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Platelet-leukocyte interplay during vascular disease

Open AccessPublished:May 10, 2020DOI:https://doi.org/10.1016/j.atherosclerosis.2020.04.018

      Highlights

      • Together, inflammatory and pro-thrombotic processes cause subendothelial damage, endothelial dysfunction and atherosclerosis.
      • Platelet-leukocyte interactions foster development and progression of aneurysms and various vaso-occlusive pathologies.
      • Platelet-leukocyte interplay during vascular disease (dys)regulates leukocyte recruitment, activation and effector function.

      Abstract

      Vascular disease is a progressive inflammatory condition fuelled by an unhealthy lifestyle of physical inactivity, cholesterol-rich diet, and smoking. Together with endogenous factors such as age, gender, and autoimmune status, an unhealthy lifestyle fosters a pro-inflammatory and pro-thrombotic milieu, which can lead to endothelial dysfunction, atherosclerotic plaque formation and vascular obstruction or degradation of the subendothelial matrix.
      Platelet-leukocyte interplay represents an important feature in this context. Platelets get activated in a pro-inflammatory and pro-thrombotic microenvironment and readily interact with innate and adaptive immune cells alike. Even though platelet affinity for physical cell-cell contact is highest with monocytes/macrophages and neutrophils, platelets also avidly interact with lymphocytes by soluble mediators.
      Platelet-leukocyte crosstalk regulates essential immune responses, supporting leukocyte recruitment at sites of vascular insult, promoting proliferation and differentiation of leukocytes and enhancing pro-inflammatory effector functions such as cytokine and reactive oxygen production. However, under certain conditions platelet-leukocyte interplay also dampens the inflammatory process.
      Crosstalk of platelet and leukocytes thus represents a driving force in vascular disease. In this review, we highlight the impact of various risk factors for vascular disease on platelet-leukocyte interactions and discuss the underlying mechanisms of platelet-mediated changes in immune responses and the effect of immune cells on the haemostatic system. As the underlying pathologies differ between vascular diseases, we summarize our current knowledge on platelet-leukocyte interplay in chronic vascular diseases such as abdominal aortic aneurysm, peripheral and coronary artery disease as well as acute vascular diseases such as ischaemic stroke and venous thromboembolism.

      Graphical abstract

      Keywords

      1. Introduction

      Many vascular diseases represent slowly progressing inflammatory pathologies, which are spurred by an unhealthy lifestyle, where smoking, high cholesterol, and high blood pressure lead to vessel injuries and plaque formation, which eventually rupture. Platelets sense these ruptures, become activated, and form aggregates, which can restrict and block blood flow or cause embolisation at distant sites. This can lead to diminished oxygen supply in vital organs, like heart, lungs, or brain.
      Over the past decade increasing evidence emerged demonstrating that platelets are not only involved in the fatal events of vascular diseases but also play a crucial role in disease development and progression. Platelets are sensitive sentinels that become activated and/or hyper-reactive in response to various stimuli, including inflammatory stress such as infections, but also smoking or hormonal changes. Upon activation they not only release their granules (containing a plethora of effector molecules that promote coagulation, inflammation, and angiogenesis) and form aggregates with other platelets, they also interact with leukocytes, which mutually modulates effector functions of both cell types.

      2. Platelet-leukocyte interactions in immune response and haemostasis

      Hetero-aggregate formation of platelets and leukocytes regularly occurs in healthy individuals, but is enhanced during various inflammatory settings including vascular diseases. Upon interaction platelets and leukocytes activate each other, enabling leukocytes to interfere with the haemostatic system and platelets to interfere with the immune system. The extent of the entwined nature of the haemostatic and the immune system only became apparent during the past couple of years, while from an evolutionary perspective the cross-talk between the two systems is immemorial as they were initially not separated (Fig. 1). In invertebrates a single cell is responsible to coordinate both systems – the haemocyte. Like immune cells, haemocytes express pattern recognition receptors and scavenger receptors, contain antimicrobial peptides, and are able to phagocytose pathogens. Like platelets, haemocytes are able to aggregate, thereby walling off invading microbes but also sealing injuries. They release pro-coagulatory factors in response to injuries and infections [
      • Cerenius L.
      • Soderhall K.
      Coagulation in invertebrates.
      ], and stabilize the clot mesh.
      Figure thumbnail gr1
      Fig. 1The immune system and the haemostatic system – evolutionary perspective.
      In invertebrates, the haemocyte fulfils both immune and haemostatic functions. By expressing pattern recognition and scavenger receptors, haemocytes sense inflammatory stimuli such as lipopolysaccharide (LPS) and β-1,3-glucans to phagocytose microbes and release antimicrobial peptides that lyse pathogens. Haemocytes also aggregate upon stimulation and release coagulatory factors, thereby walling off invading pathogens and sealing injuries. In vertebrates, cells became specialized and immune and haemostatic functions divided. Platelets ensure haemostasis by enforcing aggregation and coagulation, innate leukocytes like neutrophils and monocytes kill a broad spectrum of pathogens by phagocytosis, release of granule content/cytokines or formation of neutrophil extracellular traps (NETs) and lymphocytes are able to launch pathogen-specific responses via distinctive receptors or immunoglobulins (Ig). However, the immune and haemostatic systems are still closely interconnected as e.g. platelets also perform immune functions by phagocytosing pathogens, releasing antimicrobial peptides and modulating leukocyte function, while leukocyte-derived NETs, microvesicles (MV) and tissue factor (TF) promote thrombosis and coagulation.
      In vertebrates, the two systems seem to be divided, as cells became specialized. Leukocytes are responsible for the immune system, while platelets govern the haemostatic system. Upon activation, platelets form aggregates to diminish blood loss upon injury. Leukocytes became experts in performing various host defence mechanisms: innate immune cells like neutrophils and monocytes/macrophages efficiently phagocytose pathogens and kill and lyse invaders via release of antimicrobial peptides or reactive oxygen species (ROS). Even more focused, lymphocytes are specialized in efficiently fighting viruses and bacteria and mediate long lasting humoral defence via specific receptors or antibodies.
      However, platelets also contain antimicrobial peptides in their granules and are able to ensnare and take up pathogens. Moreover, platelets can interact with leukocytes, leading to their recruitment and activation and thereby boost immune responses [
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      Leukocytes in turn can influence the haemostatic system in a process termed immunothrombosis. Neutrophils form neutrophil extracellular traps (NETs) in response to pathogenic stressors. These insoluble NETs, consisting of expelled nuclear DNA and proteins, capture and kill bacteria with antimicrobial proteins and allow leukocytes to act more efficiently [
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      ]. Apart from neutrophils also macrophages, eosinophils, and mast cells release their DNA content and undergo etosis in evolving coronary thrombosis. While NETs are most abundant in early thrombosis, macrophage extracellular traps (METs) dominate during late thrombosis [
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      In vivo coagulation is initiated by tissue factor (TF), which is one of the driving forces of microthrombus formation. Normally, TF is expressed in an encrypted form on the cell membrane in subendothelial tissue. Upon inflammatory processes, also monocytes express TF, thereby causing immunothrombotic events. Apart from being membrane associated, TF can also be found on microvesicles shed from monocytes, which further contributes to thrombus formation [
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      3. Regulation of platelet leukocyte interactions

      Direct interaction of platelets with different types of leukocytes often precedes platelet-mediated changes of immune functions. Platelet-leukocyte aggregates (PLA), in particular those with monocytes (PMA) and neutrophils (PNA), are increased in many thrombo-inflammatory diseases and have therefore been proposed as an attractive and easily accessible prognostic and/or diagnostic marker. Molecular mediators of direct and indirect platelet-leukocytes crosstalk have been reviewed in detail elsewhere [
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      Fig. 2
      Fig. 2Platelet-leukocyte interplay regulates immune and haemostatic responses.
      During inflammatory settings, platelets interact with leukocytes via release of soluble mediators or by direct cell-cell contact, with highest binding affinity for monocytes/macrophages, followed by neutrophils and lymphocytes. Platelets promote leukocyte recruitment by facilitating chemotaxis, endothelial rolling and adhesion and extravasation. Interaction with platelets also enhances monocyte survival and differentiation to CD16-expressing subsets, foam cells or CXCL4-induced M4 macrophages as well as neutrophil phagocytosis and lymphocyte activation, proliferation and immunoglobulin production. Depending on the specific condition platelet-leukocyte interplay further modulates the inflammatory process by conferring either pro- or anti-inflammatory effects, including modulation of cyto-/chemokines release, oxidative burst and lymphocyte development. Crosstalk of platelets with leukocytes also affects haemostasis by triggering expression or release pro-coagulatory and pro-thrombotic factors such as TF as well as METs, NETs, and EETs, but also by converting exogenous PGH2 to platelet-inhibiting PGI2. Act. CD11b: activated CD11b; ATP: adenosine triphosphate; C5a: complement component 5a; CCL: C–C motif chemokine ligand; CD: cluster of differentiation; CLEC-2: C-type lectin-like 2; CXCL: C-X-C motif chemokine ligand; EET: eosinophil extracellular trap; EMMPRIN: extracellular matrix metalloproteinase inducer; FXa: coagulation factor Xa; GP: glycoprotein; HMGB1: high-mobility group box 1; 5-HT: 5-hydroxytryptamine/serotonin; Ig: immunoglobulin; IL: interleukin; IFN-α: interferon α; JamA: junctional adhesion molecule A; LTB4: leukotriene B4; MCP-1: monocyte chemoattractant protein 1; MET: macrophage extracellular trap; MIF: macrophage migration inhibitory factor; MIP-1β: macrophage inflammatory protein 1β; MMP: matrix metalloproteinase; MPO: myeloperoxidase; MV: microvesicles; NET: neutrophil extracellular trap; PAF: platelet-activating factor; PDGF: platelet-derived growth factor; PGH2: prostaglandin H2; PMV: platelet microvesicles; PS: phosphatidylserine; ROS: reactive oxygen species; TF: tissue factor; TGF-β: transforming growth factor β; Th: helper T-cell; TNF-α: tumour necrosis factor α; Treg: regulatory T-cell; TREM1: triggering receptor expressed on myeloid cells 1.
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      • Green F.H.
      • Kubes P.
      Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood.
      ,
      • Maugeri N.
      • Rovere-Querini P.
      • Baldini M.
      • Baldissera E.
      • Sabbadini M.G.
      • Bianchi M.E.
      • Manfredi A.A.
      Oxidative stress elicits platelet/leukocyte inflammatory interactions via HMGB1: a candidate for microvessel injury in sytemic sclerosis.
      ,
      • Assinger A.
      • Laky M.
      • Schabbauer G.
      • Hirschl A.M.
      • Buchberger E.
      • Binder B.R.
      • Volf I.
      Efficient phagocytosis of periodontopathogens by neutrophils requires plasma factors, platelets and TLR2.
      ,
      • Vanichakarn P.
      • Blair P.
      • Wu C.
      • Freedman J.E.
      • Chakrabarti S.
      Neutrophil CD40 enhances platelet-mediated inflammation.
      ,
      • Perazzio S.F.
      • Soeiro-Pereira P.V.
      • Dos Santos V.C.
      • de Brito M.V.
      • Salu B.
      • Oliva M.L.V.
      • Stevens A.M.
      • de Souza A.W.S.
      • Ochs H.D.
      • Torgerson T.R.
      • Condino-Neto A.
      • Andrade L.E.C.
      Soluble CD40L is associated with increased oxidative burst and neutrophil extracellular trap release in Behcet's disease.
      ,
      • Lo S.C.
      • Hung C.Y.
      • Lin D.T.
      • Peng H.C.
      • Huang T.F.
      Involvement of platelet glycoprotein Ib in platelet microparticle mediated neutrophil activation.
      ]. However, similar to platelet-monocyte interplay, the impact of platelet-neutrophil interactions is not straightforward, as platelet-derived ATP inhibits neutrophil oxidative burst [
      • Lecut C.
      • Faccinetto C.
      • Delierneux C.
      • van Oerle R.
      • Spronk H.M.
      • Evans R.J.
      • El Benna J.
      • Bours V.
      • Oury C.
      ATP-gated P2X1 ion channels protect against endotoxemia by dampening neutrophil activation.
      ]. Vice versa, activated neutrophils produce lipoxin A4 and express CCR5 which scavenges platelet-derived CCL3 and CCL5, thereby limiting the chemotactic effect of platelets on leukocytes [
      • Brancaleone V.
      • Gobbetti T.
      • Cenac N.
      • le Faouder P.
      • Colom B.
      • Flower R.J.
      • Vergnolle N.
      • Nourshargh S.
      • Perretti M.
      A vasculo-protective circuit centered on lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 operative in murine microcirculation.
      ].
      Platelet-lymphocyte interaction is far less prominent and platelets barely interact directly, especially with B-lymphocytes. Interactions of platelets with activated T-lymphocytes involve CD62P, GPIIb/IIIa, and CD40L, with serotonin and CXCL4 being the most important soluble mediators that influence lymphocyte effector functions [
      • Li N.
      • Ji Q.
      • Hjemdahl P.
      Platelet-lymphocyte conjugation differs between lymphocyte subpopulations.
      ,
      • Leon-Ponte M.
      • Ahern G.P.
      • O'Connell P.J.
      Serotonin provides an accessory signal to enhance T-cell activation by signaling through the 5-HT7 receptor.
      ,
      • Liu C.Y.
      • Battaglia M.
      • Lee S.H.
      • Sun Q.H.
      • Aster R.H.
      • Visentin G.P.
      Platelet factor 4 differentially modulates CD4+CD25+ (regulatory) versus CD4+CD25- (nonregulatory) T cells.
      ]. Platelets play an important role in lymphocyte trafficking to secondary lymphoid organs and – depending on the lymphocyte subtype and the model employed – platelets either diminish or enhance T-lymphocyte functions [
      • Lalor P.
      • Nash G.B.
      Adhesion of flowing leucocytes to immobilized platelets.
      ,
      • Diacovo T.G.
      • Catalina M.D.
      • Siegelman M.H.
      • von Andrian U.H.
      Circulating activated platelets reconstitute lymphocyte homing and immunity in L-selectin-deficient mice.
      ,
      • Iannacone M.
      • Sitia G.
      • Narvaiza I.
      • Ruggeri Z.M.
      • Guidotti L.G.
      Antiplatelet drug therapy moderates immune-mediated liver disease and inhibits viral clearance in mice infected with a replication-deficient adenovirus.
      ,
      • Fleischer J.
      • Grage-Griebenow E.
      • Kasper B.
      • Heine H.
      • Ernst M.
      • Brandt E.
      • Flad H.D.
      • Petersen F.
      Platelet factor 4 inhibits proliferation and cytokine release of activated human T cells.
      ,
      • Shi G.
      • Field D.J.
      • Ko K.A.
      • Ture S.
      • Srivastava K.
      • Levy S.
      • Kowalska M.A.
      • Poncz M.
      • Fowell D.J.
      • Morrell C.N.
      Platelet factor 4 limits Th17 differentiation and cardiac allograft rejection.
      ,
      • Gerdes N.
      • Zhu L.
      • Ersoy M.
      • Hermansson A.
      • Hjemdahl P.
      • Hu H.
      • Hansson G.K.
      • Li N.
      Platelets regulate CD4(+) T-cell differentiation via multiple chemokines in humans.
      ]. Mice deficient for either CXCL4 or platelets show exaggerated immune responses to cardiac transplantation [
      • Shi G.
      • Field D.J.
      • Ko K.A.
      • Ture S.
      • Srivastava K.
      • Levy S.
      • Kowalska M.A.
      • Poncz M.
      • Fowell D.J.
      • Morrell C.N.
      Platelet factor 4 limits Th17 differentiation and cardiac allograft rejection.
      ]. Moreover, platelet-B-lymphocyte crosstalk via CD40−CD40L has been shown to induce isotype switching and fosters immunoglobulin production [
      • Elzey B.D.
      • Tian J.
      • Jensen R.J.
      • Swanson A.K.
      • Lees J.R.
      • Lentz S.R.
      • Stein C.S.
      • Nieswandt B.
      • Wang Y.
      • Davidson B.L.
      • Ratliff T.L.
      Platelet-mediated modulation of adaptive immunity. A communication link between innate and adaptive immune compartments.
      ]. Lymphocytes can also influence platelet functions as lymphocytes use platelet-derived prostaglandin H2 (PGH2) to synthesize PGI2, which represents a potent platelet inhibitor [
      • Wu K.K.
      • Papp A.C.
      • Manner C.E.
      • Hall E.R.
      Interaction between lymphocytes and platelets in the synthesis of prostacyclin.
      ].

      4. Effects of cardiovascular risk factors on platelet-leukocyte interplay

      Several cardiovascular risk factors influence platelet function and platelet-leukocyte interactions, thereby fostering disease development and progression.
      The risk for having a myocardial infarction or stroke peaks in the morning [
      • Muller J.E.
      • Stone P.H.
      • Turi Z.G.
      • Rutherford J.D.
      • Czeisler C.A.
      • Parker C.
      • Poole W.K.
      • Passamani E.
      • Roberts R.
      • Robertson T.
      • et al.
      Circadian variation in the frequency of onset of acute myocardial infarction.
      ]. This is accompanied by increased platelet activation, an effect independent of sleep/wake cycles and other behavioural influences [
      • Scheer F.A.
      • Michelson A.D.
      • Frelinger 3rd, A.L.
      • Evoniuk H.
      • Kelly E.E.
      • McCarthy M.
      • Doamekpor L.A.
      • Barnard M.R.
      • Shea S.A.
      The human endogenous circadian system causes greatest platelet activation during the biological morning independent of behaviors.
      ]. Increased agonist-induced platelet-aggregation is most likely caused by increased sympathetic nervous system activity and elevated levels of catecholamines in blood [
      • Budkowska M.
      • Lebiecka A.
      • Marcinowska Z.
      • Wozniak J.
      • Jastrzebska M.
      • Dolegowska B.
      The circadian rhythm of selected parameters of the hemostasis system in healthy people.
      ]. Moreover, platelet numbers are highest in the afternoon and studies using genetically modified mice showed that in vivo platelet production is affected by the circadian transcription factor circadian locomotor output cycles kaput (CLOCK) that regulates expression of thrombopoietin [
      • Tracey C.J.
      • Pan X.
      • Catterson J.H.
      • Harmar A.J.
      • Hussain M.M.
      • Hartley P.S.
      Diurnal expression of the thrombopoietin gene is regulated by CLOCK.
      ].
      With age platelet counts decrease, while platelet reactivity increases. Increased ROS and activation of the mammalian target of rapamycin (mTOR) pathway seem to be responsible for these effects, which alter platelet transcriptome and function and lead to exaggerated inflammation. Also platelet-leukocyte interactions are altered in aged individuals as platelet-monocyte interactions result in significantly more cytokines due to elevated levels of platelet granzyme A [
      • Montenont E.
      • Rondina M.T.
      • Campbell R.A.
      Altered functions of platelets during aging.
      ].
      Women have higher platelet counts and enhanced platelet aggregation compared to men [
      • Budkowska M.
      • Lebiecka A.
      • Marcinowska Z.
      • Wozniak J.
      • Jastrzebska M.
      • Dolegowska B.
      The circadian rhythm of selected parameters of the hemostasis system in healthy people.
      ]. Although platelets express oestrogen receptors [
      • Nealen M.L.
      • Vijayan K.V.
      • Bolton E.
      • Bray P.F.
      Human platelets contain a glycosylated estrogen receptor beta.
      ], this effect is not dependent on the menstrual cycle or the use of oral contraceptives [
      • Berlin G.
      • Hammar M.
      • Tapper L.
      • Tynngard N.
      Effects of age, gender and menstrual cycle on platelet function assessed by impedance aggregometry.
      ]. However, PLA formation is affected by oestrogen levels as the number of PLAs peaks on day 14 of menstrual cycle [
      • Rosin C.
      • Brunner M.
      • Lehr S.
      • Quehenberger P.
      • Panzer S.
      The formation of platelet-leukocyte aggregates varies during the menstrual cycle.
      ]. When comparing female and male patients with CVD, in vivo as well as in vitro agonist-induced PLA formation was significantly pronounced in females [
      • Gremmel T.
      • Kopp C.W.
      • Eichelberger B.
      • Koppensteiner R.
      • Panzer S.
      Sex differences of leukocyte-platelet interactions and on-treatment platelet reactivity in patients with atherosclerosis.
      ].
      Similar to other cardiovascular risks, platelet function and platelet reactivity are related to cardiorespiratory fitness. A sedentary lifestyle is associated with platelet hyperreactivity while a physically active lifestyle dramatically reduces cardiovascular mortality and also platelet activation. However, vigorous exercise transiently increases the risk for myocardial infarction and results in platelet activation and hyperreactivity and subsequent platelet-leukocyte interactions [
      • Heber S.
      • Assinger A.
      • Pokan R.
      • Volf I.
      Correlation between cardiorespiratory fitness and platelet function in healthy women.
      ].
      Obesity increases platelet hyperreactivity, leading to increased mean platelet volume, platelet microvesicle formation, enhanced thromboxane B2 metabolites, sCD62P, and platelet-derived CD40L. These alterations can be explained by altered exposure and/or increased expression of surface receptors for agonists and adhesion molecules, decreased membrane fluidity, altered platelet metabolism as well as disruptions in intra-platelet signalling, and increased oxidative stress. Further, chronic inflammation as observed in obese patients leads to imbalanced haemostasis as endogenous anticoagulant mechanisms get dysregulated, including tissue factor pathway inhibitor, anti-thrombin, and protein C [
      • Badimon L.
      • Bugiardini R.
      • Cenko E.
      • Cubedo J.
      • Dorobantu M.
      • Duncker D.J.
      • Estruch R.
      • Milicic D.
      • Tousoulis D.
      • Vasiljevic Z.
      • Vilahur G.
      • de Wit C.
      • Koller A.
      Position paper of the European Society of Cardiology-working group of coronary pathophysiology and microcirculation: obesity and heart disease.
      ].
      Smoking is a major risk factor for the development of CVD as smoke and tobacco components deliver huge amounts of oxidants and toxins that favour the development of inflammation, oxidative stress, and thrombosis. Moreover, leukocyte and endothelial functions are altered, causing platelet abnormalities such as accelerated platelet turnover and increased platelet activation [
      • Assinger A.
      • Schmid W.
      • Volf I.
      Decreased VASP phosphorylation in platelets of male and female smokers of young age.
      ].
      Patients with autoimmune diseases such as anti-phospholipid syndrome, systemic lupus erythematosus, and rheumatoid arthritis show increased levels of circulating PLAs and have a significantly higher risk to develop CVD [
      • Joseph J.E.
      • Harrison P.
      • Mackie I.J.
      • Isenberg D.A.
      • Machin S.J.
      Increased circulating platelet-leucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis.
      ]. Studies in mice showed that prolonged systemic inflammation after intra-abdominal sepsis accelerates the development of atherosclerosis [
      • Kaynar A.M.
      • Yende S.
      • Zhu L.
      • Frederick D.R.
      • Chambers R.
      • Burton C.L.
      • Carter M.
      • Stolz D.B.
      • Agostini B.
      • Gregory A.D.
      • Nagarajan S.
      • Shapiro S.D.
      • Angus D.C.
      Effects of intra- abdominal sepsis on atherosclerosis in mice.
      ], corroborating a link between acute cardiovascular events and prior infection in humans [
      • Meier C.R.
      • Jick S.S.
      • Derby L.E.
      • Vasilakis C.
      • Jick H.
      Acute respiratory-tract infections and risk of first-time acute myocardial infarction.
      ]. Moreover, severe trauma and sepsis were associated with enhanced formation of PLAs [
      • Ogura H.
      • Kawasaki T.
      • Tanaka H.
      • Koh T.
      • Tanaka R.
      • Ozeki Y.
      • Hosotsubo H.
      • Kuwagata Y.
      • Shimazu T.
      • Sugimoto H.
      Activated platelets enhance microparticle formation and platelet-leukocyte interaction in severe trauma and sepsis.
      ]. Furthermore, platelet activation and platelet-leukocyte interplay have emerged as new regulators in allergic disease [
      • Pitchford S.C.
      • Yano H.
      • Lever R.
      • Riffo-Vasquez Y.
      • Ciferri S.
      • Rose M.J.
      • Giannini S.
      • Momi S.
      • Spina D.
      • O'Connor B.
      • Gresele P.
      • Page C.P.
      Platelets are essential for leukocyte recruitment in allergic inflammation.
      ], while at the same time allergic disease is associated with increased thromboembolic events [
      • Undas A.
      • Ciesla-Dul M.
      • Drazkiewicz T.
      • Potaczek D.P.
      • Sadowski J.
      Association between atopic diseases and venous thromboembolism: a case-control study in patients aged 45 years or less.
      ]. Platelets mediate systemic mast cell activation and IgE production [
      • Karhausen J.
      • Choi H.W.
      • Maddipati K.R.
      • Mathew J.P.
      • Ma Q.
      • Boulaftali Y.
      • Lee R.H.
      • Bergmeier W.
      • Abraham S.N.
      Platelets trigger perivascular mast cell degranulation to cause inflammatory responses and tissue injury.
      ,
      • Tian J.
      • Zhu T.
      • Liu J.
      • Guo Z.
      • Cao X.
      Platelets promote allergic asthma through the expression of CD154.
      ] which in turn may promote platelet activation via the FcεRI [
      • Hasegawa S.
      • Pawankar R.
      • Suzuki K.
      • Nakahata T.
      • Furukawa S.
      • Okumura K.
      • Ra C.
      Functional expression of the high affinity receptor for IgE (FcepsilonRI) in human platelets and its' intracellular expression in human megakaryocytes.
      ], thus conferring an increasingly pro-thrombotic microenvironment.

      5. Platelet-leukocyte interplay in development and progression of chronic vascular disease

      Mutual regulation of platelets and different leukocyte subsets favours the development and exacerbation of vascular abnormalities that underlie chronic vascular diseases such as abdominal aortic aneurysm, peripheral and coronary artery disease. Molecular determinants and immunomodulatory effects of platelet-leukocyte crosstalk is summarized in Fig. 3.
      Fig. 3
      Fig. 3Platelet-leukocyte interplay in the development and progression of vascular disease.
      Mutual regulation of platelets and leukocytes fosters development and progression of vascular abnormalities that underlie chronic and acute vascular disease. Coronary artery disease is associated with elevated circulating PMAs and PNAs and increased levels of CD16-negative classical monocytes. Platelets induce formation of M4 macrophages and enhance inflammatory cytokine release as well as T-cell proliferation and activation, thus exacerbating atherosclerotic plaque formation and instability which ultimately results in plaque rupture, thrombosis and vessel occlusion. In abdominal aortic aneurysm circulating PMAs are increased and platelets promote monocyte/macrophage recruitment and release of cytokines and MMPs, neutrophil NET formation and oxidative burst and T-cell activation and accumulation, thereby contributing to lamina degradation, vessel dilatation and risk of rupture. Peripheral artery disease is associated with platelet activation and strongly increased circulating PMAs and PNAs. Platelet-leukocyte interplay augments the pro-inflammatory microenvironment in atherosclerotic lesions by regulating leukocyte differentiation, cytokine release, recruitment and NET formation which exacerbates lesion size and decreases the arterial lumen. Acute ischaemic stroke is followed by a period of post-ischaemic microvascular dysfunction and loss of integrity of the blood-brain barrier. Circulating PMAs, PNAs and PLyAs are increased during this phase and platelet-leukocyte interactions mediate leukocyte recruitment and activation, thus aggravating disease severity and infarct volume. Venous thromboembolism is associated with increased circulating PMAs, PNAs and PLyAs. Augmented platelet activation is essential for recruitment of innate leukocytes, NET formation and expression of cytokines and TF. These platelet-mediated effects further promote hypercoagulability and endothelial dysfunction which are critical factors for deep vein thrombosis and subsequent pulmonary embolism. NET: neutrophil extracellular trap; PLyA: platelet-lymphocyte aggregate; PMA: platelet-monocyte aggregate; PNA: platelet-neutrophil aggregate; ROS: reactive oxygen species; TF: tissue factor.

      5.1 Abdominal aortic aneurysm (AAA)

      Chronic inflammation of the aortic vasculature may lead to endothelial damage and degradation of subendothelial vessel wall components and subsequent progressive dilatation of the vessel lumen may eventually culminate in life-threatening arterial rupture. This process can be exacerbated by recurring low-key infections [
      • Alsac J.M.
      • Delbosc S.
      • Rouer M.
      • Journe C.
      • Louedec L.
      • Meilhac O.
      • Michel J.B.
      Fucoidan interferes with Porphyromonas gingivalis-induced aneurysm enlargement by decreasing neutrophil activation.
      ]. Aneurysms can occur in all segments of the aorta, but are most commonly found in the abdominal aorta. Intraluminal thrombi (ILT) are prone to develop within the lumen of the aneurysm; however, platelets not only contribute to AAA pathology by forming thrombi, they have also been found to further vessel wall dilatation.
      AAA patients show increased platelet infiltration in aortic tissue [
      • Liu O.
      • Jia L.
      • Liu X.
      • Wang Y.
      • Wang X.
      • Qin Y.
      • Du J.
      • Zhang H.
      Clopidogrel, a platelet P2Y12 receptor inhibitor, reduces vascular inflammation and angiotensin II induced- abdominal aortic aneurysm progression.
      ] along with increased circulating PMAs [
      • Allen N.
      • Barrett T.J.
      • Guo Y.
      • Nardi M.
      • Ramkhelawon B.
      • Rockman C.B.
      • Hochman J.S.
      • Berger J.S.
      Circulating monocyte-platelet aggregates are a robust marker of platelet activity in cardiovascular disease.
      ], underlining the importance of direct platelet binding to monocytes/macrophages. In vitro platelets augment macrophage transmigration and MMP activity [
      • Liu O.
      • Jia L.
      • Liu X.
      • Wang Y.
      • Wang X.
      • Qin Y.
      • Du J.
      • Zhang H.
      Clopidogrel, a platelet P2Y12 receptor inhibitor, reduces vascular inflammation and angiotensin II induced- abdominal aortic aneurysm progression.
      ], which is associated with vessel wall degradation. The CD40L-CD40 axis has been implicated in aneurysm formation and in mouse models CD40L deficiency reduces inflammatory chemo-/cytokines as well as macrophage infiltration, lowering AAA incidence and risk of rupture of formed AAAs [
      • Kusters P.J.H.
      • Seijkens T.T.P.
      • Beckers L.
      • Lievens D.
      • Winkels H.
      • de Waard V.
      • Duijvestijn A.
      • Lindquist Liljeqvist M.
      • Roy J.
      • Daugherty A.
      • Newby A.
      • Gerdes N.
      • Lutgens E.
      CD40L deficiency protects against aneurysm formation.
      ]. Similar results were found in CD62P-deficient mice [
      • Hannawa K.K.
      • Cho B.S.
      • Sinha I.
      • Roelofs K.J.
      • Myers D.D.
      • Wakefield T.J.
      • Stanley J.C.
      • Henke P.K.
      • Upchurch Jr., G.R.
      Attenuation of experimental aortic aneurysm formation in P- selectin knockout mice.
      ] and upon pharmaceutically targeting CD62P, which also dampened neutrophil oxidative burst, NET formation and apoptosis [
      • Alsac J.M.
      • Delbosc S.
      • Rouer M.
      • Journe C.
      • Louedec L.
      • Meilhac O.
      • Michel J.B.
      Fucoidan interferes with Porphyromonas gingivalis-induced aneurysm enlargement by decreasing neutrophil activation.
      ]. Platelets regulate leukocyte recruitment in AAA indirectly via release of the chemokines CXCL4 and CCL5, which are increased in plasma of AAA patients, but are also present in high levels in luminal layers of ILTs. Accordingly, platelets and neutrophils co-localize in luminal ILT layers [
      • Houard X.
      • Touat Z.
      • Ollivier V.
      • Louedec L.
      • Philippe M.
      • Sebbag U.
      • Meilhac O.
      • Rossignol P.
      • Michel J.B.
      Mediators of neutrophil recruitment in human abdominal aortic aneurysms.
      ]. Inhibition of CXCL4-CCL5 heterodimers before or after induction of experimental AAA has proven efficient to prevent development of AAA or halt its progression, respectively [
      • Iida Y.
      • Xu B.
      • Xuan H.
      • Glover K.J.
      • Tanaka H.
      • Hu X.
      • Fujimura N.
      • Wang W.
      • Schultz J.R.
      • Turner C.R.
      • Dalman R.L.
      Peptide inhibitor of CXCL4-CCL5 heterodimer formation, MKEY, inhibits experimental aortic aneurysm initiation and progression.
      ]. This suggests that platelet inhibition could represent a viable treatment option for AAA patients.
      Indeed, in murine AAA models inhibition of cyclooxygenase by aspirin or blocking of the ADP receptor P2Y12 by clopidogrel impedes platelet accumulation and reduces plasma levels of CXCL4, CCL5 and other chemo-/cytokines as well as MMPs, which is accompanied by impaired macrophage recruitment. Inhibiting platelet-mediated modulation of immune responses thereby slows lamina degradation and vessel dilatation and increases survival in mice [
      • Liu O.
      • Jia L.
      • Liu X.
      • Wang Y.
      • Wang X.
      • Qin Y.
      • Du J.
      • Zhang H.
      Clopidogrel, a platelet P2Y12 receptor inhibitor, reduces vascular inflammation and angiotensin II induced- abdominal aortic aneurysm progression.
      ,
      • Owens 3rd, A.P.
      • Edwards T.L.
      • Antoniak S.
      • Geddings J.E.
      • Jahangir E.
      • Wei W.Q.
      • Denny J.C.
      • Boulaftali Y.
      • Bergmeier W.
      • Daugherty A.
      • Sampson U.K.A.
      • Mackman N.
      Platelet inhibitors reduce rupture in a mouse model of established abdominal aortic aneurysm.
      ], but treatment with aspirin or P2Y12 blockers also reduces risk of rupture and dissection in AAA patients [
      • Owens 3rd, A.P.
      • Edwards T.L.
      • Antoniak S.
      • Geddings J.E.
      • Jahangir E.
      • Wei W.Q.
      • Denny J.C.
      • Boulaftali Y.
      • Bergmeier W.
      • Daugherty A.
      • Sampson U.K.A.
      • Mackman N.
      Platelet inhibitors reduce rupture in a mouse model of established abdominal aortic aneurysm.
      ].
      Analysis of patient ILTs showed that local T cells are highly activated and express high levels of CCR5, which is a receptor for CCL5 and might suggest a role of platelets in the regulation of lymphocyte traffic in AAA thrombi [
      • Sagan A.
      • Mrowiecki W.
      • Mikolajczyk T.P.
      • Urbanski K.
      • Siedlinski M.
      • Nosalski R.
      • Korbut R.
      • Guzik T.J.
      Local inflammation is associated with aortic thrombus formation in abdominal aortic aneurysms. Relationship to clinical risk factors.
      ]. Indeed, histological analysis of tissue sections from patients with AAA revealed increased numbers of platelets and T lymphocytes in aneurysm sites along with augmented angiogenesis [
      • Liu O.
      • Jia L.
      • Liu X.
      • Wang Y.
      • Wang X.
      • Qin Y.
      • Du J.
      • Zhang H.
      Clopidogrel, a platelet P2Y12 receptor inhibitor, reduces vascular inflammation and angiotensin II induced- abdominal aortic aneurysm progression.
      ]. Further, extreme values of platelet to lymphocyte ratio (PLR) are associated with a higher risk for complications after AAA surgical repair [
      • Lareyre F.
      • Carboni J.
      • Chikande J.
      • Massiot N.
      • Voury-Pons A.
      • Umbdenstock E.
      • Jean- Baptiste E.
      • Hassen-Khodja R.
      • Raffort J.
      Association of platelet to lymphocyte ratio and risk of 30-day postoperative complications in patients undergoing abdominal aortic surgical repair.
      ]. Patients with metabolic syndrome showed increased levels of CXCR6 on platelet bound circulating CD8+ lymphocytes, which promotes adhesion to the dysfunctional arterial endothelium and might be involved in adverse cardiovascular events. Moreover, infiltration of CD8+ CXCR6+ lymphocytes is significantly increased in the AAA lesions of ApoE-deficient mice [
      • Collado A.
      • Marques P.
      • Escudero P.
      • Rius C.
      • Domingo E.
      • Martinez-Hervas S.
      • Real J.T.
      • Ascaso J.F.
      • Piqueras L.
      • Sanz M.J.
      Functional role of endothelial CXCL16/CXCR6-platelet- leucocyte axis in angiotensin II-associated metabolic disorders.
      ]. While platelet-lymphocyte aggregates have not been reported yet in AAA patients, platelets and neutrophils co-localize in ILTs and platelet inhibition reduces leukocyte infiltration in an animal AAA model [
      • Houard X.
      • Touat Z.
      • Ollivier V.
      • Louedec L.
      • Philippe M.
      • Sebbag U.
      • Meilhac O.
      • Rossignol P.
      • Michel J.B.
      Mediators of neutrophil recruitment in human abdominal aortic aneurysms.
      ,
      • Dai J.
      • Louedec L.
      • Philippe M.
      • Michel J.B.
      • Houard X.
      Effect of blocking platelet activation with AZD6140 on development of abdominal aortic aneurysm in a rat aneurysmal model.
      ].
      In addition to acquired AAA, several congenital human connective tissue diseases such as Marfan syndrome and Ehlers-Danlos syndrome are characterized by increased propensity to develop aortic aneurysms and increased risk of aortic dissection. However, while patients with Marfan syndrome display increased platelet activation [
      • Kornhuber K.T.I.
      • Seidel H.
      • Pujol C.
      • Meierhofer C.
      • Roschenthaler F.
      • Pressler A.
      • Stockl A.
      • Nagdyman N.
      • Neidenbach R.C.
      • von Hundelshausen P.
      • Halle M.
      • Holdenrieder S.
      • Ewert P.
      • Kaemmerer H.
      • Hauser M.
      Hemostatic abnormalities in adult patients with Marfan syndrome.
      ], Ehlers-Danlos syndrome is associated with increased bleeding risk and impaired platelet function [
      • Busch A.
      • Hoffjan S.
      • Bergmann F.
      • Hartung B.
      • Jung H.
      • Hanel D.
      • Tzschach A.
      • Kadar J.
      • von Kodolitsch Y.
      • Germer C.T.
      • Trobisch H.
      • Strasser E.
      • Wildenauer R.
      Vascular type Ehlers-Danlos syndrome is associated with platelet dysfunction and low vitamin D serum concentration.
      ]. Thus, the potential contributions of platelets and platelet-leukocyte interactions to congenital aneurysm disorders appear to me multifaceted and remain to be elucidated.

      5.2 Peripheral artery disease (PAD)

      Patients with PAD suffer from reduced blood flow to the distal extremities caused by narrowing of the arterial lumen due to presence of atherosclerotic plaques. The key feature of the disease is intermittent claudication with walking caused by temporary ischaemia of the leg muscles. Additionally, PAD serves as a marker for systemic atherosclerosis and is associated with high cardiovascular risk. While arteries aim to preserve blood flow by vasodilation at early stages of PAD, narrowing of arterial flow lumen by atherosclerotic plaques cannot be prevented in later stages and is accompanied with haemodynamic consequences involving emboli at sides of arterial bifurcations [
      • Zemaitis M.R.
      • Boll J.M.
      • Dreyer M.A.
      Peripheral Arterial Disease StatPearls Treasure Island.
      ]. Thereby, PAD and coronary artery disease (CAD) are major causes of vaso-occlusive diseases including embolism, stroke and deep vein thrombosis (DVT).
      As with all atherosclerotic disorders, monocytes and macrophages play a pivotal role in PAD and modulation of their pro-inflammatory and pro-atherogenic functions by platelets contributes to disease progression. Patients with PAD display raised pro-thrombotic platelet activation as evidenced by increased levels of surface and soluble CD62P, GPIIb/IIIa activation, and enhanced aggregation [
      • Gremmel T.
      • Xhelili E.
      • Steiner S.
      • Koppensteiner R.
      • Kopp C.W.
      • Panzer S.
      Response to antiplatelet therapy and platelet reactivity to thrombin receptor activating peptide-6 in cardiovascular interventions: differences between peripheral and coronary angioplasty.
      ,
      • Woollard K.J.
      • Kling D.
      • Kulkarni S.
      • Dart A.M.
      • Jackson S.
      • Chin-Dusting J.
      Raised plasma soluble P-selectin in peripheral arterial occlusive disease enhances leukocyte adhesion.
      ]. Furthermore, circulating PMAs and PNAs are strongly elevated in PAD with PMA levels exceeding those observed in CAD [
      • Gremmel T.
      • Xhelili E.
      • Steiner S.
      • Koppensteiner R.
      • Kopp C.W.
      • Panzer S.
      Response to antiplatelet therapy and platelet reactivity to thrombin receptor activating peptide-6 in cardiovascular interventions: differences between peripheral and coronary angioplasty.
      ,
      • Dopheide J.F.
      • Rubrech J.
      • Trumpp A.
      • Geissler P.
      • Zeller G.C.
      • Bock K.
      • Dunschede F.
      • Trinh T.T.
      • Dorweiler B.
      • Munzel T.
      • Radsak M.P.
      • Espinola-Klein C.
      Leukocyte-platelet aggregates-a phenotypic characterization of different stages of peripheral arterial disease.
      ]. Use of blocking antibodies revealed that binding of platelets to neutrophils or monocytes/macrophages prominently involves the CD62P-PSGL-1 axis [
      • Woollard K.J.
      • Kling D.
      • Kulkarni S.
      • Dart A.M.
      • Jackson S.
      • Chin-Dusting J.
      Raised plasma soluble P-selectin in peripheral arterial occlusive disease enhances leukocyte adhesion.
      ,
      • Dann R.
      • Hadi T.
      • Montenont E.
      • Boytard L.
      • Alebrahim D.
      • Feinstein J.
      • Allen N.
      • Simon R.
      • Barone K.
      • Uryu K.
      • Guo Y.
      • Rockman C.
      • Ramkhelawon B.
      • Berger J.S.
      Platelet-derived MRP-14 induces monocyte activation in patients with symptomatic peripheral artery disease.
      ]. Platelet CD62P and macrophage PSGL-1 expression can be further amplified by platelet-derived myeloid-related protein 14 (MRP-4), which is increased in platelets and plasma of PAD patients [
      • Dann R.
      • Hadi T.
      • Montenont E.
      • Boytard L.
      • Alebrahim D.
      • Feinstein J.
      • Allen N.
      • Simon R.
      • Barone K.
      • Uryu K.
      • Guo Y.
      • Rockman C.
      • Ramkhelawon B.
      • Berger J.S.
      Platelet-derived MRP-14 induces monocyte activation in patients with symptomatic peripheral artery disease.
      ].
      Platelet crosstalk with neutrophils and monocytes/macrophages augments the pro-inflammatory microenvironment in PAD lesions by fostering leukocyte activation, migration and release of inflammatory cytokines. Specifically, platelet-derived sCD62P was found to promote neutrophil adhesion and activation of Mac-1 [
      • Diacovo T.G.
      • Catalina M.D.
      • Siegelman M.H.
      • von Andrian U.H.
      Circulating activated platelets reconstitute lymphocyte homing and immunity in L-selectin-deficient mice.
      ], and platelet-derived MRP-14 entices macrophage migration and induction of interleukin-6 (IL-6), tumour necrosis factor α (TNF-α), and CCL2 (monocyte chemotactic protein 1, MCP-1) in vitro [
      • Dann R.
      • Hadi T.
      • Montenont E.
      • Boytard L.
      • Alebrahim D.
      • Feinstein J.
      • Allen N.
      • Simon R.
      • Barone K.
      • Uryu K.
      • Guo Y.
      • Rockman C.
      • Ramkhelawon B.
      • Berger J.S.
      Platelet-derived MRP-14 induces monocyte activation in patients with symptomatic peripheral artery disease.
      ]. Coincidentally, in critical limb ischaemia (CLI), a precariously advanced manifestation of PAD, upregulation of platelet CD62P expression is accompanied by increased PMA formation and plasma CCL2 [
      • Cleanthis M.
      • Bhattacharya V.
      • Smout J.
      • Ashour H.
      • Stansby G.
      Platelet monocyte aggregates and monocyte chemoattractant protein-1 are not inhibited by aspirin in critical limb ischaemia.
      ]. Additionally, elevated plasma sCD40L, which is primarily derived from platelets, contributes to immunomodulation in PAD patients with enlarged lesions, however, this does not coincide with increased CCL2 or C-reactive protein (CRP) [
      • Lee W.J.
      • Sheu W.H.
      • Chen Y.T.
      • Liu T.J.
      • Liang K.W.
      • Ting C.T.
      • Lee W.L.
      Circulating CD40 ligand is elevated only in patients with more advanced symptomatic peripheral arterial diseases.
      ]. Agonist-induced platelet CD62P expression also associates with increased cell-free DNA and citrullinated histone H3, pointing towards a strong link between platelet activation and NET formation in PAD [
      • Demyanets S.
      • Stojkovic S.
      • Mauracher L.M.
      • Kopp C.W.
      • Wojta J.
      • Thaler J.
      • Panzer S.
      • Gremmel T.
      Surrogate markers of neutrophil extracellular trap formation are associated with ischemic outcomes and platelet activation after peripheral angioplasty and stenting.
      ].
      Disease exacerbation of PAD is further associated with monocyte differentiation to CD16−expressing subsets. In CLI the fraction of intermediate (CD14++ CD16+) and non-classical (CD14+ CD16+) subsets as well as platelet aggregates with them are increased relative to both controls and patients with intermittent claudication [
      • Dopheide J.F.
      • Rubrech J.
      • Trumpp A.
      • Geissler P.
      • Zeller G.C.
      • Bock K.
      • Dunschede F.
      • Trinh T.T.
      • Dorweiler B.
      • Munzel T.
      • Radsak M.P.
      • Espinola-Klein C.
      Leukocyte-platelet aggregates-a phenotypic characterization of different stages of peripheral arterial disease.
      ], suggesting that monocyte differentiation is regulated by platelets in advanced PAD.
      Anti-platelet drugs and physical activity are commonly recommended to reduce risk for CVD. However, neither circulating PMAs nor IL-6 or CRP were reduced in PAD patients upon low-dose aspirin or by exercise training when provided additionally to best medical treatment [
      • Allen N.
      • Barrett T.J.
      • Guo Y.
      • Nardi M.
      • Ramkhelawon B.
      • Rockman C.B.
      • Hochman J.S.
      • Berger J.S.
      Circulating monocyte-platelet aggregates are a robust marker of platelet activity in cardiovascular disease.
      ,
      • Schlager O.
      • Hammer A.
      • Giurgea A.
      • Schuhfried O.
      • Fialka-Moser V.
      • Gschwandtner M.
      • Koppensteiner R.
      • Steiner S.
      Impact of exercise training on inflammation and platelet activation in patients with intermittent claudication.
      ]. In a large cohort study of more than 2000 PAD patients, increased PLR was significantly associated with risk for cardiovascular endpoints [
      • Gary T.
      • Pichler M.
      • Belaj K.
      • Hafner F.
      • Gerger A.
      • Froehlich H.
      • Eller P.
      • Rief P.
      • Hackl G.
      • Pilger E.
      • Brodmann M.
      Platelet-to-lymphocyte ratio: a novel marker for critical limb ischemia in peripheral arterial occlusive disease patients.
      ].

      5.3 Coronary artery disease (CAD)

      CAD is caused by vascular obstructive lesions leading to adverse cardiovascular events such as myocardial infarction, stroke and cardiovascular death. Typically, rupture or erosion of an atheroma causes local thrombus formation and thereby limits local blood supply to cardiomyocytes. Besides classical risk factors such as hyperlipidaemia, diabetes, and hypertension, the extent and severity of atherosclerosis, plaque vulnerability, and the likelihood of thrombus formation and propagation determine the probability of a cardiovascular event [
      • Fox K.A.A.
      • Metra M.
      • Morais J.
      • Atar D.
      The myth of 'stable' coronary artery disease.
      ]. Rupture-prone plaques are characterized by a large necrotic core and a thin fibrous cap accompanied by decreased numbers of smooth muscle cells.
      Platelet activation pivotally regulates CAD and PNAs and PMAs are elevated in patients with diagnosed CAD [
      • Nijm J.
      • Wikby A.
      • Tompa A.
      • Olsson A.G.
      • Jonasson L.
      Circulating levels of proinflammatory cytokines and neutrophil-platelet aggregates in patients with coronary artery disease.
      ,
      • Furman M.I.
      • Benoit S.E.
      • Barnard M.R.
      • Valeri C.R.
      • Borbone M.L.
      • Becker R.C.
      • Hechtman H.B.
      • Michelson A.D.
      Increased platelet reactivity and circulating monocyte-platelet aggregates in patients with stable coronary artery disease.
      ]. Accordingly, surface markers of platelet activation that can mediate direct cellular interactions increase with disease progression. During stable CAD surface CD62P is upregulated in peripheral venous blood [
      • Furman M.I.
      • Benoit S.E.
      • Barnard M.R.
      • Valeri C.R.
      • Borbone M.L.
      • Becker R.C.
      • Hechtman H.B.
      • Michelson A.D.
      Increased platelet reactivity and circulating monocyte-platelet aggregates in patients with stable coronary artery disease.
      ] and in coronary arteries [
      • Yong A.S.
      • Pennings G.J.
      • Chang M.
      • Hamzah A.
      • Chung T.
      • Qi M.
      • Brieger D.
      • Behnia M.
      • Krilis S.A.
      • Ng M.K.
      • Lowe H.C.
      • Kritharides L.
      Intracoronary shear-related up-regulation of platelet P-selectin and platelet-monocyte aggregation despite the use of aspirin and clopidogrel.
      ], along with platelet EMMPRIN (CD147) expression [
      • Yong A.
      • Pennings G.
      • Wong C.
      • Javadzadegan A.
      • Brieger D.
      • Lowe H.
      • Qi M.
      • Behnia M.
      • Krilis S.
      • Kritharides L.
      Intracoronary upregulation of platelet extracellular matrix metalloproteinase inducer (CD147) in coronary disease.
      ]. Analysis of monocyte subsets revealed that classical monocytes (CD14++ CD16) are increased in stable CAD while non-classical monocytes are decreased; however platelet binding is increased in all subsets [
      • Czepluch F.S.
      • Kuschicke H.
      • Dellas C.
      • Riggert J.
      • Hasenfuss G.
      • Schafer K.
      Increased proatherogenic monocyte-platelet cross-talk in monocyte subpopulations of patients with stable coronary artery disease.
      ]. These alterations are accompanied by an enhanced pro-inflammatory milieu as evidenced by augmented monocyte CD11b expression [
      • Yong A.S.
      • Pennings G.J.
      • Chang M.
      • Hamzah A.
      • Chung T.
      • Qi M.
      • Brieger D.
      • Behnia M.
      • Krilis S.A.
      • Ng M.K.
      • Lowe H.C.
      • Kritharides L.
      Intracoronary shear-related up-regulation of platelet P-selectin and platelet-monocyte aggregation despite the use of aspirin and clopidogrel.
      ] and heightened levels of IL-6 and CRP as well as the endogenous IL-1β inhibitor IL-1 receptor antagonist (IL-1RA) [
      • Nijm J.
      • Wikby A.
      • Tompa A.
      • Olsson A.G.
      • Jonasson L.
      Circulating levels of proinflammatory cytokines and neutrophil-platelet aggregates in patients with coronary artery disease.
      ], which suggests induction of anti-inflammatory mechanisms to limit excessive inflammation.
      Platelet CD40L expression was noted in acute coronary syndrome (ACS) and increased with lesion complexity and vessel occlusion [
      • Lee Y.
      • Lee W.H.
      • Lee S.C.
      • Ahn K.J.
      • Choi Y.H.
      • Park S.W.
      • Seo J.D.
      • Park J.E.
      CD40L activation in circulating platelets in patients with acute coronary syndrome.
      ]. Further, while CCL5 is not elevated in stable CAD [
      • Czepluch F.S.
      • Kuschicke H.
      • Dellas C.
      • Riggert J.
      • Hasenfuss G.
      • Schafer K.
      Increased proatherogenic monocyte-platelet cross-talk in monocyte subpopulations of patients with stable coronary artery disease.
      ], platelet-derived chemokines CCL5, CXCL4 and CXCL4L1 (PF4 variant 1, PF4V1) are increased in ACS [
      • Blanchet X.
      • Cesarek K.
      • Brandt J.
      • Herwald H.
      • Teupser D.
      • Kuchenhoff H.
      • Karshovska E.
      • Mause S.F.
      • Siess W.
      • Wasmuth H.
      • Soehnlein O.
      • Koenen R.R.
      • Weber C.
      • von Hundelshausen P.
      Inflammatory role and prognostic value of platelet chemokines in acute coronary syndrome.
      ]. In line with this, CXCL4-induced M4 macrophages accumulate in coronary plaques and their prevalence is associated with aggravated plaque instability [
      • Erbel C.
      • Wolf A.
      • Lasitschka F.
      • Linden F.
      • Domschke G.
      • Akhavanpoor M.
      • Doesch A.O.
      • Katus H.A.
      • Gleissner C.A.
      Prevalence of M4 macrophages within human coronary atherosclerotic plaques is associated with features of plaque instability.
      ].
      Moreover, numbers of circulating pro-inflammatory T helper lymphocytes are increased in patients with stable CAD and T lymphocytes comprise up to 20% of plaque cells [
      • Liuzzo G.
      • Kopecky S.L.
      • Frye R.L.
      • O'Fallon W.M.
      • Maseri A.
      • Goronzy J.J.
      • Weyand C.M.
      Perturbation of the T-cell repertoire in patients with unstable angina.
      ]. CD40L predominantly released from platelets is associated with cardiovascular events and could be the stimulus for CD40L-CD40 activation of T-cells [
      • Alber H.F.
      • Frick M.
      • Suessenbacher A.
      • Doerler J.
      • Schirmer M.
      • Stocker E.M.
      • Dichtl W.
      • Pachinger O.
      • Weidinger F.
      Effect of atorvastatin on circulating proinflammatory T- lymphocyte subsets and soluble CD40 ligand in patients with stable coronary artery disease-- a randomized, placebo-controlled study.
      ]. In patients with stable CAD the PLR was associated with severity of coronary atherosclerosis and inflammation [
      • Akboga M.K.
      • Canpolat U.
      • Yayla C.
      • Ozcan F.
      • Ozeke O.
      • Topaloglu S.
      • Aras D.
      Association of platelet to lymphocyte ratio with inflammation and severity of coronary atherosclerosis in patients with stable coronary artery disease.
      ], and a high PLR was shown to predict in-hospital and long-term mortality of patients with myocardial infarction or acute heart failure [
      • Azab B.
      • Shah N.
      • Akerman M.
      • McGinn Jr., J.T.
      Value of platelet/lymphocyte ratio as a predictor of all-cause mortality after non-ST-elevation myocardial infarction.
      ,
      • Ugur M.
      • Gul M.
      • Bozbay M.
      • Cicek G.
      • Uyarel H.
      • Koroglu B.
      • Uluganyan M.
      • Aslan S.
      • Tusun E.
      • Surgit O.
      • Akkaya E.
      • Eren M.
      The relationship between platelet to lymphocyte ratio and the clinical outcomes in ST elevation myocardial infarction underwent primary coronary intervention.
      ,
      • Ye G.L.
      • Chen Q.
      • Chen X.
      • Liu Y.Y.
      • Yin T.T.
      • Meng Q.H.
      • Liu Y.C.
      • Wei H.Q.
      • Zhou Q.H.
      The prognostic role of platelet-to-lymphocyte ratio in patients with acute heart failure: a cohort study.
      ]. The PLR takes two critical parameters into account: first, the association between high platelet counts and major adverse cardiovascular outcomes in patients with CAD [
      • Nikolsky E.
      • Grines C.L.
      • Cox D.A.
      • Garcia E.
      • Tcheng J.E.
      • Sadeghi M.
      • Mehran R.
      • Lansky A.J.
      • Na Y.
      • Stone G.W.
      Impact of baseline platelet count in patients undergoing primary percutaneous coronary intervention in acute myocardial infarction (from the CADILLAC trial).
      ] and second, low lymphocyte counts that have been associated with worse outcome in patients with CAD [
      • Zouridakis E.G.
      • Garcia-Moll X.
      • Kaski J.C.
      Usefulness of the blood lymphocyte count in predicting recurrent instability and death in patients with unstable angina pectoris.
      ,
      • Acanfora D.
      • Gheorghiade M.
      • Trojano L.
      • Furgi G.
      • Pasini E.
      • Picone C.
      • Papa A.
      • Iannuzzi G.L.
      • Bonow R.O.
      • Rengo F.
      Relative lymphocyte count: a prognostic indicator of mortality in elderly patients with congestive heart failure.
      ]. Both parameters are measures for inflammation as cytokines can decrease lymphocyte counts due to lymphocyte apoptosis on the one hand and increased platelet count due to promotion of megakaryopoiesis on the other hand.
      Additionally, increased PMAs and PNAs, but not platelet-lymphocyte aggregates were associated with development of the no-reflow phenomenon in patients with myocardial infarction who underwent percutaneous coronary intervention [
      • Ren F.
      • Mu N.
      • Zhang X.
      • Tan J.
      • Li L.
      • Zhang C.
      • Dong M.
      Increased platelet- leukocyte aggregates are associated with myocardial No-reflow in patients with ST elevation myocardial infarction.
      ]. Furthermore, circulating PMAs and plasma CRP levels correlate and are elevated in unstable CAD, especially in patients with heightened troponin levels [
      • Zhang S.Z.
      • Jin Y.P.
      • Qin G.M.
      • Wang J.H.
      Association of platelet-monocyte aggregates with platelet activation, systemic inflammation, and myocardial injury in patients with non-st elevation acute coronary syndromes.
      ], underlining the importance of platelet-leukocyte interactions for CAD progression and disease severity. Thus, anti-platelet therapy with clopidogrel and aspirin is one of the pillars in the treatment of patients with CAD and was shown to reduce the risk of recurrent adverse cardiovascular effects by 20% [
      • Rade J.J.
      Platelet function testing in patients with coronary artery disease: is the Who and the when any clearer than the what and the what then?.
      ]. Additionally, CAD therapeutics that do not primarily target platelets such as statins are able to reduce circulating PMAs and PNAs as well as myocardial damage, even though not all studies could demonstrates effects on platelet degranulation or aggregation [
      • Sexton T.
      • Wallace E.L.
      • Smyth S.S.
      Anti-thrombotic effects of statins in acute coronary syndromes: at the intersection of thrombosis, inflammation, and platelet- leukocyte interactions.
      ,
      • Sexton T.R.
      • Wallace E.L.
      • Macaulay T.E.
      • Charnigo R.J.
      • Evangelista V.
      • Campbell C.L.
      • Bailey A.L.
      • Smyth S.S.
      The effect of rosuvastatin on thromboinflammation in the setting of acute coronary syndrome.
      ,
      • Moscardo A.
      • Valles J.
      • Latorre A.
      • Madrid I.
      • Santos M.T.
      Reduction of platelet cytosolic phospholipase A2 activity by atorvastatin and simvastatin: biochemical regulatory mechanisms.
      ]. Similarly, application of β-blockers is also associated with decreased PLA formation [
      • Lee S.
      • Durstberger M.
      • Eichelberger B.
      • Kopp C.W.
      • Koppensteiner R.
      • Panzer S.
      • Gremmel T.
      beta-blockers are associated with decreased leucocyte-platelet aggregate formation and lower residual platelet reactivity to adenosine diphosphate after angioplasty and stenting.
      ]. These findings further underline that modulation of platelet-leukocyte interplay may have tremendous therapeutic potential in CVD.

      6. Platelet-leukocyte interplay in the acute phase of vascular diseases

      Progressive vascular dysfunction often culminates in acute thrombotic events which can be accompanied by embolisation and subsequent organ damage, e.g. upon acute ischaemic stroke or venous thromboembolism. Platelet-leukocyte crosstalk modulates primary thrombosis as well as acute inflammatory responses which favours secondary thrombosis at distant sites of embolisation. Effects of platelet-leukocyte interactions and involved molecular mediators are summarized in Fig. 3.

      6.1 Acute ischaemic stroke (AIS)

      Local thrombosis or translocation of an embolus to a cerebral artery or vein may cause vessel obstruction and acute ischaemia in the brain, resulting in hypoxia and tissue damage, which manifests as loss of neurological and motor function. Although stroke can also be of haemorrhagic aetiology, ischaemia accounts for up to 87% of stroke events [
      • Albertson M.
      • Sharma J.
      Stroke: current concepts.
      ]. However, cerebral hypoperfusion and subsequent ischaemia causes not only neuronal damage but also loss of integrity of the blood-brain barrier (BBB) due to dysfunction of the cerebral microvasculature, which may further exacerbate brain injury.
      Platelet-leukocyte interplay contributes to the different stages of ischaemic stroke by modulating the inflammatory response as well as vascular dysfunction. In particular, direct cellular interactions between platelets and innate leukocytes are implicated with increased circulating levels of PNAs and PMAs during the acute phase of stroke [
      • Ishikawa T.
      • Shimizu M.
      • Kohara S.
      • Takizawa S.
      • Kitagawa Y.
      • Takagi S.
      Appearance of WBC-platelet complex in acute ischemic stroke, predominantly in atherothrombotic infarction.
      ]. While PNAs are increased within the first three days after the ischaemic event, PMAs are mainly detectable at day two [
      • Marquardt L.
      • Anders C.
      • Buggle F.
      • Palm F.
      • Hellstern P.
      • Grau A.J.
      Leukocyte- platelet aggregates in acute and subacute ischemic stroke.
      ]. These data are corroborated by in vivo studies employing a rodent model of transient midline cerebral artery occlusion (MCAO) followed by a period of reperfusion that leads to formation of PLAs in the circulation [
      • Ritter L.S.
      • Stempel K.M.
      • Coull B.M.
      • McDonagh P.F.
      Leukocyte-platelet aggregates in rat peripheral blood after ischemic stroke and reperfusion.
      ] as well as endothelial rolling and adhesion of leukocytes and platelets [
      • Ishikawa M.
      • Cooper D.
      • Arumugam T.V.
      • Zhang J.H.
      • Nanda A.
      • Granger D.N.
      Platelet-leukocyte-endothelial cell interactions after middle cerebral artery occlusion and reperfusion.
      ]. Notably, neutrophil adhesion is required for subsequent platelet sequestration [
      • Ishikawa M.
      • Cooper D.
      • Arumugam T.V.
      • Zhang J.H.
      • Nanda A.
      • Granger D.N.
      Platelet-leukocyte-endothelial cell interactions after middle cerebral artery occlusion and reperfusion.
      ], indicating that platelet dysfunction is not the primary trigger of post-ischaemic vascular dysfunction.
      Platelet-leukocyte interplay is suspected to increase leukocyte activation and recruitment, thereby fostering inflammation and promoting tissue damage. Indeed, L-selectin shedding [
      • Htun P.
      • Fateh-Moghadam S.
      • Tomandl B.
      • Handschu R.
      • Klinger K.
      • Stellos K.
      • Garlichs C.
      • Daniel W.
      • Gawaz M.
      Course of platelet activation and platelet-leukocyte interaction in cerebrovascular ischemia.
      ] and plasma CCL2 levels are increased in patients upon acute stroke [
      • Garlichs C.D.
      • Kozina S.
      • Fateh-Moghadam S.
      • Handschu R.
      • Tomandl B.
      • Stumpf C.
      • Eskafi S.
      • Raaz D.
      • Schmeisser A.
      • Yilmaz A.
      • Ludwig J.
      • Neundorfer B.
      • Daniel W.G.
      Upregulation of CD40-CD40 ligand (CD154) in patients with acute cerebral ischemia.
      ] and PNA formation is associated with upregulation of neutrophil CD11b in murine MCAO [
      • Ritter L.S.
      • Stempel K.M.
      • Coull B.M.
      • McDonagh P.F.
      Leukocyte-platelet aggregates in rat peripheral blood after ischemic stroke and reperfusion.
      ].
      Platelet-leukocyte interactions via CD62P and CD40L-CD40, which are upregulated in platelets of patients suffering from acute stroke or transient ischaemic attack [
      • Lukasik M.
      • Dworacki G.
      • Michalak S.
      • Kufel-Grabowska J.
      • Watala C.
      • Kozubski W.
      Chronic hyper-reactivity of platelets resulting in enhanced monocyte recruitment in patients after ischaemic stroke.
      ], promote brain injury by mediating leukocyte recruitment, furthering BBB permeability and increasing infarct volume [
      • Ishikawa M.
      • Cooper D.
      • Arumugam T.V.
      • Zhang J.H.
      • Nanda A.
      • Granger D.N.
      Platelet-leukocyte-endothelial cell interactions after middle cerebral artery occlusion and reperfusion.
      ,
      • Ishikawa M.
      • Vowinkel T.
      • Stokes K.Y.
      • Arumugam T.V.
      • Yilmaz G.
      • Nanda A.
      • Granger D.N.
      CD40/CD40 ligand signaling in mouse cerebral microvasculature after focal ischemia/reperfusion.
      ]. Additionally, platelet integrins support PLA formation which can be abrogated by GPIIb/IIIa inhibition [
      • Ritter L.S.
      • Stempel K.M.
      • Coull B.M.
      • McDonagh P.F.
      Leukocyte-platelet aggregates in rat peripheral blood after ischemic stroke and reperfusion.
      ]. Blocking or inhibition of platelet adhesion receptor GPIb in mice diminishes neutrophil and macrophage infiltration into the brain as well as expression of pro-inflammatory cytokines, thereby reducing BBB dysfunction and infarct volume and ameliorating neurological deficits [
      • Schuhmann M.K.
      • Guthmann J.
      • Stoll G.
      • Nieswandt B.
      • Kraft P.
      • Kleinschnitz C.
      Blocking of platelet glycoprotein receptor Ib reduces "thrombo-inflammation" in mice with acute ischemic stroke.
      ,
      • Chen C.
      • Li T.
      • Zhao Y.
      • Qian Y.
      • Li X.
      • Dai X.
      • Huang D.
      • Pan T.
      • Zhou L.
      Platelet glycoprotein receptor Ib blockade ameliorates experimental cerebral ischemia-reperfusion injury by strengthening the blood-brain barrier function and anti-thrombo-inflammatory property.
      ].
      In addition to direct interactions, platelets may modulate leukocyte function by soluble mediators such as sCD62P and sCD40L which are also increased in patients upon acute stroke [
      • Lukasik M.
      • Dworacki G.
      • Michalak S.
      • Kufel-Grabowska J.
      • Watala C.
      • Kozubski W.
      Chronic hyper-reactivity of platelets resulting in enhanced monocyte recruitment in patients after ischaemic stroke.
      ]. Further, deficiency of CCL5 blunts leukocyte and platelet sequestration in mice and reduces plasma levels of IL-12, IL-6, and IL-10 as well as tissue infarction [
      • Terao S.
      • Yilmaz G.
      • Stokes K.Y.
      • Russell J.
      • Ishikawa M.
      • Kawase T.
      • Granger D.N.
      Blood cell-derived RANTES mediates cerebral microvascular dysfunction, inflammation, and tissue injury after focal ischemia-reperfusion.
      ]. Levels of CCL5 are increased in murine ischaemic brain and inhibition of CXCL4-CCL5 prevents cerebral infiltration of innate leukocytes and ameliorates disease severity [
      • Fan Y.
      • Xiong X.
      • Zhang Y.
      • Yan D.
      • Jian Z.
      • Xu B.
      • Zhao H.
      MKEY, a peptide inhibitor of CXCL4-CCL5 heterodimer formation, protects against stroke in mice.
      ].
      While the molecular interplay between platelets and leukocytes in stroke is only partially unveiled, platelet-leukocyte interactions clearly contribute to AIS progression and severity. Enhanced PMA formation is associated with worse clinical outcome after stroke and may be suitable as predictive marker [
      • Lukasik M.
      • Dworacki G.
      • Kufel-Grabowska J.
      • Watala C.
      • Kozubski W.
      Upregulation of CD40 ligand and enhanced monocyte-platelet aggregate formation are associated with worse clinical outcome after ischaemic stroke.
      ]. Accordingly, interference with platelet-leukocyte crosstalk is an attractive target for prevention of AIS. However, aspirin has failed to show clear effects on platelet-leukocyte interactions in stroke as one study reports reduced PMA formation upon treatment of patients with acute cerebral infarction [
      • Cao Y.J.
      • Wang Y.M.
      • Zhang J.
      • Zeng Y.J.
      • Liu C.F.
      The effects of antiplatelet agents on platelet-leukocyte aggregations in patients with acute cerebral infarction.
      ] while another study found reduced PMAs only in healthy controls treated with aspirin, but not in acute stroke patients [
      • Lukasik M.
      • Dworacki G.
      • Michalak S.
      • Kufel-Grabowska J.
      • Golanski J.
      • Watala C.
      • Kozubski W.
      Aspirin treatment influences platelet-related inflammatory biomarkers in healthy individuals but not in acute stroke patients.
      ]. Nevertheless, new generation anti-platelet drugs such as clopidogrel might prove to be more efficient at interfering with platelet-leukocyte binding than aspirin [
      • Cao Y.J.
      • Wang Y.M.
      • Zhang J.
      • Zeng Y.J.
      • Liu C.F.
      The effects of antiplatelet agents on platelet-leukocyte aggregations in patients with acute cerebral infarction.
      ].

      6.2 Venous thromboembolism (VTE)

      VTE is the third major cardiac disease and includes deep vein thrombosis (DVT) and pulmonary embolism (PE). In contrast to their arterial counterparts, venous thrombi usually form in deep veins of the legs and pelvis under low shear conditions via a complex interplay of platelets, innate immune cells, endothelial cells, and the subsequent activation of the plasmatic coagulation system, rendering them rich in fibrin and erythrocytes. The main causal factors of DVT are commonly summarized as alterations or stasis of blood flow, hypercoagulability and endothelial dysfunction, termed “Virchow's Triad” [
      • Koupenova M.
      • Kehrel B.E.
      • Corkrey H.A.
      • Freedman J.E.
      Thrombosis and platelets: an update.
      ].
      Prolonged periods of physical inactivity e.g. due to illness, long-distance flights, or pregnancy, favour stasis of blood flow, thereby creating local hypoxia especially in the valvular pockets. Together with accumulated ROS, hypoxia incites cell activation and expression of adhesion molecules on leukocytes, endothelial cells [
      • Budnik I.
      • Brill A.
      Immune factors in deep vein thrombosis initiation.
      ], and platelets [
      • Xu Y.
      • Ouyang X.
      • Yan L.
      • Zhang M.
      • Hu Z.
      • Gu J.
      • Fan X.
      • Zhang L.
      • Zhang J.
      • Xue S.
      • Chen G.
      • Su B.
      • Liu J.
      Sin1 (Stress-Activated protein kinase-interacting protein) regulates ischemia-induced microthrombosis through integrin alphaIIbbeta3-mediated outside-in signaling and hypoxia responses in platelets.
      ], and loosens tight-junctions of the endothelial layer, which allows for platelet penetration into the subendothelium [
      • Budnik I.
      • Brill A.
      Immune factors in deep vein thrombosis initiation.
      ]. Platelet recruitment is mainly dependent on GPIbα but also involves C-type lectin-like 2 (CLEC-2), as podoplanin expression (the only known ligand for CLEC-2) in the murine tunica media and adventitia is upregulated during thrombus formation [
      • Payne H.
      • Ponomaryov T.
      • Watson S.P.
      • Brill A.
      Mice with a deficiency in CLEC-2 are protected against deep vein thrombosis.
      ,
      • von Bruhl M.L.
      • Stark K.
      • Steinhart A.
      • Chandraratne S.
      • Konrad I.
      • Lorenz M.
      • Khandoga A.
      • Tirniceriu A.
      • Coletti R.
      • Kollnberger M.
      • Byrne R.A.
      • Laitinen I.
      • Walch A.
      • Brill A.
      • Pfeiler S.
      • Manukyan D.
      • Braun S.
      • Lange P.
      • Riegger J.
      • Ware J.
      • Eckart A.
      • Haidari S.
      • Rudelius M.
      • Schulz C.
      • Echtler K.
      • Brinkmann V.
      • Schwaiger M.
      • Preissner K.T.
      • Wagner D.D.
      • Mackman N.
      • Engelmann B.
      • Massberg S.
      Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.
      ].
      Platelet interaction with leukocytes is essential for development and progression of venous thrombosis. Patients with VTE or PE display increased platelet activation and circulating PLAs, as well as leukocyte activation and raised levels of inflammatory cytokines such as CCL5 and CCL2 [
      • Chirinos J.A.
      • Heresi G.A.
      • Velasquez H.
      • Jy W.
      • Jimenez J.J.
      • Ahn E.
      • Horstman L.L.
      • Soriano A.O.
      • Zambrano J.P.
      • Ahn Y.S.
      Elevation of endothelial microparticles, platelets, and leukocyte activation in patients with venous thromboembolism.
      ]. In a mouse model of inferior vena cava flow restriction, platelets adhered either to the intact endothelium or to adherent leukocytes in a GPIbα-dependent manner within 2 h, with as much as at least one platelet per adherent leukocyte [
      • von Bruhl M.L.
      • Stark K.
      • Steinhart A.
      • Chandraratne S.
      • Konrad I.
      • Lorenz M.
      • Khandoga A.
      • Tirniceriu A.
      • Coletti R.
      • Kollnberger M.
      • Byrne R.A.
      • Laitinen I.
      • Walch A.
      • Brill A.
      • Pfeiler S.
      • Manukyan D.
      • Braun S.
      • Lange P.
      • Riegger J.
      • Ware J.
      • Eckart A.
      • Haidari S.
      • Rudelius M.
      • Schulz C.
      • Echtler K.
      • Brinkmann V.
      • Schwaiger M.
      • Preissner K.T.
      • Wagner D.D.
      • Mackman N.
      • Engelmann B.
      • Massberg S.
      Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.
      ]. Interaction of platelet GPIb and CD62P with neutrophil CD18 and PSGL-1, respectively, promotes NET release [
      • Carestia A.
      • Kaufman T.
      • Rivadeneyra L.
      • Landoni V.I.
      • Pozner R.G.
      • Negrotto S.
      • D'Atri L.P.
      • Gomez R.M.
      • Schattner M.
      Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets.
      ] and thus a pro-thrombotic environment.
      Recently, platelet-derived high-mobility group box 1 (HMGB1) has been identified as a potential target for DVT therapy. Platelets are the main source of circulating HMGB1, which triggers recruitment of innate leukocytes, NET formation, and monocyte TF expression. Leukocyte-oxidized HMGB1 in turn promotes platelet activation and aggregation, thereby potentiating platelet CD62P expression, HMGB1 release, and amplifying NET formation and monocyte recruitment and activation [
      • Stark K.
      • Philippi V.
      • Stockhausen S.
      • Busse J.
      • Antonelli A.
      • Miller M.
      • Schubert I.
      • Hoseinpour P.
      • Chandraratne S.
      • von Bruhl M.L.
      • Gaertner F.
      • Lorenz M.
      • Agresti A.
      • Coletti R.
      • Antoine D.J.
      • Heermann R.
      • Jung K.
      • Reese S.
      • Laitinen I.
      • Schwaiger M.
      • Walch A.
      • Sperandio M.
      • Nawroth P.P.
      • Reinhardt C.
      • Jackel S.
      • Bianchi M.E.
      • Massberg S.
      Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice.
      ,
      • Dyer M.R.
      • Chen Q.
      • Haldeman S.
      • Yazdani H.
      • Hoffman R.
      • Loughran P.
      • Tsung A.
      • Zuckerbraun B.S.
      • Simmons R.L.
      • Neal M.D.
      Deep vein thrombosis in mice is regulated by platelet HMGB1 through release of neutrophil-extracellular traps and DNA.
      ].
      Anti-platelet therapy with aspirin protects from DVT following limb surgery [
      • Mistry D.A.
      • Chandratreya A.
      • Lee P.Y.F.
      A systematic review on the use of aspirin in the prevention of deep vein thrombosis in major elective lower limb orthopedic surgery: an update from the past 3 years.
      ] and high platelet counts are an independent risk predictor for VTE in cancer patients [
      • Simanek R.
      • Vormittag R.
      • Ay C.
      • Alguel G.
      • Dunkler D.
      • Schwarzinger I.
      • Steger G.
      • Jaeger U.
      • Zielinski C.
      • Pabinger I.
      High platelet count associated with venous thromboembolism in cancer patients: results from the Vienna Cancer and Thrombosis Study (CATS).
      ]. Moreover, interplay between platelets and immune cells was shown in idiopathic DVT where CX3CR1-expressing platelet-bound CD8+ lymphocytes were significant increased and served as prognostic marker for long-term adverse cardiovascular events [
      • Furio E.
      • Garcia-Fuster M.J.
      • Redon J.
      • Marques P.
      • Ortega R.
      • Sanz M.J.
      • Piqueras L.
      CX3CR1/CX3CL1 Axis mediates platelet-leukocyte adhesion to arterial endothelium in younger patients with a history of idiopathic deep vein thrombosis.
      ].
      Most preclinical data are generated from venous thrombosis models that (partially) ligate the inferior vena cava. They consistently show that especially the interplay of platelets and neutrophils with coagulation factor XII (FXII) is of critical importance; however, a different model that silences the endogenous anticoagulants anti-thrombin and protein C, indicates that TF and platelets are the rate-limiting factors, not FXII and neutrophils. Inhibition of TF significantly affected venous thrombosis, while FXII inhibition did not. Platelets are recruited early during disease progression and contribute to immune cell accumulation and stabilization. While monocytic TF induces initial fibrin formation, platelets mediate fibrin accumulation in more advanced stages. Nonetheless, this model breaks with several accepted assumptions by proposing a seemingly minor role of neutrophils and von Willebrand factor, which might be due to the lack of surgical injury and subsequent local inflammation [
      • Heestermans M.
      • Salloum-Asfar S.
      • Streef T.
      • Laghmani E.H.
      • Salvatori D.
      • Luken B.M.
      • Zeerleder S.S.
      • Spronk H.M.H.
      • Korporaal S.J.
      • Kirchhofer D.
      • Wagenaar G.T.M.
      • Versteeg H.H.
      • Reitsma P.H.
      • Renne T.
      • van Vlijmen B.J.M.
      Mouse venous thrombosis upon silencing of anticoagulants depends on tissue factor and platelets, not FXII or neutrophils.
      ].
      PE is the most common cause of pulmonary infarction and caused by blockage of pulmonary arteries due to blood clots traveling from deep veins. PE has a 2-fold increased mortality rate compared to DVT [
      • Saghazadeh A.
      • Rezaei N.
      Inflammation as a cause of venous thromboembolism.
      ] and can be caused by major surgery, trauma or periods of immobility. Moreover, it is assumed that inflammatory mediators contribute to its development, which is supported by increased frequency of PE in patients with chronic inflammatory disorders such as rheumatoid arthritis as well as by a correlation between PE and the inflammatory marker CRP [
      • Folsom A.R.
      • Lutsey P.L.
      • Astor B.C.
      • Cushman M.
      C-reactive protein and venous thromboembolism. A prospective investigation in the ARIC cohort.
      ]. Moreover, elevated PLR is associated with all-cause mortality in acute PE patients [
      • Phan T.
      • Brailovsky Y.
      • Fareed J.
      • Hoppensteadt D.
      • Iqbal O.
      • Darki A.
      Neutrophil-to- lymphocyte and platelet-to-lymphocyte ratios predict all-cause mortality in acute pulmonary embolism.
      ]. Murine models have tried to shed light on the complex role of platelet-leukocyte crosstalk in PE. However, while platelet-leukocyte interplay mainly appears to confer pro-inflammatory effects in the context of vascular dysfunction, platelet-derived amyloid precursor protein has been identified to limit PE secondary to DVT by downregulating NET formation and thrombus growth, whereas arterial thromboembolism was not affected [
      • Canobbio I.
      • Visconte C.
      • Momi S.
      • Guidetti G.F.
      • Zara M.
      • Canino J.
      • Falcinelli E.
      • Gresele P.
      • Torti M.
      Platelet amyloid precursor protein is a modulator of venous thromboembolism in mice.
      ].

      7. Summary and perspectives

      Taken together platelet-leukocyte interplay modulates the development and progression of various chronic as well as acute vascular diseases. While we increasingly learn more about the complex interplay of the haemostatic and the immune system and the role of platelet-leukocyte interplay therein, there is still much to be learned on the role of these interactions at the onset and progression of specific vascular diseases. Moreover, effects of anti-platelet and anti-inflammatory therapies on platelet-leukocyte interplay in this context are understudied. A clear understanding of the role of platelet-leukocyte interactions in vascular disease could provide a basis to adapt current patient therapy and help to prevent the fatal consequences of this disease.

      Financial Support

      This work was supported by grants from the Austrian Science Fund ( P32064 ) to A.A. and the Austrian National Bank ( OENB18450 ) to W.C.S. M.M. is supported by a Max-Kade Award.

      Declaration of competing interest

      The authors declare that they do not have anything to disclose regarding conflict of interest with respect to this manuscript.

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