Platelets are small, anuclear, megakaryocyte-derived discoid fragments of 2–3 μm in diameter. Apart from their classical function in thrombosis and hemostasis , it is now widely recognized that platelets have a significant function in inflammatory and immune responses [2,3,4▪,5▪,6–8]. After leaving the bone marrow platelets to circulate in the blood system macrophages in the liver and spleen will clear them in a period of 5–9 days [9–11]. Their classical role in thrombosis and hemostasis was recently reviewed in detail by several experts in the field [1,5▪,12▪,13]. Furthermore, recent studies implicated platelets as essential effectors in sepsis , experimental autoimmune encephalomyelitis , allergy, rheumatoid arthritis , host defense during bacterial infection , and cancer . In addition to their role in these disease models, platelets display a particular versatile ability to modulate immune responses in atherosclerosis [19–22]. Specifically, platelets facilitate the recruitment of inflammatory cells to inflamed lesion sites and dysfunctional endothelium  by interacting with endothelial cells , circulating leukocytes [25,26] (monocytes , neutrophils [28,29], dendritic cells [30,31], and T cells ), and progenitor cells  (Fig. 1). This crosstalk induces leukocyte activation, adhesion, transmigration as well as formation of platelet–leukocytes aggregates (PLAs) [34,35]. Platelets contribute to atherosclerosis and modulate immune responses via various surface molecules  such as glycoproteins [36–44], costimulatory molecules (CD40,CD40L) [45–54], cell adhesion molecules (CAMS) (e.g. P-selectin) [55–59,60▪▪], junctional adhesion molecules (e.g. JAM-A) [61▪▪,62▪▪,63,64], Toll-like receptors (TLRs) [65–72], chemokine receptors [6,73▪], scavenger receptors , and protease-activated receptors (PARs)  (Fig. 1). In addition, platelets release upon activation considerable amounts of cytokines [e.g. interleukin-1 beta (IL-1β) [75,76] or transforming growth factor beta (TGF-β) [77,78]], chemokines (e.g. CXCL4 or CCL5) [73▪], and other contents [e.g. ADP, thromboxane A2 (TXA2), serotonin, platelet-derived growth factor (PDGF) , and soluble CD40 ligand (sCD40L) [46,80–83]] partly from preformed granules . Emerging work suggests a strong link between thrombosis and innate immune cells, particularly monocytes, neutrophils, and dendritic cells. Innate immune cells can initiate and propagate fibrin formation, induce neutrophil extracellular trap (NET) formation (which comprise matrix DNA and histones), and trigger platelet activation during development of thrombosis [1,14,84–88] (Fig. 1). A recent study reported that neutrophils scan for activated platelets to transmigrate and initiate inflammation [60▪▪]. The concept of immunothrombosis, which is increasingly recognized as an independent line of host defense, is reviewed excellently by others .
Since several overview articles recently discussed platelets under inflammatory conditions in general, this article highlights only a few topics of platelet biology in more detail. In particular, recent developments in specific platelet-signaling pathways (e.g. CD40-CD40L dyad, or JAM-A) as well as platelet–leukocyte crosstalk will be summarized. Furthermore, we will discuss direct effects of statins on platelets and interactions between lipid metabolism and platelets. Lastly, therapeutic options for the above-mentioned pathways and the role of platelets in novel cardiovascular imaging approaches will be considered.
Atherosclerosis is a chronic inflammatory disease of large-sized and medium-sized arteries, which is caused by complex immune and metabolic processes. Initial lipid accumulation, subsequent cellular activation inducing the recruitment of immune cells, transformation of monocytes into foam cells, and various immune reactions mediated by T cells, B cells, granulocytes, monocytes, and dendritic cells lead to a dysfunctional endothelium during an asymptomatic process which starts in childhood and progresses with age . Atherosclerosis is the primary underlying cause of cardiovascular disease (CVD) such as ischemic heart disease and stroke. In 2011, CVD still accounted for 31.3% (786 641) of all 2.5 million deaths, or approximately one in every three deaths in the USA. Finally, in 2011 the estimated annual costs for CVD and stroke were $320.1 billion highlighting the economic burden in addition to the personal calamities .
In addition to leukocytes and vascular cells platelets contribute to atherosclerosis during all stages by secretion of chemokines [6,25,73▪,90▪,91] and other inflammatory mediators [20,92–94]. One of the most important functions of platelets during atherogenesis, apart from their role in coagulation, lies in the interaction with endothelial cells and leukocyte activation and recruitment .
LIPID METABOLISM AND PLATELET BIOLOGY
LDL cholesterol levels correlate with CVD [89,95,96]. Recent studies evidence a functional link between platelet biology and lipid metabolism [97▪▪]. Notably, the amount of platelet-bound oxidized LDL (oxLDL) is increased in patients with acute coronary syndromes . Carnevale et al.[99▪] showed that platelets can oxidize LDL-particles in vitro via NADPH oxidase 2 (NOX2)-derived oxidative stress. OxLDL-particles in turn amplify platelet activation via receptors such as CD36, or lectin-like oxLDL receptor-1 (LOX-1) (Fig. 1). Interestingly, platelets of NOX2-hereditary deficient patients showed reduced oxLDL production compared with platelets from healthy individuals. NOX2 antagonists, CD36-blocking, or LOX1-blocking peptides inhibited platelet activation significantly. Stimulation with ‘ApoB100 danger-associated signal 1’, a native peptide derived from Apolipoprotein B-100 of LDL, induces platelet activation, degranulation, adhesion, and release of proinflammatory cytokines [100▪]. Platelets also mediate oxLDL-induced monocyte extravasation, platelet–monocyte aggregate (PMA) formation, and foam cell formation and enhance neutrophil transmigration. Of note, platelet inhibition by clopidogrel or aspirin is effective in reducing oxLDL uptake and PMA formation [101,102▪]. Platelet CD36 promotes atherosclerotic inflammatory processes through its interaction with oxLDL and is involved in thrombus formation following atherosclerotic plaque rupture . Specific oxLDL-CD36 interactions induce platelet activation, upregulation of P-selectin, CD40L, and intraplatelet reactive oxygen species (ROS) . Whole-body deficiency of CD36 in mice causes reduced atherosclerotic plaque formation [105,106].
3-Hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitors (statins), a drug class primarily used to lower plasma cholesterol concentrations in the prevention of myocardial infarction and stroke, can act as antithrombotic and reduce platelet activation via platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-signaling and decrease microparticle shedding from endothelium, platelets, and inflammatory cells [107–109]. Atorvastatin delays murine platelet activation and reduces thrombin generation and expression of tissue factor, P-selectin, and integrin αIIbβ3 on platelet-derived microparticles in patients with peripheral arterial occlusive disease [110,111]. In addition, atorvastatin can inhibit platelet CD40-mediated and CD40L-mediated thrombin generation, independently of its cholesterol-lowering effect . Notably atorvastatin reduces the proadhesive and prothrombotic endothelial cell phenotype caused by cocaine consumption . Atorvastatin enhances interactions between endothelial nitric oxide synthase (eNOS) and caveolin-1, thus increasing endothelial cell nitric oxide production . In addition, statins can directly increase platelet nitric oxide production by upregulating platelet eNOS in turn inhibiting platelet recruitment. Furthermore, statins lower CXCL4 and CXCL7 plasma levels, inhibit signaling of the thrombin receptor PAR-4, and inhibit NOX2, which mediates oxidation of LDL particles by platelets [110,114–116]. Prior to simvastatin therapy hyperlipidemic patients show a significantly higher percentage of P-selectin-positive platelets and higher reactivity to thrombin compared with healthy control individuals. Simvastatin therapy reduces P-selectin expression on platelets and soluble P-selectin (sP-selectin) levels . The ARMYDA-ACS (Atorvastatin for Reduction of Myocardial Damage During Angioplasty) clinical trial showed that atorvastatin pretreatment improves outcomes in patients with acute coronary syndromes (ACS) undergoing early percutaneous coronary intervention . Sexton et al.[119▪] demonstrated that a high dose of rosuvastatin administered early can lead to significantly lower PLAs in patients with ACS. The SPARCL (Stroke Prevention by Aggressive Reduction in Cholesterol Levels) trial showed that atorvastatin reduces the risk of ischemic stroke and other cardiovascular events, however, treated patients displayed a higher risk of suffering intracranial hemorrhage. This side-effect, nonetheless, was not confirmed by others [120,121]. The acute benefits of statins in the setting of ACS might lead to an inclusion of high-dose statin therapy as a part of the standard of care for the initial management of ACS .
Several studies suggest that cholesterol efflux acts as a crucial regulator of myelopoiesis and atherogenesis [97▪▪,123,124,125▪,126,127]. Murphy et al. were able to connect two seemingly separate research areas, platelet generation and cholesterol metabolism. The absence of ATP-binding cassette sub-family G member 4 (ABCG4), a cholesterol transporter highly expressed in bone marrow megakaryocyte progenitors, led to increased megakaryocyte production, thrombocytosis, and atherogenesis. ABCG4-deficient megakaryocyte progenitor cells showed defective cholesterol efflux accompanied by increased proliferation. Of note, cholesterol is a key component of cell membranes and in mammalian cells its metabolism is closely regulated by several feedback mechanisms to prevent dyslipidemia. ABCG4 in megakaryocytes prevents thrombocytosis via thrombopoietin (TPO)-TPO receptor (c-MPL) degradation thereby impairing c-MPL signaling (Fig. 2). In the absence of ABCG4 megakaryocyte progenitor cells showed an increased cell surface expression of c-MPL and reduced activity of the cholesterol-sensitive Src family kinase Lyn. The reduced activity of Lyn kinase caused an interruption of the negative-feedback loop suppressing c-MPL expression. Tolimidone, an allosteric Lyn kinase activator, in turn reduced c-MPL levels on ABCG4-deficient megakaryocyte progenitor cells. Interestingly, Murphy et al. showed that HDL infusions could reduce platelet counts in LDL receptor-deficient (Ldlr−/−) mice, suggesting that HDL infusions may offer a new approach to tackle thrombocytosis and atherothrombotic events. In addition lack of ABCB6, likewise highly expressed on megakaryocyte progenitor cells, leads to thrombocytosis, enhanced proinflammatory platelet activity, and accelerated atherosclerosis in Ldlr−/− mice. Furthermore, ABCB6 deficiency increased platelet counts and mean platelet volume. Platelets of Abcb6−/− mice contained more CCL5 which was paralleled by increased plasma CCL5 levels. Additionally, the number of PLAs in Abcb6−/− bone marrow-transplanted mice was increased, which resulted in enhanced leukocyte activation [129▪]. Taken together Murphy et al. showed that not only ABCG4, but also ABCB6 modulates thrombopoiesis and thus atherogenesis.
PLATELET JUNCTIONAL ADHESION MOLECULES IN ATHEROSCLEROSIS
The JAM family is a subclass within the immunoglobulin (Ig) superfamily and mediates leukocyte-endothelial contact and regulates cell polarity [62▪▪,130,131]. JAM-C and JAM-A were initially identified on platelets . In addition to expression on platelets , JAM-A is also found on endothelial cells , leukocytes , and CD34+ progenitor cells. JAM-A mediates cell polarity , leukocyte trafficking, and recruitment. Apart from that, JAM-A has a barrier function and modulates stem cell adhesion and differentiation [133,135–137]. On endothelial cells, JAM-A is a component of the tight junction and upon inflammatory stimuli (e.g. oxLDL) translocates from its baso-lateral position to the apical surface to interact with blood leukocytes [138,139]. The role of JAM-A appears highly cell-type specific. Total body JAM-A deficiency yields no significant effects on the extent of atherosclerosis. Although JAM-A on leukocytes protects from atherosclerosis, endothelial JAM-A promotes plaque formation by guiding leukocytes to sites of plaque development [62▪▪]. Recently, Karshovska et al.[61▪▪] showed increased atherosclerotic plaque formation in apolipoprotein E (ApoE)-deficient mice with platelet-specific inactivation JAM-A (trJama−/−Apoe−/−) compared with control (trJama+/+Apoe−/−) mice. Platelet JAM-A deficiency accelerated early-stage atherosclerosis corroborating a previously reported role of JAM-A in controlling activation of platelets [140,141]. Plasma levels of platelet-derived chemokines CCL5 and CXCL4 were elevated in trJama−/−Apoe−/− mice accompanied by increased capacity to bind neutrophils and monocytes [61▪▪]. In addition, lack of platelet JAM-A caused a prothrombotic phenotype, increased aggregation, and c-Src activation, which could be abolished by the inhibition of integrin αIIbβ3 signaling in vitro. Upon platelet activation JAM-A is phosphorylated [142,143], and in resting platelets JAM-A acts as an endogenous inhibitor of integrin αIIbβ3 by attenuating c-Src-dependent ‘outside-in’ signaling of integrin αIIbβ3 via recruitment of the c-Src-inhibiting kinase (CSK) [140,141,144]. Upon binding of αIIbβ3 by its ligand fibrinogen, JAM-A is dephosphorylated by protein tyrosine phosphatase nonreceptor type 1 (PTPN1) causing a CSK and JAM-A dissociation from the c-Src/αIIbβ3-complex (Fig. 2). Karshovska et al.[61▪▪] were also able to inhibit PTPN1, which is responsible for the dephosphorylation of JAM-A and enhanced platelet activation. Similar approaches might find their way into translational antithrombotic research in the future.
PLATELET CD40-CD40L COSTIMULATORY PATHWAY
The CD40-CD40L dyad plays an important role in atherosclerosis [20,45,46] and neointima formation [145,146▪]. CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily  that is activated by CD40 ligand (CD40L, also known as CD154), a 39 kDa transmembrane glycoprotein and member of the TNF super family, which is present on human platelets at a rate of approximately 1600 copies/platelet . CD40L also interacts with leukocyte integrin alpha M (Mac-1) [149,150] and can be shed as a truncated and soluble form (sCD40L, 18 kDa) . CD40L is highly expressed on activated platelets which are the main source of sCD40L . sCD40L is biologically active and binds CD40 as a trimeric ligand followed by activation of nuclear factor (NF)-κB and subsequent induction of inflammation [152–154]. CD40L shedding is mediated by matrix metalloproteinase 2 and 9, ADAM10 as well as integrin αIIbβ3 [155–158]. Elevated sCD40L levels are found in patients with CVD and may predict the presence and extent of ACS . Inhibition of CD40  or CD40L  not only reduces atherosclerotic plaque size but also yields a less inflammatory and more fibrotic plaque phenotype . Furthermore, CD40L deficiency ameliorates adipose tissue inflammation in diet-induced obesity by increasing the number of regulatory T cells (Tregs), a T cell subset known to control inflammation and also to protect from atherosclerosis [53,162], in obese adipose tissue [163,164]. In contrast, CD40 deficiency aggravates adipose tissue inflammation and causes metabolic dysregulation [165▪]. Gavins et al. showed that mice deficient in either CD40 or CD40L are protected against microvascular thrombosis after treatment with lipopolysaccharide (LPS). In brain arterioles, a CD40 deficiency will completely, whereas CD40L deficiency will only partially, prevent accelerated thrombosis induced by LPS administration. In venules, on the other hand, LPS-induced thrombus formation was completely prevented in Cd40l−/− mice, whereas CD40 deficiency provided no significant protection . CD40 and CD40L are not only expressed on leukocytes but also on platelets, endothelial cells, and other cells present in the atherosclerotic plaque. Activated platelets bind via CD40L endothelial and leukocytic CD40 [48,49] (Fig. 1). The platelet–endothelium interaction can induce the release of prothrombotic mediators (e.g. tissue factor, TF), chemokines (e.g. CCL2 and CCL5), matrix metalloproteases (e.g. MMP-1, MMP-2, MMP-3, and MMP-9), and upregulation of adhesion molecules (e.g. VCAM-1 and ICAM-1) by endothelial cells (Fig. 1). Apart from the subsequent leukocyte recruitment and enhanced atherogenesis, interactions between platelet CD40L or sCD40L and endothelial CD40 can lead to reduced synthesis of nitric oxide by endothelial cells . Huo et al. first demonstrated that Apoe−/− mice receiving injection of activated platelets showed exacerbated atherosclerotic plaque formation in comparison with untreated Apoe−/− mice. We were able to demonstrate that injection of activated CD40L-deficient platelets into Apoe−/− mice prevents PLA formation and reduces atherogenesis when compared with transfusions of activated wild-type platelets . Injection of activated CD40L-deficient platelets caused in contrast to the injection of wild-type platelets no decrease of Tregs. Moreover, we showed recently that platelet CD40L modulates thrombus growth through phosphatidylinositol 3-kinase β rather than via CD40 and IκB kinase α [167▪]. Others demonstrated that platelet bound or recombinant sCD40L stimulates human-cultured endothelial cells to release ultralarge vWF multimers, which are rapidly proteolyzed into smaller units .
ANTIPLATELET DRUGS AND PROSPECT
Current antiplatelet therapies are mostly based on the direct modulation of platelet function via ADP receptor antagonists or inhibitors of cyclooxygenase (COX)-1, integrin αIIbβ3, and enzymes involved in the coagulation cascade [20,169–171]. Interestingly, a recent study failed to demonstrate a benefit of low-dose aspirin therapy for Japanese patients aged more than 60 years [172,173]. This and other studies illustrate the persisting need for alternative approaches to modulate platelet physiology and function. As already reported, inhibition of PTPN1 [61▪▪], which dephosphorylates JAM-A and enhances platelet activation, might provide a new alternative approach to control platelet activity. We showed that specific inhibition with small-molecule inhibitors  of CD40–TRAF6 (TNF receptor associated factor 6, an adaptor molecule involved in CD40 signaling) interaction improved insulin sensitivity, reduced adipose tissue inflammation, and decreased hepatosteatosis [175▪,176]. This effect on leukocytic target cells could be at least in part evoked by platelet-expressed CD40L. Accordingly targeted treatments of selective signaling pathways may constitute a promising strategy to reduce platelet-driven contribution to atherosclerosis without encountering thrombotic complications, which were observed in early clinical studies of systemic CD40/CD40L inhibition. Although many clinical trials of treatments that increase HDL, for example via CETP inhibitors torcetrapib and dalcetraoib or ER-niacin, failed [177–179], Murphy et al. showed that recombinant HDL (rHDL) infusion reduced platelet counts in Ldlr−/− mice. HDL infusion may offer a new approach to treat thrombocytosis and reduce atherothrombotic events. The HDL infusion therapies in development are currently at a crossroad [180–183]. The potent CETP inhibitors evacetrapib and anacetrapib are currently in phase 3 trials for chronic cardiovascular risk reduction, reports are expected in 2015 and 2017. Apart from HDL injection, Murphy et al. showed that oral application of tolimidone, an allosteric Lyn kinase activator, which decreases thrombopoietin receptor (c-MPL) expression on megakaryocyte progenitor cells, reduced thrombocytosis and atherogenesis. Therapeutic inhibition of ABCG4  or ABCB6 [129▪] might provide a new alternative to treat patients with thrombocytopenia.
Recent findings show the potential role of platelets and immune cells in noninvasive cardiovascular imaging [184,185,186▪,187,188]. MRI approaches successfully identified atherosclerotic plaques in the carotid artery. In recent years, substantial advances were made in the development of targeted magnetic agents for cardiovascular imaging [189–192,193▪]. von Elverfeldt et al. were able to image the extent of myocardial ischemia/reperfusion injury after coronary vessel occlusion using a contrast agent, consisting of microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody directed against activated platelets, via MRI. Jacobin-Valat et al. used the Versatile UltraSmall SuperParamagnetic Iron Oxide (VUSPIO) platform, based on 7.5-nm-sized magnetic cores (maghemite γ-Fe2O3), that enhances contrast in MRI. VUSPIO conjugated to the humanized αIIbβ3 antibody rIgG4 TEG4 forms the TEG4-VUSPIO complex, which could selectively accumulate around activated platelets within atherosclerotic lesions. Despite potential challenges and limitations on the way (e.g. human antihuman antibody responses ) these efforts open a perspective to identify culprit locations of atherothrombotic activity.
In this article, we have described the versatile functions of platelets in atherosclerosis. The increasing recognition of platelets as immune modulators offers new possibilities beyond classical anticoagulation to target specific platelet–leukocyte and platelet–endothelium interactions to limit atherosclerosis. Recent findings furthermore clarify the direct impact of lipid metabolism and cholesterol metabolism on platelet physiology. The novel utilization of platelets in cardiovascular imaging shows the great potential of platelets beyond their former unspectacular role as ‘discoid fragments’ in biomedicine.
The authors thank Holger Winkels and Christina Bürger for critical editing of the manuscript.
Financial support and sponsorship
Research of the authors is supported by the Deutsche Forschungsgemeinschaft (SFB1123-A5 to NG, SFB1123-A2 to PvH, and SFB1123-A1 & B4 to CW). MA is supported by a doctoral stipend from the Deutsches Zentrum für Herzkreislaufforschung (DZHK).
Conflicts of interest
There are no conflicts of interest.
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