Platelets in Inflammation and Atherogenesis

Platelets contribute to processes beyond thrombus formation and may play a so far underestimated role as an immune cell in various circumstances. This review outlines immune functions of platelets in host defense, but also how they may contribute to mechanisms of infectious diseases. A particular emphasis is placed on the interaction of platelets with other immune cells. Furthermore, this article outlines the features of atherosclerosis as an inflammatory vascular disease highlighting the role of platelet crosstalk with cellular and soluble factors involved in atheroprogression. Understanding, how platelets influence these processes of vascular remodeling will shed light on their role for tissue homeostasis beyond intravascular thrombosis. Finally, translational implications of platelet-mediated inflammation in atherosclerosis are discussed.


INTRODUCTION
The vasculature is one of the large networks of the human body and, thus, it needs to be well protected by immune mechanisms. When tissue is injured, the wound is paralleled by a severance of the vascular network, as well. Disruption of the endothelial monolayer lining all vessels from the inside triggers a process referred to as thrombus formation, a well regulated and complex cascade of events (1). During thrombus formation, other systems located within the vasculature can be activated, the most prominent of which is the immune system and inflammation being a part of its innate response. The inflammatory response to tissue injury triggers various events, which allow for defense against possible intruders and initiate the healing process (2). Simultaneously and in close proximity, platelets are recruited to the wound to restore endothelial integrity; they are activated and initiate thrombus formation. Given the close spatiotemporal relationship of these molecular processes, it is not surprising that growing evidence suggests that platelets are not only effectors of thrombus formation, but actively participate in inflammation and other processes related to tissue remodeling (3).

PLATELETS PRESERVE VASCULAR INTEGRITY
Repair of vascular damage while simultaneously preserving the patency of narrow capillaries is a complex task and requires a finely tuned machinery of pro-as well as anti-thrombotic mechanisms. Platelets are the key cells of primary hemostasis and thrombus formation. They mediate thrombus formation through cellular and soluble factors [recently also reviewed in Ref. (1,(4)(5)(6)(7)]. GPIbα is a platelet transmembrane receptor associated with GPIX and GPV (8). GPIbα binding to von-Willebrand-factor (vWF) initiates primary hemostasis (8). In a shear-dependent fashion, GPIbα binding to vWF immobilized on collagen enables initial platelet rolling, which precedes all further steps of thrombus formation (9). Except at sites of high shear rates (10), stable platelet adhesion requires additional contribution of GPVI and integrins (11). GPVI is one of the platelet collagen receptors. It provides strong mechanic adhesion but also serves as the primary inducer of platelet activation mediated by its FcRγ-chain (12,13). Amongst other signaling events, activation of GPVI causes an elevation of intracellular Ca 2+ and subsequent platelet shape change (14). Platelet activation is paralleled by the secretion of soluble factors from platelet granules, the most important of which are ADP and TxA2 (15)(16)(17), as they activate platelets in an autocrine fashion (18). C-type lectin-like type II (CLEC-2) supports GPVI as it sustains a similar signaling pathway as the one induced by the GPVI/FcRγ complex (19,20). In fact, CLEC-2 was found to be of particular importance for stable aggregate formation under flow conditions (21). The thrombotic activity of platelets is regulated by controlling the surface density of these major receptors by ectodomain shedding (22). A central process in platelet-mediated thrombus formation is integrin activation, as integrins connect the ECM to the platelets' cytoskeleton and enable platelet aggregate formation (11). The integrin α IIb β 3 has the ability to"crosslink"platelets via fibrinogenbridging (23), thus stabilizing the forming thrombus. Due to its central importance, its inside-out activation is referred to as the "final common pathway of platelet activation" (24). The activation of α IIb β 3 is mediated by the classical platelet agonists ADP or TxA2, and interfering with these pathways was successfully transferred into patient treatment (25). Similar to other integrins, α IIb β 3 promotes "outside-in" signaling as well as platelet spreading and clot retraction (26). Finally, platelets also interact with the coagulation system in various ways stabilizing the developing thrombus by fibrin formation, which provides for provisional wound closure (27-29).

"NON-CLASSICAL" PLATELET FUNCTIONS
Although traditionally not conceived as immune cells, platelets hold important functions in the immune response, particularly in innate immunity (30-32). In both host defense and preservation of vascular functions, platelets are helpful in some and harmful in www.frontiersin.org other conditions (33). In the following, we will aim to exemplify how platelets mediate effects beyond thrombus formation.

PLATELETS IN HOST DEFENSE
Platelets contribute to pathogen recognition by interacting with immune cells, but also by interacting with the bacteria themselves (34-41). Recently, it was demonstrated that platelet-rich plasma (PRP) inhibited the growth of bacteria (42). The various receptor interactions involved in this platelet-bacteria crosstalk were already reviewed elsewhere (43, 44). For instance, platelets are able to recognize CpG islands upon thrombin activation and subsequent TLR9 expression (40). Furthermore, platelets react to fungal infections in vitro and in vivo (45). Finally, platelet "nuclear functions" are increasingly uncovered and recognized (2). Via transcription of mRNA and post-transcriptional modification (46-48), platelets seem to contribute to the inflammasome by producing IL-1β (49), they are involved in modulating NFκB (50) and may influence endothelial polarization by miRNA secretion (51).

PLATELETS CONTRIBUTING TO MECHANISMS OF INFECTIONS
On the other hand, there are a number of reports describing platelets as an important element in the progression of infections (52). The platelet receptor CLEC-2 has been shown to facilitate the entry of HI-viruses (53), and platelets contribute to disease progression via CD40L (54, 55). Furthermore, platelets are involved in the progression of HBV-infection and other viral diseases (56, 57) by the recruitment of cytotoxic T-cells (CTL) to the liver (58) or other organs in a serotonin-dependent manner (59). Verschoor et al. could recently show that platelets are a relevant factor in the process of immune evasion by intracellular bacteria such as Listeria (38). Furthermore, platelets play an important role in infections by Leishmania (60) and in the pathogenesis of Hantavirus infection (61). A fact, which complicates the picture even more, is that platelets can also modulate the function of further cells involved in the response to infections -the leukocyte.

PLATELET CROSSTALK WITH IMMUNE CELLS
One of the main immune mechanisms of platelets is their capability to recruit leukocytes to sites of infection and inflammation (32, 62). P-selectin-PSGL-1 binding (63,64), ICAM1 (51), and GPIbα (65) play an important role in how platelets bring other immune cells to the scene (66), particularly under high shear conditions (67). Platelets have the ability to form aggregates with neutrophils (68). In periodontitis, aggregate formation of platelets and neutrophils (NPA) enhances neutrophil phagocytosis in a TLR-2-dependent manner (69). In acute lung injury, NPA formation mediates neutrophil extravasation (70) and platelet-derived platelet factor 4 (PF4) fostered neutrophil survival in a model of arterial occlusion (71). Furthermore, platelet-leukocyte aggregates can be used as a diagnostic tool, for example as a parameter to assess sepsis severity (72). Another recently discovered way, how platelets modulate neutrophil function is their involvement in neutrophil extracellular trap (NET) formation to ensnare intruders (73). Platelet TLR-4 (74) as well as platelet β-defensins have been implicated in NET formation (75,76). Specifically, plateletinduced NET formation may play a role in viral infections (77) or transfusion-related lung injury (78). Rossaint et al. have recently proposed that simultaneous activation of neutrophils via Mac-1 outside-in signaling and Gαi engagement by platelet-derived RANTES-PF4 heterodimers is required for NET formation (79). Interestingly, platelets seem to form especially stable aggregates with monocytes (80), and activated platelets induce an inflammatory monocyte phenotype (81). As this process seems to be partially independent of P-selectin interaction with PSGL-1, paracrine mechanisms to strengthen platelet-monocyte aggregate formation have been proposed as an alternative mechanism (81). Plateletmonocyte interaction seems to be of functional relevance, as their formation increases the number of circulating monocytes with a higher affinity for adhesion to the endothelium (82). Furthermore, activated platelets are taken up by monocytes which induces enhancement of cytokine release from macrophages (83). Other authors, however, report on anti-inflammatory effects of plateletmonocyte interaction (84)(85)(86) via CXCR5 engagement of CXCL13 on monocytes (84) or, following experimental sepsis, by inhibition of macrophage tumor necrosis factor α (TNF-α) and IL-6 secretion (85,86). Thus, the effect of platelets on monocytes appears to be context-dependent.
Via P-selectin PSGL-1 interaction, platelets can form aggregates with lymphocytes (PLA), as well. Platelet interaction with T-cells, B-cells, and NK-cells induces their homing, activation, and recruitment as recently reviewed (87). Platelets may even serve as a bridge directing T-cells to the endothelium (88). Furthermore, platelets modulate lymphocyte function via direct cell-cell interaction as well as soluble mediators (87). In rheumatoid arthritis patients, platelet binding to lymphocytes promoted activationinduced proliferation as well as IL-17 and interferon-γ production by GPVI positive CD4+ T-cells (89). Serotonin from platelet vesicles may also stimulate T-lymphocytes (90). Through release of PF4 and CCL5, platelets can enhance cytokine production in CD4+ T-cells (34). Furthermore, in HCV infection, platelet CCL5 causes upregulation of T-lymphocyte helper cells type 1 (Th1) (91). Finally, platelets may also enhance T-cell-mediated germinal center formation and release of specific IgGs from B-cells via CD40L signaling (35, 41). In fact, platelets may substitute CD40L when few CD40L-positive T-cells are present to stimulate B-cell maturation (92). However, platelets may also induce anti-inflammatory effects. PF4 released from platelets leads to an increase in regulatory T-lymphocytes (Tregs) (93) and limits Th17 differentiation (94). Interestingly, T-lymphocytes can activate platelets, which amplify the release of CCL5 (95). Addressing antigen-presenting cells, there are a number of ways in which platelets interact with dendritic cells (DCs). For instance, platelets can recruit DC via Mac-1 interaction with JAM-C and can activate them inducing platelet phagocytosis and subsequently apoptosis of DCs (96). High shear rates may be a trigger for platelets to recruit DCs and promote their maturation (97). In fact, direct contact of platelets and DCs seems to induce other effects than crosstalk via soluble factors suggesting that platelets have the ability to differentially regulate a DC response (98). This conclusion is supported by recent findings demonstrating that platelets can enhance DC-mediated Th-2 cell response in allergy by secreted RANKL (99). Platelets can, however, also impair DC differentiation or reduce DC production of proinflammatory cytokines IL-12p70 and TNF-α (100).

PLATELETS IN ATHEROSCLEROSIS
Atherosclerosis is a chronic inflammatory disease featuring various complex processes contributing to its pathophysiology and the development of the atherosclerotic plaque over decades. Figure 1 summarizes the steps contributing to fatty streak formation, inflammation, progression of the plaque, and finally plaque rupture.

PLATELET CONTRIBUTION TO ATHEROSCLEROSIS
In atherosclerosis, platelets are known to contribute to early steps of this chronic vascular pathology (Figure 2) such as endothelial dysfunction (101,102), but also to final events such as rupture of the vulnerable plaque [(103), see also Figure 4]. For instance, platelets participate in atherogenesis by chemokine release (79,104,105), surface association of oxLDL (106), direct cell-cell interaction (107,108), release of microparticles (109), and provision of inflammatory mediators [(110), see also Figure 3]. Platelets within the atherosclerotic plaque may remain activated for a long time providing for proinflammatory IL-1β production [(111), see also Figure 3]. One of the most considered functions of platelets in atherosclerosis is the recruitment of leukocytes via direct receptor-ligand interactions or augmented by released factors such as chemokines [(112, 113), see also previous sections and Figure 3]. The role of a particular leukocyte subtype -DCs, FIGURE 1 | Pathophysiology of atherosclerosis. During atherogenesis, a plaque forms on the luminal side of the arterial wall through a complex process involving lipid accumulation, cellular activation inducing the transformation and differentiation of monocytes into foam cells, and various immune reactions mediated by T-and B-cells, neutrophils, granulocytes (neutrophils), and dendritic cells (DCs). Hence, the progression of atherosclerosis is driven by inflammation, although some of these inflammatory cells/factors may also mediate atheroprotection under certain conditions. At later stages, the atherosclerotic plaque develops a necrotic core with areas of neovascularization, a thin fibrous cap, and scattered calcifications. Finally, plaque rupture exposes the thrombogenic atherosclerotic core inducing platelet activation and, subsequently, initiation of the coagulation cascade. Ab, antibody; Hb, hemoglobin; HSP, heat shock protein; NET, neutrophil extracellular trap; PF4, platelet factor 4; SMC, smooth muscle cell; Treg, regulatory T-cell; VALT, vascular-associated lymphatic tissue.
the classical antigen presenting cells of our body -has been emphasized recently in the context of atherosclerosis and, interestingly, platelets interact with DCs (Figure 3). In fact, GPIb-Mac-1 interaction may be an interesting signaling mechanism in the context of platelet-DC crosstalk modulating atheroprogression (114,115). This is of particular importance, as DCs have been proposed to play a significant role in the different steps of atherosclerosis (116).

PLATELETS IN THE COMPLEMENT SYSTEM AND ATHEROSCLEROSIS
A further part of our innate immune response, the complement system receives increasing attention in the context of atherosclerosis. This cascade of soluble plasma proteins constitutes a phylogenetically very old part of the inherited immune system (117). Complement activation is important for inflammatory conditions associated with vascular injury (118,119). Most interestingly, platelets were reported to express a number of complement receptors relevant for platelet function and their crosstalk with the local microenvironment (120)(121)(122). Several complement components can be bound to the platelet surface (123,124). We found that expression of complement anaphylatoxin receptors on platelets showed a strong and positive correlation with platelet activation markers such as P-selectin in patients with atherosclerosis (125). Further mechanistic studies will have to address the relevance of this association. In a recent review, the literature on platelets and potential intersection points with the complement system in diverse settings was summarized (126). Additional profound studies are needed to differentiate our understanding of the intersection points of platelet activation with the immunological elements of atherogenesis such as endothelial inflammation, leukocyte recruitment, antigen presentation, chemokine and cytokine production, or complement activation.

PLAQUE RUPTURE
At later stages, the atherosclerotic core becomes hypoxic inducing the outgrowth of vasa vasorum from the adventitia toward the intima [ (127,128), see also Figure 4]. As a consequence, fragile and leaky vessels form, which facilitate further invasion of immune cells and release of soluble factors into the surrounding atherosclerotic tissue (129,130). Moreover, red blood cells get stuck in the plaque liberating hemoglobin and iron (131). These mechanisms ultimately result in plaque destabilization (132). Foam cells produce tissue factor (133), and as soon as the thrombogenic lipid core is exposed to the lumen, fibrin generation is initiated (134)(135)(136). In parallel, platelets as well as the coagulation cascade become activated (137,138). Plaque rupture in the region of a thin fibrous cap is the final event, how atherosclerosis causes acute vascular complications such as myocardial infarction or stroke (139)(140)(141)(142)(143).

TRANSLATIONAL RELEVANCE
The fact that inflammation plays a key role in central steps of atherosclerosis (31, 144), is increasingly integrated into clinical Frontiers in Immunology | Inflammation considerations. Accordingly, this hypothesis is addressed by two current trials, the CANTOS trial launched in 2011 (145) and the CIRT trial (146) using immunosuppressants to treat FIGURE 4 | Mechanisms, how platelets affect plaque rupture. Plaque rupture is tailored by platelets in several ways: platelets are known to modulate angiogenesis, which is a decisive factor for plaque stability. Platelets have also been found to promote smooth muscle cell (SMC) progenitor recruitment via CXCR4-SDF-1α signaling. SMCs are responsible for generating extracellular matrix stabilizing the fibrous cap of the plug. Thrombus formation in areas of the ruptured fibrous cap exposing components of the extracellular matrix is initiated by platelets. Future investigations will also have to test, whether platelets enter the plaque tissue and affect tissue remodeling. Ab, antibody; CD40L, CD40 ligand (CD154); DC, dendritic cell; IL-1α, interleukin 1α; JAM-C, junctional adhesion molecule type C; Mac-1, macrophage-1 antigen; MIF, macrophage migratory inhibitory factor; miRNA, microRNA; NFκB, nuclear factor kappa-light-chain-enhancer of activated B-cells; PF4, platelet factor 4; PSGL-1, P-selectin glycoprotein ligand-1; RANKL, receptor activator of nuclear factor kappa-B ligand; RANTES, chemokine ligand 5 (CCL5); TLRs, Toll-like receptors; Treg, regulatory T-cell.
atherosclerosis. Earlier in this article, we have aimed at depicting the importance of platelets for inflammation in atherosclerosis. Inhibition of platelet-mediated inflammation may already be everyday clinical practice considering the use of aspirin for the treatment of cardiovascular disease (147). As an irreversible inhibitor of the enzyme cyclooxygenase, aspirin is a mild inhibitor of platelet function (148). Despite its widespread use, the definite role of aspirin in the prevention of atherosclerosis and atherosclerosis-related diseases is still under discussion (147). There is evidence from preclinical studies that aspirin is able to inhibit the initiation (149) and even the progression of experimental atherosclerosis (150) via its effect on prostaglandin synthesis but also by other mechanisms such as the modulation of endothelial NO synthesis (151), NFκB signaling (152), CRP, or soluble CD40 ligand (sCD40L) (153). Clinical studies on the use of aspirin for primary prevention of atherosclerosis, however, have also yielded negative results [recently reviewed by Gaziano and Greenland (147)]. A large study in a Japanese population over 60 years of age could demonstrate no benefit of low-dose aspirin therapy (154,155). In patients at low risk for cardiovascular events, the use of aspirin needs to be weighed very carefully against an elevated risk of bleeding events or even hemorrhagic stroke (147).

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For the ADP-receptor antagonist clopidogrel, reports on an effect in the context of atherosclerosis exist, too. In animal models, clopidogrel has the ability to slow down the inflammatory progression of atherosclerosis (156,157). Clopidogrel reduces platelet activation as measured by P-selectin expression and other inflammatory markers (158), while others stress that important inflammatory markers such as hsCRP are not affected (159, 160). On the other hand, platelet-leukocyte aggregate formation is inhibited more effectively by clopidogrel compared to aspirin (161,162). In contrast, another group reported that under therapy with clopidogrel, the expression of some inflammatory chemokines may even be increased in peripheral blood mononuclear cells in patients with coronary artery disease (163). On the platelet surface, a number of inflammatory receptors may represent potential targets for new therapeutic approaches such as CXCL4, CCL5, CD40 ligand, PSGL-1 (164). Further targets may be platelet-activating factor (PAF) (165) or Annexin A5 (166). Finally, we have already described the evidence on complement receptor involvement in atherosclerosis (126,167). This class of receptors are involved in a large number of inflammatory processes (117) and are also expressed on platelets (120)(121)(122). There are a number of substances targeting different parts of the complement system which are evaluated in different stages of clinical trials in conditions such as age-related macular degeneration or hereditary angioedema (168,169). Some have even established themselves as first-line treatment such as eculizumab for paroxysmal nocturnal hemoglobinuria (170,171). It is tempting to speculate, that these substances might be worth an evaluation in the context of atherosclerosis, as well.
Moreover, biomarkers of atherosclerosis are of great importance from a clinical point of view. Due to the high prevalence of cardiovascular disease, it is vital to identify which patient is at particular risk for adverse cardiovascular events and would benefit from preventive diagnostic or therapeutic interventions. A number of platelet surface receptors may be promising candidates to consider in this context. A prominent example is soluble CD40 ligand (sCD40L) released from platelets (172). In a number of settings such as on hospital admission of patients with acute coronary syndrome or in patients undergoing primary angioplasty, sCD40L levels appear to have predictive capacity (173)(174)(175). Some authors even discuss sCD40L as a therapeutic target (176). Apart from sCD40L, soluble P-selectin released from platelets is referred to as a further potential platelet-derived biomarker (177,178).
Considering the achievements of platelet research over the last two decades with a bounty of platelet-targeted drugs, which found their way into everyday clinical practice, platelets and plateletassociated molecular mechanisms offer potential translational applications.

CONCLUSION
In conclusion, platelets -conceived as immune cells and mediators of vascular/tissue remodeling -have a strong impact on atherosclerosis through inflammatory mechanisms discussed here on the basis of selected cellular or soluble platelet-derived mediators. The net effect of platelet-mediated inflammation may be an atheropromoting one, although these anucleate cells may mediate distinct atheroprotective mechanisms, as well. Future investigations will have to identify these specific platelet aspects to enable us to develop better diagnostic markers and therapeutic approaches with fewer undesired side effects.