Platelets as an inter‐player between hyperlipidaemia and atherosclerosis

Platelet hyperreactivity and hyperlipidaemia contribute significantly to atherosclerosis. Thus, it is desirable to review the platelet–hyperlipidaemia interplay and its impact on atherogenesis. Native low‐density lipoprotein (nLDL) and oxidized LDL (oxLDL) are the key proatherosclerotic components of hyperlipidaemia. nLDL binds to the platelet‐specific LDL receptor (LDLR) ApoE‐R2′, whereas oxLDL binds to the platelet‐expressed scavenger receptor CD36, lectin‐type oxidized LDLR 1 and scavenger receptor class A 1. Ligation of nLDL/oxLDL induces mild platelet activation and may prime platelets for other platelet agonists. Platelets, in turn, can modulate lipoprotein metabolisms. Platelets contribute to LDL oxidation by enhancing the production of reactive oxygen species and LDLR degradation via proprotein convertase subtilisin/kexin type 9 release. Platelet‐released platelet factor 4 and transforming growth factor β modulate LDL uptake and foam cell formation. Thus, platelet dysfunction and hyperlipidaemia work in concert to aggravate atherogenesis. Hypolipidemic drugs modulate platelet function, whereas antiplatelet drugs influence lipid metabolism. The research prospects of the platelet–hyperlipidaemia interplay in atherosclerosis are also discussed.


Introduction
Platelet hyperactivity/thrombosis and hyperlipidaemia are the two leading risk factors for atherosclerosis [1][2][3][4][5][6].Hyperlipidaemia is the classical cause of atherosclerosis; high levels of lowdensity lipoprotein (LDL) and total cholesterol lead to excess lipid deposition and exaggerate inflammatory responses in the arterial vessel walls [1,2].Platelets are the primary contributor to the thrombotic mechanisms and are involved in all stages of atherosclerosis [4,5].Platelet hyperreactivities have long been recognized in atherothrombotic diseases [4,5].These pathophysiological features of atherosclerosis indicate that lipid-lowering and antiplatelet therapies are the two cornerstones of atherosclerotic disease management.A good understanding of the interactions between platelet hyperactivity and hyperlipidaemia during atherogenesis is important for therapeutic improvements and novel drug development for atherothrombotic diseases.This review provides an overview of how hyperlipidaemia influences platelet physiology and how platelets regulate lipid metabolism.Future research prospects for this subject are also discussed.

Platelet physiology
Platelets are the smallest blood cells derived from megakaryocytes in the bone marrow.Quiescent platelets circulate as discoid anucleated cells in large numbers (0.5-4 µM in diameter, 1.5-4 × 10 11 /L) with the total cell mass only next to erythrocytes.Platelets are the principal cells involved in haemostasis and thrombosis.They continuously patrol and readily detect the inflamed and/or injured sites in the vasculature.Platelets instantly respond through cellular activation in the form of adhesion, secretion and aggregation and therefore exert their primary function to seal/cover the damaged vessel, control bleeding (haemostasis) and, if uncontrolled, to build up thrombosis [7].
Platelets are very sensitive cells that swiftly respond to their surrounding environment and interact with adjacent and distant cells via direct cell-cell contact and soluble mediators.In addition to haemostasis and thrombosis, platelets are closely involved in multiple physiological and pathophysiological processes, such as coagulation, inflammation, immunity, tissue regeneration, angiogenesis and lipid metabolism [8][9][10].

Platelet dysfunction in hyperlipidaemia
Dyslipidaemia refers to abnormal blood levels of triglycerides, cholesterol and phospholipids, which are carried into the bloodstream by very LDLs (VLDLs), LDLs, high-density lipoproteins (HDLs) and chylomicrons.Hyperlipidaemia with high LDL levels is the most important risk factor for atherogenesis, and the interplay between elevated LDL levels and disturbed platelet function is thus the subject of the present review.
Hyperlipidaemia is associated with platelet hyperactivity [11].Thus, hyperlipidaemia augments platelet activation in vivo, as evidenced by enhanced thromboxane A 2 (TXA 2 ) biosynthesis in platelets, with a positive correlation between urinary excretion of the thromboxane metabolite TXB 2 and plasma lipid levels [12].Elevated plasma levels of platelet-released serotonin mirror the elevation of lipid levels [11]; soluble P-selectin (sP-Sel), a marker largely reflecting platelet activation in vivo, is also elevated in patients [13].Moreover, hyperlipidaemia enhances platelet adhesion to vessel walls and thrombus formation in vivo [14].Platelets from patients with hyperlipidaemia also show enhanced responsiveness to in vitro stimulation.Hence, platelet adhesion increases under flow conditions [15], and platelet aggregation is enhanced in response to multiple platelet agonists [16].Further supporting the notion of hyperlipidaemia-induced platelet dysfunction, reduced lipid levels by lipidlowering therapies have been shown to attenuate platelet dysfunction [11,17].
LDL particles circulate in the blood and are present in vascular tissues as native LDL (nLDL) and oxidized LDL (oxLDL).Both nLDL and oxLDL regulate various aspects of platelet function.In general, nLDL and oxLDL enhance platelet reactivity in a concentration-and time-dependent manners [18,19]; nLDL mildly, whereas oxLDL more intensely enhances platelet adhesion, secretion, aggregation and procoagulant activities [18,20].Using a deep-learning morphometry approach, it was shown that both nLDL and oxLDL alter but yet differentially regulate platelet spreading and migration on extracellular matrix protein-coated surfaces [21].OxLDL has a prominent pathological impact on atherosclerotic lesion development and evokes strong inflammatory responses in the vessel wall through the activation, differentiation and proliferation of vessel wall cells, such as endothelial and smooth muscle cells, as well as infiltrated inflammatory cells, such as macrophages, T cells and neutrophils [1,2,22].The same applies to platelets.nLDL has mild effects on platelet activation, whereas oxLDL augments platelet activation more markedly [20].The effects of nLDL and oxLDL on platelet activation have been reported with different potencies, sometimes even with opposite effects, in various studies [23,24].This effect variance can be attributed to multiple factors, such as the preparation of nLDL/oxLDL, platelet sample type and the experimental setup.For example, washed platelets are the most sensitive to elucidate the effects of nLDL/oxLDL, platelet-rich plasma is modestly sensitive and whole blood is less prone to nLDL/oxLDL augmentation, highlighting the buffering effect of plasma and the physiological milieu on oxLDL.It is also known that hypochloride-oxLDL is a more potent stimulus for platelet activation than copper-oxLDL [25,26].These aspects of nLDL/oxLDL-induced platelet activation were thoroughly reviewed and interpreted in a recent review [27].Moreover, the pathophysiological importance of oxLDL likely lies in its capacity to prime/sensitize platelets to the stimulation of platelet primary agonists, such as thrombin, collagen and adenosine diphosphate, although oxLDL per se has been reported to induce platelet activation [26,28].
Integrin GPIIb/IIIa (α IIb β 3 , CD41/CD61) is the best known fibrinogen/fibrin receptor on platelets.It has also been suggested to be a receptor for nLDL on platelets [18], which was supported by the colocalization of fibrinogen and immunogold-labelled LDL on the platelet membrane using immunoelectron microscopy [35].However, other studies have challenged the notion that GPIIb/IIIa is an LDLR.For instance, platelet LDL-binding capacity is only approximately 1/10th of the GPIIb/IIIa molecule intensity per platelet, and nLDL binding is not altered in GPIIb/IIIa-deficient platelets from patients with Glanzmann's thrombasthenia [18,31,36].The notion of GPIIb/IIIa as an nLDL receptor also needs to be considered, as the scavenger receptor of LDL, CD36, is constitutively associated with GPIIb/IIIa [37].The latter likely explains why GPIIb/IIIa has been suggested to be an nLDL receptor.

Platelet stimulation by oxLDL
In contrast to the selective binding of nLDL to the platelet-specific LDLR ApoE-R2 , oxLDL selectively binds to scavenger receptors.Scavenger receptors are a large and diverse family of receptors expressed on the cell surface.The discovery of this superfamily was initiated by the identification of LDLR (currently also termed ApoB/E-R) by Drs. Brown and Goldstein in the 1970s [29].Scavenger receptors are characterized by ligand binding and uptake that do not trigger the downregulation of receptor expression through LDLR recycling procedures [29].Platelet-expressing scavenger receptors include CD36, lectin-type oxLDL receptor 1 (LOX-1) and scavenger receptor class A 1 (SR-A1).CD36 (GPIV) is a class B scavenger receptor constitutively expressed on platelets [38].As the primary receptor of oxLDL on platelets [39], oxLDL-CD36 ligation stimulates multiple signalling pathways (Fig. 1) [20,27,40].Notably, CD36, upon ligation by oxLDL, induces the activation of the Src kinases Fyn and Lyn.The latter activates phospholipase Cγ 2 (PLCγ 2 ), triggers calcium mobilization and protein kinase C (PKC) and enhances MAPK activation, leading to platelet activation, observed as platelet shape change, adhesion, aggregation and secretion.oxLDL-CD36 ligation also stimulates p38 MAPK and PLA 2 , which liberate AA from membrane phospholipids, initiate TXA 2 synthesis via COX-1 and result in platelet activation [20,37].Moreover, CD36 ligation-stimulated Fyn/Lyn signalling activates guanine nucleotide exchange factors Vav1 and Vav3 (Vav1/3) as well as MAPK kinase 4 (Mkk4) and c-Jun N-terminal kinase (JNK), leading to platelet aggregation and secretion [27,41,42].oxLDL-CD36 ligation also evokes reactive oxygen species (ROS) production.Scavenger ligation can activate membrane-embedded NOX (nicotinamide adenine dinucleotide phosphate [NADPH] oxidases) either directly or via Fyn/Lynspleen tyrosine kinase (Syk)-phosphatidylinositol 3-kinase (PI3K)-PKC signalling [38,40,43].Activated NOX in platelets produces and releases ROS either intracellularly or extracellularly.Intraplatelet ROS modulate intracellular signalling and act as secondary messengers in platelet activation.Extracellular ROS exert their effects on platelets in an autocrine manner or on adjacent cells of other cell types in a paracrine manner.For example, CD36 signalling stimulates hydrogen peroxide production, which oxidizes the cysteines of platelet proteins, including Src family kinases, and primes platelets, thereby aggravating thrombosis in dyslipidaemia [19].
Another oxLDL receptor in the platelets, SR-A1, often functions in cooperation with CD36 [37].This is evidenced by the observation that oxLDL binding-induced p38 signalling was arrested not by individuals but by the joint blockade of SR-A1 and/or CD36, and that single receptor deficiency did not show, but double deficiency of SR-A1 and CD36 demonstrated impaired platelet responses to oxLDL [37].SR-A1 ligation by oxLDL induces p38 signalling and also stimulates Mkk4 and JNK signalling, resulting in platelet aggregation and secretion (Fig. 1) [20,37,42].
Platelets can also bind oxLDL via LOX-1, a transmembrane protein and a class E scavenger receptor [44].In quiescent platelets, LOX-1 is embedded in the membranes of the platelet α-granules.Upon activation, it is translocated to the platelet surface and is available for oxLDL ligation [44].The characteristics of the activation-dependent expression of LOX-1 imply that oxLDL-LOX-1 ligation exerts its effects on the amplification of platelet activation rather than priming or initiating platelet activation.oxLDL-LOX-1 ligation stimulates intracellular signalling through the IκB kinase 2 (IKK2)nuclear factor-κB (NFκB)-JNK pathway and via the PI3K and Akt signalling pathways (Fig. 1), subsequently enhancing platelet aggregation and secretion [27,45,46].In addition to platelets, LOX-1 is also expressed in multiple cell types involved in atherothrombotic diseases, including endothelial cells, macrophages and smooth muscle cells.The broad cellular expression of LOX-1 makes its ligation by oxLDL impact on multiple steps of atherosclerotic progression.The latter has been proven by several in vivo studies using murine models with LOX-1 interventions and has prompted therapeutic developments and clinical trials targeting LOX-1 inhibition for atherothrombotic diseases [47].

Alternation of platelet membrane lipid composition by oxLDL
Crosstalk occurs between the platelets and LDL particles when they are in close contact.LDL binding to platelets not only induces platelet activation but also donates its phospholipids.Platelets incubated with LDL particles receive phosphatidylcholine, phosphatidylethanolamine and sphingomyelin from LDL, thus altering their membrane phospholipid composition [48].This is further supported by evidence showing that LDL particles from obese patients have higher levels of total cholesterol, triglycerides and lipid hydroperoxides than those from healthy controls and that incubation with obese LDL can enrich the membrane lipids of healthy platelets, thus decreasing Na + /K + adenosine triphosphatase (ATPase) activity and membrane fluidity of healthy platelets [49].

Platelet influences on lipid metabolisms
Platelets circulate in the blood and are in constant contact with nLDLs and oxLDLs.Binding of nLDLs and oxLDLs to various platelet receptors primes and stimulates platelet activation.Platelets, in turn, may influence multiple aspects of lipid metabolism (Fig. 2).

Platelet-derived ROS reinforces LDL oxidation
Platelet activation by oxLDL or other platelet agonists promotes ROS production in platelets (Figs. 1  and 2).Platelet-derived ROS can then, among other effects, contribute to the oxidation of LDL [50].Indeed, it has been shown that incubation of activated platelets with both purified LDL and homogenized atherosclerotic plaque markedly elevates the oxidation levels of LDL particles, and that the oxidation of LDL is significantly hampered by NOX inhibition [51].A recent study also showed that LDL and oxLDL uptake by platelets enhances mitochondrial ROS production, facilitates ROSmediated LDL oxidation and further elevates the intraplatelet levels of oxLDL [52].

Platelets promote foam cell formation and lipid accumulation
Platelets bind to nLDL via ApoE-R2 and engage oxLDL with their scavenger receptors, CD36, SR-A1 and LOX-1.Although it is not entirely clear whether platelets internalize their surface-bound nLDL/oxLDL particles, nLDL/oxLDL-bound platelets undergo cellular activation as described above.Activated and nLDL/oxLDL-bound platelets present themselves to macrophages and sacrifice themselves for phagocytosis [53,54].Platelet phagocytosis by macrophages converts the latter into lipid-laden cells that disintegrate platelets, digest platelet-carried lipoproteins, deposit large amounts of cholesterol esters in the cytoplasm and subsequently develop into foam cells (Fig. 2) [54][55][56][57].Moreover, platelets can actively infiltrate atherosclerotic plaques via the leakage of newly formed microvessels [58].These infiltrated platelets provide further prey for macrophages in the plaque, enhancing the sequential processes of phagocytosis, platelet disintegration, foam cell formation, foam cell apoptosis and, eventually, the release and deposition of cytoplasmic lipids and lipid core formation in the plaques.

Platelet-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) contributes to LDL metabolism
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of LDL metabolism.PCSK9 is synthesized and released mainly by the liver, brain, kidney and intestine.PCSK9 is expressed in multiple cell types, including hepatocytes and platelets, and is found in intra-and extracellular spaces.It regulates LDL metabolism by controlling LDLR disintegration and recycling.When released onto the cell surface, PCSK9 binds to the epidermal growth factor-like repeat A domain of LDLR to form the trimolecular complex LDL-LDLR-PCSK9, initiating an extracellular route of LDLR degradation [59].Upon internalization, the LDLR of the bimolecular complex LDL-LDLR unloads its cargo LDL to lysosomes for digestion, returns to the cell surface and prepares for another round of LDL endocytosis.In contrast, PCSK9 docking of the trimolecular complex LDL-LDLR-PCSK9 prevents LDLR separation from its cargo and leads to the degradation of the triad by lysosomes [60].Moreover, cytoplasmic PCSK9 can also steer the intracellular route of LDLR degradation, in which PCSK9 binds and shuttles LDLR from the trans-Golgi network directly to the lysosomes for degradation [61].Hence, PCSK9 elevates the extracellular and circulating LDL levels by promoting LDLR degradation in lysosomes and reducing LDLR numbers during LDL endocytosis [59].
PCSK9 deficiency leads to impaired platelet function and reduced thrombosis.Thus, PCSK9 deficient mice have reduced platelet aggregation and secretion and decreased ferric chloride injuryinduced carotid artery thrombus formation [62], indicating the close involvement of PCSK9 in platelet activity.Indeed, PCSK9 directly and dosedependently enhances platelet aggregation, dense granule (ATP) secretion, α-granule secretion/Pselectin expression, platelet spreading and clot retraction by stimulating CD36 signalling [63].Platelets also express PCSK9, as evidenced by the fact that platelets are positive for PCSK9 detection by flow cytometry [59,64].Moreover, elevated PCSK9 serum levels (including platelet-released PCSK9 during maximal platelet activation of blood clotting) are associated with higher platelet reactivity and may be a predictor of ischaemic events in patients with acute coronary syndrome [65].Data from a large cohort of patients with coronary artery disease confirm that platelets contain significant amounts of PCSK9 and that platelet PCSK9 expression is closely correlated with platelet secretion [64].Moreover, platelet-released PCSK9 appears to be functionally active because the release of activated platelets promotes platelet migration, and platelet co-culture enhances monocyte differentiation into macrophages and foam cells, both of which are hampered by PCSK9 neutralization [64].Given that plasma PCSK9 levels in patients with coronary artery disease are positively correlated with platelet counts [66], it is conceivable that platelet-derived PCSK9 may significantly contribute to PCSK9 levels in the circulation.
PCSK9 not only has critical regulatory roles in LDLR trafficking but has also emerged as a versatile player apart from its primary role in regulating LDLR degradation [67].PCSK9 can bind to the lipid receptors VLDLR, ApoE-R2 and CD36 and promote their degradation, albeit with a much lower efficiency than that of LDLR [68,69].PCSK9 is closely linked to inflammation, as evidenced by increased PCSK9 expression during monocyte and smooth muscle cell activation and macrophage development [70,71].In contrast, PCSK9 can also stimulate the expression of proinflammatory cytokines and chemokines, for example interleukin-1β (IL-1β), IL-6, tumour necrosis factor α, macrophage inflammatory protein-2 alpha, CXCL2 and monocyte chemoattractant protein-1 (MCP-1), of macrophages [72].Furthermore, PCSK9 expression in the vasculature is altered in response to shear stress and haemodynamics.Thus, low shear stress is enhanced, and high shear stress attenuates PCSK9 expression in smooth muscle cells and endothelial cells in concert with ROS production [71], which may be implicated in atherogenesis in arterial channels with low shear stress.Haemodynamics and proinflammatory status jointly regulate PCSK9 expression of vascular cells via NFκB signalling and proinflammatory cytokines, such as IL-1β, IL-6 and MCP-1 [73].It is likely that platelet-derived PCSK9 exerts its effects on hepatocytes and thus contributes to the regulation of hepatocytic LDLR expression and LDL levels in the blood [67].However, this requires further investigation.

Platelet-derived cytokines and chemokines regulate LDL metabolism
Platelets contain a number of growth factors and cytokines/chemokines, such as platelet-derived growth factor, transforming growth factor β (TGFβ) and platelet factor 4 (PF4), in their α-granules.Platelet activation by nLDL/oxLDL and other platelet agonists triggers the release of soluble mediators that may regulate various physiological and pathophysiological processes, such as thrombosis, coagulation, inflammation and angiogenesis (Fig. 2) [5,9].Platelet factor 4. PF4 is a 70-amino acid cationic protein released from αgranules upon platelet activation.PF4 is known for its primary role in binding and neutralizing heparins, thus promoting coagulation [74].PF4 is a chemokine (C-X-C motif ligand 4, CXCL4) [75] and an antiangiogenic factor [76].Moreover, PF4 influences several aspects of LDL metabolism.PF4 inhibits LDL from binding to the surface of cells expressing LDLR [77].Further studies have shown that PF4 binds to the ligand-binding domain of LDLR and that PF4 does not bind or compete with the same binding site of LDL but rather induces a conformational change and a lower affinity for the LDL-binding site.PF4 engagement partially inhibits LDL binding to its receptor, LDLR, but more profoundly inhibits the internalization and degradation of LDL [78].PF4 appears to inhibit internalization and degradation by forming and retaining the PF4-LDLR-LDL complex on the cell surface, which may enhance the formation of oxLDL [78].In contrast to the inhibition of LDL-LDLR ligation, PF4 enhances the binding of oxLDL to macrophages.Hence, PF4 directly binds to oxLDL and subsequently enhances oxLDL binding to macrophages through cooperation with macrophage-expressing glycosaminoglycans [79].However, PF4 deficiency may have limited effects on LDL levels in mice, as suggested by the insignificant changes in non-HDL cholesterol levels but significantly increased HDL levels [80].Therefore, the pro-atherosclerotic effects of PF4 appear to involve complex regulations exerted by PF4, including the regulation of lipoprotein catabolism [78,80], coagulation and thrombosis and inflammation [5,81].
Transforming growth factor β. Platelets contain and release, upon activation, a large amount of TGFβ.Platelets are the principal source of TGFβ in circulation, and platelet-derived TGFβ accounts for about 70% TGFβ in plasma [82].
TGFβ is an essential cytokine that regulates a broad range of physiological and pathophysiological processes, from development to wound healing, immunity to cancer progression and lipid metabolisms.Hence, TGFβ regulates the metabolisms of the energy-storing lipid triglycerides and the cellular structure lipids, such as sphingolipids and phospholipids.TGFβ-stimulated Smad2/3 signalling enhances unsaturated fatty acid synthesis by elevating the expression of stearoyl-CoA desaturase, the rate-limiting enzyme in unsaturated fatty acid synthesis [83].In contrast, TGFβ signalling blockade promotes fatty acid release from triglycerides and fatty acid oxidation.The latter is evidenced by the observation that ablation of the TGFβ signalling molecule Smad2/3 increases brown adipogenesis within white adipose tissue, enhances mitochondria biogenesis in adipocytes and results in elevations in fatty acid oxidation and energy production of mitochondria [84].Moreover, TGFβ signalling can enhance the synthesis of sphingolipids by elevating the expression and activities of the enzymes involved in sphingolipid synthesis [85].
TGFβ also modulates the metabolisms of lipoproteins.Thus, TGFβ has been shown to increase the binding, uptake and degradation of LDL in vascular smooth muscle cells.These effects are exerted by increasing LDLR expression via the pertussis toxin-sensitive G-protein-dependent signal transduction pathway [86].As for macrophages, TGFβ exerts an opposite effect; TGFβ inhibits macrophage uptake of modified LDL through a Smad signalling-dependent mechanism, and TGFβ signalling attenuates the expression of lipoprotein lipase and the LDL scavenger receptors CD36, scavenger receptor-A and scavenger receptor-B [87].It is important to note that the effects of TGFβ on lipoprotein catabolism are cell-type-specific and may have a different impact on atherogenesis.TGFβ-enhanced LDL uptake in vascular smooth muscle cells may facilitate LDL-derived cholesterol metabolisms in the cells.TGFβ-attenuated LDL uptake in macrophages can inhibit foam cell formation and possibly contribute to the antiatherosclerotic impacts of TGFβ.

Impact of thrombocytopaenia and thrombothemia on lipid levels
Platelets play an important role in lipid metabolism.Thus, it is possible that blood lipid levels may be affected by pathophysiologically low and high concentrations of platelets, namely immune thrombocytopaenia (ITP, platelet count <10 11 /L) and essential thrombothemia (ET, also termed primary thrombothemia, platelet count >4.5 × 10 11 /L).Indeed, recent retrospective studies have shown that ITP patients were associated with dyslipidaemia with higher and lower HDL-C levels than in controls, and that more than half of ITP patients had hyperlipidaemia [88,89].Moreover, dyslipidaemia persisted in ITP patients after depriving them of the confounder of higher blood lipid levels and corticosteroid therapy [88], providing solid evidence supporting that thrombocytopaenia may induce hyperlipidaemia.The latter may contribute to ITP-increased cardiovascular risk, as shown by a recent population-based retrospective cohort study [90], apart from other ITP-related pro-atherothrombotic dysfunctions, such as platelet hyperreactivity and enhanced neutrophil extracellular trap formation [91].Interestingly, ET patients, who had excessively high platelet counts, were also found to have a markedly increased prevalence of dyslipidaemia, including hyper-LDL cholesterolemia and hypertriglyceridemia [92,93].These observations highlight the interesting phenomenon that both ITP and ET are associated with hyperlipidaemia.Delineation of the underlying mechanisms is required and is intriguing.As discussed above, the two major platelet-derived mediators, PF4 and TGFβ, contextdependently regulate lipoprotein metabolism and may exert opposite regulatory effects on different cells [77-79, 86, 87].Given that ITP patients may have a 70% reduction of circulating TGFβ [82], the markedly decreased TGFβ levels may significantly hamper its effect on facilitating LDL binding to LDLR and LDL uptakes [86] and result in the prevalence of hyperlipidaemia in ITP patients.In contrast, ET may increase the PF4 levels by 70 times [94].Extremely high PF4 levels may aggravate the inhibition of LDL binding to and uptake by cells [77,78] and subsequently lead to dyslipidaemia in ET patients [92,93].However, the above deduction must be tested and approved in experimental settings, and other mechanisms may also be responsible for ITP-and ET-related dyslipidaemia.

Platelet-hyperlipidaemia interplays and atherosclerosis
Atherosclerosis is a chronic vascular disease with dyslipidaemia, inflammation and thrombosis [3,6].Hyperlipidaemia and platelet hyperreactivity are risk factors and indicators of atherosclerosis.Hyperlipidaemia, especially elevated oxLDL levels, may stimulate platelets through various mechanisms [27].Hyperlipidaemia alone induces mild platelet activation.Therefore, the pathophysiological significance of hyperlipidaemia-induced platelet activation may involve priming platelets and aggravating their responsiveness to other platelet agonists and/or pro-atherosclerotic risk factors throughout the chronic process of atherosclerotic lesion development (Fig. 2).The priming effects may be of particular pathophysiological importance at the advanced stage of atherosclerosis because they initiate and accelerate thrombotic events at the sites of erosional and/or ruptured atherosclerotic plaques and accelerate the clinical manifestations of atherosclerotic ischaemia.
Platelets and platelet-derived mediators have complex influences on lipoprotein metabolism, for example, showing context-dependent inhibition or enhancement of LDL uptake by different cells or events in the same type of cells [78,79].Platelet activation is pro-atherosclerotic.This concept has been well established in earlier studies showing that repeated injection of activated platelets enhances atherosclerosis [95] and that both functional blockade and genetic deficiency of platelet adhesion molecules attenuate lesion development in murine atherosclerotic models [96,97].Supporting the significance of platelet-hyperlipidaemia interactions in atherosclerosis, there is clinical evidence showing that platelet hyperreactivity is correlated with hyperlipidaemia, namely LDL levels, and that platelet counts and platelet-released PCSK9 are correlated with LDL levels in patients with atherosclerotic coronary artery disease [64].The platelet content of oxLDL is also elevated in patients with coronary artery disease, and there is enhanced oxLDL deposition in platelet-rich areas of the intracoronary thrombus in patients [52].Moreover, platelet uptake of LDL increases the oxLDL content within platelets, suggesting that platelets are closely involved in LDL oxidation.In an LDLR-deficient murine model of hyperlipidaemia, platelets have elevated oxLDL content and are associated with enhanced thrombus formation ex vivo [52].

Cross-over therapeutic effects between hypolipidemic and antiplatelet drugs
Hypolipidemic and antiplatelet treatments are the cornerstone of atherosclerotic cardiovascular disease management, in which both therapies efficiently reduce cardiovascular risks, as evidenced by the fact that every 1 mM LDL reduction equals 22% cardiovascular risk reduction [98] and that antiplatelet treatment with COX inhibitors (e.g.aspirin) and/or ADP receptor inhibitors (clopidogrel or ticagrelor) efficiently reduces cardiovascular risk in patients with coronary artery disease [99].In addition to their targeted therapeutic effects, both hypolipidemic and antiplatelet drugs exert pleiotropic effects.Hypolipidemic drugs modulate platelet function, whereas antiplatelet drugs influence lipid metabolism [100,101].
Statins, which inhibit HMG-CoA reductase and endogenous cholesterol synthesis in the liver, achieve their therapeutic effects partially via nonlipid-lowering actions, including the inhibition of platelet activation [102].The latter is evidenced by platelet hyperreactivity after statin treatment withdrawal [103].It is now clear that statins can attenuate platelet activation through multiple mechanisms, for example by reducing platelet TxA 2 and ROS production, suppressing platelet thrombin receptor PAR1 expression and hampering platelet adhesion and aggregation while elevating nitric oxide synthesis and intracellular cAMP levels [100,104].For instance, statins inhibit platelet PLA 2 activation and thus TxA 2 production and decrease platelet NADPH oxidase 2 activity and ROS production [105].Moreover, statins activate platelet peroxisome proliferator-activated receptor alpha (PPARα) and PPARγ and subsequently inhibit PKC signalling pathway and platelet aggregation [106].
PCSK9 inhibitors are another group of lipidlowering drugs.They are either humanized anti-PCSK9 antibodies or PCSK9 synthesis-blocking small interfering RNAs (siRNAs) that inhibit PCSK9-dependent LDLR degradation, enhance LDL uptake and therefore reduce LDL levels [67,107].Similar to statins, PCSK9 inhibitors have also been suggested to influence platelet functions [67,[107][108][109].Hence, a recent study showed that PCSK9 enhanced agonist-induced platelet aggregation, secretion, platelet spreading and platelet clot retraction of human platelets by directly binding to platelet CD36 and that the PCSK9 inhibitor evolocumab abolished the enhancements of PCSK9 [63].Moreover, platelet aggregation was attenuated in washed platelets resuspended in the plasma of patients with familial hypercholesterolaemia after treatment with the PCSK9 inhibitor/siRNA ezetimibe, compared to those suspended in the plasma of non-PCSK9 inhibitor-treated controls [110].There is indirect evidence that PCSK9 inhibition modulates platelet activation.In a prospective observational clinical study of patients with acute coronary syndrome, a direct correlation between PCSK9 plasma levels and platelet reactivity was found, indicating that PCSK9 may enhance platelet activation [65].In animal studies, PCSK9 enhanced thrombosis in vivo in a murine model of FeCl 3induced mesenteric arteriole thrombosis, whereas the PCSK9 inhibitor evolocumab ameliorated these effects [63].PCSK9-deficient mice showed reduced plasma levels of soluble P-selectin [111], a marker of platelet and endothelial activation.PCSK9 deficiency also attenuates venous thrombosis formation compared to that in wild-type mice after inferior vena cava ligation [112].Further clinical studies are required to demonstrate the direct effect of PCSK9 inhibitors on platelet function.
Antiplatelet drugs may also influence lipid metabolism, but their effects on platelet function seem to be less profound than those of hypolipidemic drugs.Aspirin is known to increase the expression of the scavenger receptor CD36 and scavenger receptors B type I (SR-BI) on monocytes/macrophages [113], which are the principle receptors for oxLDL and HDL, respectively.Hence, aspirin-induced increases in macrophage CD36 expression may promote oxLDL uptake by macrophages.Treatment with the ADP receptor P2Y12 antagonist ticagrelor reduced lipid deposition in the arterial vessel wall in a murine model of atherosclerosis, which was achieved by attenuating endothelial inflammation [114].The additional effects of antiplatelet drugs on lipid metabolism may be attributed to their indirect effects.For example, antiplatelet drugs inhibit platelet activation, thereby hampering the production of platelet-derived ROS.The latter may contribute to the attenuation of LDL oxidation and atherogenesis.Furthermore, platelet activation leads to PCSK9 release [64,65].Antiplatelet drug treatments attenuate platelet activation and may thus decrease platelet-released PCSK9, which can hamper LDLR degradation, enhance LDL uptake and contribute to lipid-lowering effects.However, the effects of antiplatelet drugs on lipid metabolism are mild, and caution should be exercised when interpreting the therapeutic effects of antiplatelet drugs on dyslipidaemia.

Research prospect
The platelet-hyperlipidaemia interplay has pathophysiological significance in atherosclerotic progression.Therefore, it is tempting to propose that interventions on the platelet-hyperlipidaemia interplay may be a future approach for improving the management of atherosclerotic diseases.However, it must be stressed that platelets are circulating in the constant presence of lipoproteins, including both nLDL and oxLDL, and that there are considerably high levels of platelet-derived PF4 and TGFβ in circulation and even higher concentrations at the sites of massive platelet activation.Co-existence of platelets, lipoproteins and plateletderived mediators (such as PF4 and TGFβ) is part of human physiology.Hence, special care needs to be taken to develop intervention strategies towards their interplay and not disrupt the physiological balance.
As stated above, the main effect of oxLDL/LDL on platelet function may be the priming of platelets.This indicates that there is limited scope for interventions targeting oxLDL/LDLinduced platelet activation per se.Although hyperlipidaemia-induced platelet dysfunction has been extensively investigated, the effect of platelets on lipoprotein metabolism is not well understood.Among platelet-derived regulators, PF4 and TGFβ are circulating in high concentrations and have broad impacts on cardiovascular physiology and pathophysiology.Although they are known to regulate LDL uptake and foam cell formation, there is a paucity of knowledge, and it is more interesting to further investigate if and how PF4 and TGFβ regulate lipoprotein production and metabolism.A better understanding of the mechanisms of plateletregulated lipoprotein metabolism will provide new insights into the dyslipidaemic and thrombotic mechanisms of atherosclerosis and may lay the foundation for therapeutic development targeting platelet-regulated lipid metabolism.