NETs.[51 ]
[52 ] Reciprocally, cell-free NETs induce platelet aggregation which depends on cathepsin
G.[53 ]
In summary, both activated platelets and NETs alone have been shown to play a role
in experimental atherosclerosis. However, there is interdependency as activated platelets
bind to and activate neutrophils which eventually leads to NETosis.[39 ]
[45 ]
[54 ] What is unknown so far is to what extent each component drives atherosclerosis independently.
Although it is known that platelets induce NETs which then play a role in atherosclerosis,
the concept that NETs represent a causative link between activated platelets and atherosclerosis
has yet to be proven.
Platelets and Lipids in Atherosclerosis: A Complex Relationship
Platelets and Lipids in Atherosclerosis: A Complex Relationship
Based on the LIPID MAPS classification system, lipids can be classified into eight
categories (fatty acids [FAs], glycerolipids, [glycerol-]phospholipids, sphingolipids,
sterols, prenols, saccharolipids, and polyketines).[55 ] While all these are detectable in platelets, only few examples of prenols, saccharolipids,
and polyketines have been detected and are therefore not further discussed in this
section.[56 ] Nevertheless, several prenols, saccharolipids, and polyketines have important functions
in platelet biology and have been reviewed elsewhere.[57 ]
[58 ]
[59 ]
Polyunsaturated Fatty Acids Are Regulators of Platelet Activity and Affect Atherosclerosis
FAs can harbor multiple double bonds (PUFAs, polyunsaturated FAs). The position of
the first double bond at the methyl end (omega, opposite the carboxyl group) explains
the terminology so that omega-3 (n-3) FAs such as eicosapentaenoic acid (EPA) are
differentiated from omega-6 FAs like arachidonic acid (AA). Integrated into phospholipids
of the plasma membrane, FAs influence the fluidity and stability of cell membranes.
Bulkier molecules such as the n-3 FA docosaheptaenoic acid result in greater membrane
fluidity than FAs that fit better into the membrane geometry such as EPA.[60 ] This may have important implications for the activity of platelets and other cell
types and be one of various reasons why clinical trials in cardiovascular prevention
using PUFA (consisting of mixtures) reported ambiguous results. Nevertheless, the
REDUCE-IT trial, which corroborates the significant protective effects of pure EPA
on cardiovascular events in the JELIS study, reignited the interest in using omega-3
acids in preventing atherosclerosis.[61 ]
[62 ] Various preclinical data shed some light on the manifold, incompletely understood
mechanisms, including reduced inflammation by effects on T cells and enhancement of
resolution by lipid mediators, enhanced cholesterol efflux, antioxidant properties,
and last but not least inhibitory effects on platelets.[60 ] A hint that these effects on platelet activation might be clinically relevant comes
from the tendency toward more bleeding events under EPA in REDUCE-IT.[61 ] Feeding EPA to rabbits increases the incorporation of EPA into platelets and reduces
collagen-induced platelet aggregation.[63 ] FAs are part of phospholipids in the platelet membrane and get released by cytoplasmic
phospholipase A2 to serve as a substrate for platelet cyclooxygenase (COX-1), lipoxygenase
(12-LOX), and CYP50 epoxygenases yielding various platelet inhibitors and activators.
While AA serves as a precursor for the potent platelet activator thromboxane A2 and
an opponent of the potent platelet inhibitor PGI2 (prostacyclin), EPA can be metabolized
in platelets to various inhibitory lipids like thromboxane A3.[59 ] These lipid mediators have a short half-life so that they are generated on demand.
Adding AA to whole blood rapidly induces platelet activation in an autocrine manner
via thromboxane binding to its Gi -coupled receptor TP on platelets, whereas PGI2 is produced by endothelial cells and
activates in a paracrine fashion its Gs -coupled receptor IP. Deletion of TP results in diminished platelet reactivity and
reduced atherosclerosis, whereas the knockout of IP accelerated atherosclerosis and
decreased the stability of the lesions. Hence it appears that platelet activation
through lipid mediators can be an important regulator of atherosclerosis.[64 ]
[65 ]
[66 ] The platelet-specific effects of low-dose aspirin result from irreversible inhibition
of COX-1 in platelets. Effects on PGI2 and thromboxane A2 are only the tip of the
iceberg as low-dose aspirin in humans leads to drastic changes of the platelet FA
profile and of other lipids that on top seem to vary considerably between donors.[56 ] Aspirin is established in secondary but not in primary cardiovascular prevention.
In humans low-dose aspirin completely abrogates platelet-derived thromboxane generation
without reducing C-reactive protein (CRP) levels.[67 ] Whether aspirin can reduce atherosclerosis in humans remains unclear; in light of
many disappointing results in the setting of primary prevention, the latest being
the ASPREE trial, large effects on atherogenesis seem unlikely.[68 ] A failure of low-dose aspirin to reduce residual inflammation, as assessed with
the most established prognostic risk marker in CVD, CRP, could be a possible explanation.
Sphingolipids
Sphingolipids are components of the cell membrane and regulate signaling. They comprise
a group of molecules that are derivatives of ceramide which is mainly generated at
the cytosolic side of the endoplasmic reticulum by assembling the amino acid serine
and palmitoyl-CoA. Enzymatic reactions with ceramide as a substrate result in sphingosine
and further steps produce sphingosine-1-phosphate (S1P), glucosylceramide, lactosylceramide,
and sphingomyelin, which are important signaling molecules in inflammation and atherogenesis.[69 ]
[70 ]
[71 ] Analysis of atherosclerotic plaques revealed that sphingolipids are important plaque
components and contribute to plaque inflammation and stability.[72 ]
Different to the on-demand metabolism of FA, platelets store S1P in granules and in
nongranular compartments.[73 ] Platelet activation leads to a release of S1P upon activation. As platelets express
S1P receptors such as S1PR1, this results in a positive feedback mechanism.[74 ] Moreover, expression of S1PR1 on megakaryocytes is required for normal thrombopoiesis
as S1P drives cytoplasmic extensions of megakaryocytes into bone marrow sinusoids
to shed proplatelets into the circulation.[75 ] Although S1P is considered a proinflammatory mediator, platelet-derived S1P may
have atheroprotective properties: it accelerates endothelial cell proliferation and
drives endothelial cell migration.[76 ] Sphingomyelin affects platelet reactivity through its incorporation into and formation
of specialized regions within platelet membranes, lipid rafts, where important platelet
adhesion receptors including GPVI and GPI-V-IX or scavenger receptors such as CD36
are embedded and that are required for a proper function.[57 ] Another lipid species, phospholipids, is essential in these rafts fostering signaling
through G-protein-coupled receptors by providing important second messengers.[77 ]
(Glycero-)Phospholipids
Phospholipids are the main component of cell membranes composed of glycerol derivatized
by two hydrophobic acyl groups and a polar phosphate group. Depending on the molecules
attached to the phosphate, phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine
(PE), phosphatidylserine (PS), and the phosphatidylinositol-related species (PI-PIP3)
are classified. Cleavage of phospholipids by phospholipase C generates phosphoinositide
second messengers in signal transduction (e.g., PIP, IP3 ) and diacylglycerol, whereas lysophosphatidylcholine (LPC) arises after removal of
an alkyl group of PC and a subsequent step by the phospholipase D autotaxin forms
lysophosphatidic acid (LPA).
Under resting conditions the aminophospholipids PE and PS remain located at the inner
leaflet of the platelet membrane. Platelet activation and apoptosis lead to Ca2+ -dependent activation of a phospholipid transporter, the scramblase TMEM16F, that
leads to the translocation of PS and PE to the outer leaflet thereby exposing binding
sites for Annexin V and coagulation factors.[78 ]
Furthermore, the comparison of the platelet lipid profiles from CAD patients with
matched controls reveals that LPC is weakly detectable in platelets of healthy persons,
but increased by several orders of magnitude in CAD patients and also found in vulnerable
atherosclerotic plaques.[79 ]
[80 ] LPC has been reported to be concentrated in microvesicles from activated platelets
which are important markers and factors in vascular inflammation and atherosclerosis.[81 ] Lipid profiling of platelet MV identified differences in the composition with higher
amounts of PC and LPC compared with the activated parent platelets.[80 ] Platelets express the lysophospholipid receptor G2A/GPR132 that is responsible for
platelet activation through LPC.[80 ]
LPA, a derivative of LPC, is implicated in platelet activation and atherosclerosis
signaling through G-protein-coupled receptors of the LPAR (EDG) family. LPA is produced
by platelets through phospholipase A1 and autotaxin and is one active component of
mildly oxidized LDL and atherosclerotic plaques in platelet activation.[82 ]
[83 ]
Sterols/Cholesterol
Hypercholesterolemia in humans correlates with platelet count.[84 ] As specified above, the relevance of the platelet count and platelet indices in
humans for atherosclerosis remains vague/obscure. However, antibody-induced selective
depletion of platelets inhibits atherogenesis significantly.[85 ] Inversely, it is conceivable that an increased generation of platelets by hypercholesterolemia
is atherogenic although the mechanisms how cholesterol levels modulate thrombopoiesis
are unresolved.[86 ] Reducing cholesterol and circulating LDL levels affects platelet reactivity by cholesterol-dependent
and pleiotropic, cholesterol-independent effects.[87 ] Clinically, pleiotropic, antithrombotic actions of statins have been concluded from
the JUPITER trial that showed a significant reduction of deep vein thrombosis in the
cohort treated with rosuvastatin.[88 ] Which molecular mechanisms lie behind these assumed antiplatelet effects have been
investigated in preclinical studies. Statins inhibit the synthesis of cholesterol
by blocking HMG-CoA-reductase and as a side effect other lipids such as farnesyl pyrophosphate
and geranylgeranyl pyrophosphate are missing for posttranslational prenylation of
the small GTP-binding proteins Rho and Rac so that their activity is reduced.[89 ] Rho and Rac are ubiquitously expressed and are indispensable regulators of the platelet
cytoskeleton with various effects on downstream pathways including nitric oxide signaling.[90 ] However, the size of the attributed pleiotropic effects on platelet reactivity in
vivo is difficult to separate from the effects that occur as a result of the reduction
of cholesterol: hypercholesterolemia alone increases platelet activation via binding
of LDL and oxLDL to platelet CD36.[91 ]
[92 ] Novel treatment options for hypercholesterolemia as antibodies against PCSK9 also
display antiplatelet characteristics: in a small clinical study the treatment with
PCSK9 inhibitors resulted in a decrease of platelet P-selectin, platelet aggregation,
and released proteins from α-granules like CXCL4.[93 ] Levels of circulating PCSK9 correlate with platelet activity in acute coronary syndrome
patients.[94 ]
Genetic Evidence for a Role of Platelets in Coronary Artery Disease
Genetic Evidence for a Role of Platelets in Coronary Artery Disease
Platelet Parameters
Hematologic parameters including platelet count or mean platelet volume (MPV) have
been associated with atherosclerosis and cardiovascular risk.[95 ]
[96 ] Larger platelets have been described to be more reactive and to have a greater prothrombotic
potential so that MPV has been found to be a useful prognostic parameter in MI.[95 ] Although MPV is a readily available parameter with implications to platelet function,
it is not standardized. Multiple factors including preanalytical issues such as time-dependent
swelling of platelets in EDTA affect MPV so that using MPV is not a standard in clinical
practice.[97 ] As hematologic parameters show high interindividual variability, large genome-wide
analyses of hematologic parameters have been undertaken to identify genetic variants
that influence traits of red and white blood cell counts, but also platelet indices.
[Supplementary Table S1 ] (available in the online version) gives an overview of studies reporting genetic
associations with platelet phenotypes. In 2009 a first large systematic genome-wide
meta-analysis[98 ] identified 15 loci determining the MPV that jointly explained 8.6% of the total
genetic variance in MPV, but only to 0.5% of the platelet count.[98 ] The most interesting region associated with platelet count and MPV was a haplotype
restricted to Europeans located on chromosome 12q24 comprising 10 common single nucleotide
polymorphisms (SNPs) including a nonsynonymous SNP Arg262Trp (rs3184504) in the gene
SH2B3 associated with atherosclerosis and MI (see below). This haplotype is of importance
because it significantly associates with premature CAD.[99 ] In more recent and even larger studies searching for cardiometabolic risk factors
in Europeans, more loci and variants have been discovered and refined that are more
strongly associated with platelet count and explain the variance of platelet count
to more than 8%.[100 ] Signals for platelet count were mostly found within genes for congenital (GFI1B , THPO ) or acquired (APOH ) platelet disorders, underscoring that more subtle genetic variation within genes
known to contain loss-of-function variants may reflect interindividual differences
in these complex traits.[100 ] In the last and so far most powerful study, more than 1,000 variants were identified
to define platelet indices. In a Mendelian randomization study these variants displayed
a weak, unexpectedly inverse relationship of coronary heart disease (CHD) and MPV
suggesting shared causal pathways for CHD and MPV, although the mechanisms behind
remain to be clarified.[101 ]
Taken together the results of these studies suggest that functional properties are
more important for the role of platelets in atherosclerosis than the number of platelets
([Supplementary Table S1 ], available in the online version).[101 ]
[102 ]
[103 ]
[104 ]
[105 ]
[106 ]
[107 ]
[108 ]
As a prominent example, the cytochrome P450 2C19 genotype has been associated with
a response to clopidogrel therapy.[109 ]
[110 ] Genome-wide analyses also identified several genetic variants identified with platelet
aggregation after stimulation with different agonists.[111 ] Additionally, genes or pathways involved in platelet biology or function have also
been identified to be associated with coronary atherosclerosis (for a review, see
Erdmann et al[112 ]), e.g., the SH2B3 gene and nitric oxide signaling.
SH2B3/LNK—A Coronary Artery Disease Risk Gene with a Role in Platelet Function
As stated above, the SH2B3 gene is located on 12q24 at a very complex genomic locus which shows associations
with a variety of traits, e.g., type 1 diabetes,[113 ] blood pressure,[114 ]
[115 ] celiac disease,[116 ] but also CAD[99 ] and platelet count.[98 ]
[101 ] The gene encodes LNK, an inhibitory adaptor protein regulating cytokine signaling
and cell cycle in endothelial and hematopoietic cells.[117 ]
[118 ] LNK prevents the signal transduction from a receptor tyrosine kinase to downstream
JAK2, such as the signaling from TPO via MPL that triggers thrombopoiesis. Therefore,
loss of LNK signaling results in thrombocytosis and increased platelet activation
by αIIbβ3 outside-in signaling.[117 ]
[119 ]
[120 ] In a study designed to explain the mechanism of the common CAD risk variant which
results in a loss of function of LNK, Wang et al demonstrated that this variant causes
an increase in platelet count especially under proatherogenic conditions, i.e., high-cholesterol
levels. Lnk
−/− mice displayed enhanced platelet activation, more leukocyte–platelet complexes, and
accelerated arterial thrombosis and atherosclerosis. Specifically, hypercholesterolemia
in Lnk
−/− mice led to enhanced interleukin-3/granulocyte–macrophage colony-stimulating factor
receptor signaling but also increased platelet activation.[121 ] In summary, platelet LNK is now an experimental and genetic link between, on the
one side, high cholesterol levels, high platelet counts, high platelet reactivity
and on the other side increased atherosclerotic plaque formation and MI. Enhancing
LNK to inhibit platelet generation and activation might be an innovative strategy
to reduce cardiovascular risk
Multiple Genes Involved in Nitric Oxide Signaling
Several genes which encode proteins that play a prominent role in nitric oxide signaling
have been associated with CAD in genome-wide association studies (for a review see
Wobst et al[122 ]): NOS3 , which encodes the endothelial nitric oxide synthase (eNOS),[123 ]
GUCY1A1 (formerly named GUCY1A3 ), which encodes the α1-subunit of the soluble guanylyl cyclase (sGC),[124 ]
MRVI1 , which encodes inositol 1,4,5-trisphosphate receptor-associated cyclic guanosine
monophosphate (cGMP) kinase substrate (IRAG),[125 ] and PDE5A , which encodes phosphodiesterase 5A (PDE5A), are the most prominent examples. However,
several genes encoding proteins in related pathways, e.g., PDE3A , encoding phosphodiesterase 3A,[126 ] or the genes EDN-1
[127 ]
[128 ] and EDNRA ,[124 ] encoding endothelin 1 and its receptor, respectively, have also been associated
with CAD.
In the vasculature, nitric oxide is produced by eNOS mainly in endothelial cells leading
to production of the second messenger cGMP in, e.g., vascular SMCs (VSMCs) and platelets
by sGC. Accumulation of cGMP leads to relaxation of VSMC[129 ] and inhibition of platelet aggregation, respectively.[130 ]
[131 ] One mechanism is that the elevation of endogenous NO levels leads to reducing the
thiol reductase activity of protein disulfide isomerase by S-nitrosylation which prevents
platelet aggregation, α-granule release, and thrombin generation on platelets.[132 ] These NO effects, which also have been shown to influence, e.g., vascular remodeling[133 ] or vascular inflammation,[134 ]
[135 ] are limited by the breakdown of cGMP into GMP by PDE5A. Pharmacological modulation
of these processes is used in a variety of diseases: supplementation of nitric oxide
donors to relief angina pectoris, stimulators of sGC in pulmonary hypertension and
heart failure, and PDE5A inhibitors in pulmonary hypertension and erectile dysfunction.
MRVI1 , the gene encoding IRAG, which represents a target of cGMP-dependent intracellular
signaling, has also been associated with platelet aggregation.[111 ] The variants at all of these loci, i.e., NOS3 , GUCY1A1 , PDE5A , and MRVI1 , are located in noncoding regions. However, at least for NOS3 , GUCY1A1 , and PDE5A , an association between genotype and gene expression has been reported, i.e., the
risk alleles of NOS3 and GUCY1A1 lead to reduced gene expression.[136 ]
[137 ]
[138 ] As a consequence cGMP availability is reduced whereas the PDE5A risk allele is associated with increased gene expression.[139 ]
[140 ] While the effect at NOS3 and GUCY1A1 loci has been reported to be mediated via altered promoter activity,[138 ]
[141 ] the link between genotype and gene expression at the PDE5A locus is not yet understood.
While the connection between variants involved in nitric oxide signaling and platelet
aggregation is obvious, it is still not known how exactly this pathway is involved
in plaque formation and atherosclerosis in general. Hints that the pathway might not
be only important in atherothrombotic but also preceding processes come from both
experimental studies rendering an involvement in the recruitment of inflammatory cells
to the vessel wall likely.[134 ]
[135 ] Furthermore, there is genetic evidence that at least impaired sGC activity might
primarily affect atherosclerosis: in a family with high prevalence of premature manifestation
of CAD due to a digenic mutation in GUCY1A1 and the gene encoding the chaperone protein CCTeta, four of 11 family members carrying
at least one mutation underwent PCI or coronary artery bypass surgery at a young age
but did not suffer from MI.[142 ] It will thus be a challenge to identify the underlying cellular and molecular mechanisms
to evaluate the potential of modifying this pathway in atherosclerosis. In this respect,
cumulative effects of multiple risk alleles which share effects in the NO–cGMP-signaling
pathway or respective coexpression networks may be informative.[123 ]
[124 ]
[125 ]
[139 ]
[143 ]
Despite these data suggesting a role for platelet phenotypes and/or function in atherosclerosis,
it has to be mentioned that monogenic diseases influencing platelet phenotypes have
not been associated with reduced incidence of CVDs. In Glanzmann's thrombasthenia
in particular, a disease which is a consequence of deficient αIIbβ3 integrin function
in platelets, cardiovascular events as arterial thrombosis or deep vein thrombosis
have been reported (for a review, see Nurden[144 ]). Another example is the rare Bernard–Soulier syndrome that is characterized by
defects in the VWF–receptor complex (GPIb-V-IX), where also MI has been reported.[145 ] However, these reports have to be taken with caution as it is obvious that CVDs
and in particular CAD are influenced by several further risk factors that cause the
disease despite altered platelet function.
Pharmacological Approaches Targeting Platelets in Atherosclerosis
Pharmacological Approaches Targeting Platelets in Atherosclerosis
Given the findings from basic research, inhibiting platelet function seems like a
plausible strategy to prevent atherosclerotic plaque formation and progression. Current
platelet treatment targets include COX-1, which mediates AA metabolism, P2Y12 adenosine diphosphate receptors, and the αIIbβ3 glycoprotein receptor. Whereas the
latter two are rather targeted/utilized in specific situations, e.g., after coronary
stenting or in acute coronary syndromes, the role of aspirin in both primary and secondary
prevention of atherosclerotic disease as well as in animal models has been extensively
studied.
In secondary prevention, aspirin has been shown to significantly reduce the incidence
of vascular events in patients with acute stroke (absolute reduction: 0.9%) or MI
(absolute reduction: 3.8%), previous stroke/transient ischemic attack (absolute reduction:
3.6%) or MI (absolute reduction: 3.5%), but also other high-risk situations (absolute
reduction: 2.1%).[146 ] These benefits clearly outweigh the risk of bleeding, i.e., the number needed to
treat (NNT) to prevent a serious vascular event ranges between 50 and 100, whereas
the number needed to harm ranges between 500 and 1,000 and 5,000 and 10,000 for gastrointestinal
bleeding events and hemorrhagic strokes, respectively.[147 ] The effect in secondary prevention is thought to be a consequence of preventing
atherothrombosis.[148 ]
In primary prevention of atherosclerotic plaques using aspirin, the situation seems
more complex. In animal models, several studies have suggested that AA-related pathways
and their inhibition are associated with atherosclerotic plaque formation and/or progression.
In Ldlr
−/− mice fed a Western-type diet, indomethacin, a nonselective COX inhibitor reduced
atherosclerotic plaque formation and reduced expression of, e.g., soluble intercellular
adhesion molecule and monocyte chemotactic protein-1.[149 ] That this effect is mediated via COX-1 is suggested by the fact that selective inhibition
of COX-1 but not COX-2 led to reduced atherosclerotic plaque formation in ApoE
−/− mice fed a high-cholesterol diet.[150 ] An impact of reduced thromboxane A2 , the product of COX-1-mediated AA metabolism, on atherosclerotic plaque formation
has also been shown by specifically inhibiting its receptor; in this study, however,
the unselective inhibition of COX-1 and -2 by indomethacin did not lead to reduced
atherosclerotic plaque formation.[151 ] Data from human studies do not ultimately clarify the role of aspirin in primary
prevention. Although there are early data which suggested a benefit from high-dose
aspirin in atherosclerotic plaque progression,[152 ] randomized clinical trials in healthy subjects[153 ] and meta-analyses[154 ] have not been able to show a benefit from aspirin intake that outweighs the increased
risk in bleeding. Two recent studies in diabetic or high-risk patients also failed
to prove a benefit from aspirin in primary prevention.[155 ]
[156 ] There may be a subgroup of patients without previous cardiovascular events, but
at a risk comparable to that of patients in secondary prevention (predicted 10% mortality
in 10 years) or individuals with a particular genetic background. A very interesting
example is the reduction of LDL-cholesterol and cardiovascular events using statins.
Here, it has been shown that a high genetic risk score (including 27 variants associated
with incident CHD) is associated with a stronger reduction of cardiovascular risk
by statin therapy compared with individuals with a low genetic risk score. In an analysis
of the JUPITER trial, the NNTs to prevent a cardiovascular event within 10 years were
66, 42, and 25 in the low, intermediate, and high genetic risk groups, respectively.[99 ] It is possible that in the sense of precision medicine, individuals could also be
identified to specifically benefit from antiplatelet treatment with an unspecific
drug such as aspirin if this is identified as the disease-driving pathway.[157 ] Also here, knowledge from genome-wide association studies might be useful. Whereas
the Womens' and Physician's Health Study did not show a clear benefit from aspirin
in primary prevention in the overall study population,[153 ] individuals carrying the homozygous GUCY1A3 risk genotype had a benefit from aspirin treatment with a 17% risk reduction in women
and a 51% reduction in men. In women the NNT treated to avoid one major CVD event
was 121.[158 ] Considering higher risk and stronger effects in men, the NNT for GUCY1A3 -guided prescription of aspirin could enter the range of clear benefit unless otherwise
contraindicated (NNT ≤ 100).[159 ] Surprisingly, carriers of the nonrisk allele, either heterozygous or homozygous,
did not only lack benefit from aspirin but rather experienced an increased risk compared
with placebo.[158 ] This is a peculiar observation that remains to be validated and explained. However,
it has been shown that the GUCY1A3 genotype is also associated with a response to aspirin therapy with nonrisk allele
carriers showing lower on-aspirin platelet reactivity.[160 ] One could speculate that in nonrisk allele carriers aspirin shifts platelets toward
an increased risk of bleeding which is itself—directly and indirectly via anemia—associated
with cardiovascular events.[161 ]
[162 ]
[163 ] Of note, it also needs to be taken into account that the effect of particular SNPs
on CAD risk—and thereby also the presumed effect on the responsiveness to antiplatelet
therapies— is rather mediated by altered gene expression than protein function. In
contrast to CYP2C19 alleles leading to slower metabolism of clopidogrel to active metabolites and—as
a consequence—to increased risk of ischemic events in patients with acute coronary
syndromes[109 ]
[164 ]
[165 ], the GUCY1A3 risk allele is associated with reduced expression of the gene.[138 ] The influence of reduced sGC protein levels on response to aspirin treatment is,
however, thought to be a result of changes in intracellular equilibria: as such, AA
influences platelet nitric oxide levels[166 ] and GUCY1A3 risk allele carriers presenting reduced sGC protein levels and activity might benefit
from aspirin to outweigh this effect.[160 ] While this remains speculative, such complex interactions need to be taken into
account.
Conclusion and Outlook
In this brief review we have pointed out some recent advances in the understanding
how platelets influence atherogenesis, but a comprehensive reporting on all concepts
was not within the scope. There is still a large gap to be closed between the clear
notion of platelets as inflammatory and atherogenic cellular particles derived from
experimental data and prove for this concept in humans. This is based primarily on
the fact that platelets have a dual role as drivers of atherosclerosis and executers
of arterial thrombosis after plaque rupture. Human data evaluating prognosis originate
to a large part from registries, trials, and observational studies that mainly include
symptomatic patients after hospitalization and therefore the thrombotic role of platelets
in plaque erosion and rupture masks their impact on earlier stages in plaque development.
Still, some studies were able to relate platelet phenotypes to CAD by recording PCI
or coronary artery bypass grafting in a nonacute setting. An obstacle that needs to
be overcome generally in evaluating atherosclerosis, which is a slowly progressive
disease, is the possibility to assess plaque phenotypes in asymptomatic humans over
long time ranges. High-resolution imaging such as optical coherence tomography and
IVUS are able to characterize coronary plaques, but are invasive techniques precluding
a screening of asymptomatic patients. Therefore, a translational realization of all
these interesting concepts remains challenging.
