The dual role of platelet‐innate immune cell interactions in thrombo‐inflammation

Abstract Beyond their role in hemostasis and thrombosis, platelets are increasingly recognized as key regulators of the inflammatory response under sterile and infectious conditions. Both platelet receptors and secretion are critical for these functions and contribute to their interaction with the endothelium and innate immune system. Platelet‐leukocyte interactions are increased in thrombo‐inflammatory diseases and are sensitive biomarkers for platelet activation and targets for the development of new therapies. The crosstalk between platelets and innate immune cells promotes thrombosis, inflammation, and tissue damage. However, recent studies have shown that these interactions also regulate the resolution of inflammation, tissue repair, and wound healing. Many of the platelet and leukocyte receptors involved in these bidirectional interactions are not selective for a subset of immune cells. However, specific heterotypic interactions occur in different vascular beds and inflammatory conditions, raising the possibility of disease‐ and organ‐specific pathways of intervention. In this review, we highlight and discuss prominent and emerging interrelationships between platelets and innate immune cells and their dual role in the regulation of the inflammatory response in sterile and infectious thrombo‐inflammatory diseases. A better understanding of the functional relevance of these interactions in different vascular beds may provide opportunities for successful therapeutic interventions to regulate the development, progression, and chronicity of various pathological processes.


| INTRODUCTION
Platelets are small, anucleated cell fragments derived from megakaryocytes and known for their hemostatic functions at the site of vascular lesion. In the past 2 decades, multiple roles for platelets beyond hemostasis have been shown, including in vascular permeability, inflammation, infection, and tissue repair. [1][2][3][4][5] More recently, a new role for platelets in the maintenance of vascular integrity at the site of inflammation was described, a process currently termed inflammatory hemostasis. 3,[6][7][8][9][10] Both hemostatic and immunomodulatory functions of platelets are tightly regulated by environmental cues, in particular their interaction with the endothelium and innate immune system components.
The concept of thrombo-inflammation was originally introduced to describe the role of platelets in the inflammatory response following cerebral ischemia-reperfusion injury. 11 It is currently more broadly used to describe various diseases regulated by the crosstalk between thrombosis and inflammation, such as deep vein thrombosis, stroke and atherosclerosis, and infectious diseases, such as sepsis. The common feature in thrombo-inflammatory disease is interplay of the endothelium and immune and hemostatic systems.
In these diseases, inflammation triggers thrombosis, which in return fuels the inflammatory response. Increased platelet-leukocyte interactions on the inflamed endothelium, in thrombi, and in the blood are observed in thrombo-inflammatory experimental models and in patients with atherosclerosis, acute coronary syndrome, ischemic stroke, deep vein thrombosis (DVT), and sepsis. [12][13][14][15][16][17] The level of circulating platelet-leukocyte aggregates (PLAs) is suggested to be both a sensitive biomarker for platelet activation and a novel therapeutic target. For example, platelet activation and platelet-monocyte aggregates in acute coronary syndrome are used as an early hallmark for acute myocardial infarction. 12 Furthermore, platelet-monocyte aggregates after myocardial infarction are more sensitive markers of platelet activation than P-selectin. 13 However, it is unclear whether these interactions are a cause, an active participant, or merely an epiphenomenon of the inflammatory response. In this review, we focus on established and emerging interactions between platelets and innate immune cells and discuss the evidence that links these interactions to function in disease states in different vascular beds, in particular in the maintenance of vascular integrity and inflammation.

| PLATELETS SUPPORT CLASSICAL AND INFLAMMATORY HEMOSTASIS
At the site of vascular injury and under high shear conditions, platelets roll and adhere to the exposed extracellular matrix through glycoprotein Ib (GPIb)-von Willebrand factor (VWF) and glycoprotein VI (GPVI)-collagen interactions. Platelet activation leads to platelet integrin GPIIbIIIa activation, the release of granule contents including the feedback agonist ADP, and the formation of thromboxane A 2 (TxA 2 ), promoting recruitment of circulating platelets. While the formation of a platelet plug supports classical hemostasis, uncontrolled platelet activation leads to pathogenic thrombosis. Platelet granules are an important source of pro-and anti-inflammatory mediators, for example, tumor necrosis factor-α (TNF-α); transforming growth factor-β; chemokines, for example, RANTES, platelet factor 4 (PF4), and neutrophil activating peptide 2 (NAP-2); endothelial cell modulators, for example, sphingosine-1-phosphate, and serotonin; and growth factors, for example, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). 18 The translocation of other proteins including P-selectin and CD40-ligand (CD40-L) to the platelet surface favors their interactions with immune and endothelial cells promoting vascular permeability, thrombosis, and inflammation. [19][20][21] Platelets also contain mRNAs and pre-mRNAs that are translated upon platelet activation, with interleukin (IL)-1β being the most studied. 22 Platelet granule secretion occurs instantly following platelet activation, however, platelet IL-1β is released a few hours after stimulation.
At the site of inflammation, platelets play a dual role in the maintenance of vascular integrity and inflammation. 10 Platelet recruitment and activation increase vascular permeability, leukocyte recruitment, and edema through the release of chemokines and permeability factors with key roles for PF4, RANTES, VEGF, and serotonin. 10,20,23 However, platelets also maintain integrity of the endothelial cell layer at sites of endothelial damage inflicted by neutrophil transmigration, thus preventing inflammatory bleeding. 24 Inflammatory hemostasis is primarily GPIIbIIIa independent, although in certain inflammatory environments, such as ischemiareperfusion injury in the brain, GPIIbIIIa-dependent aggregation can be required. 3,10 Many mechanisms underlie the protective role of platelets in inflammatory hemostasis including physical sealing of the damaged endothelium and secretion of soluble factors that tighten endothelial junctions. 9 Interestingly, platelets adhere predominantly at the site of endothelial cell junctions during inflammation and guide neutrophils to their extravasation sites. 25 Therefore, the location of platelet adhesion might be critical for prevention of leakage through endothelial junctions and limiting inflammatory bleeding. 6,7,9 Unlike classical hemostasis, the roles of platelet receptors and secretion in inflammatory hemostasis depend on the vascular bed and the nature of the inflammatory stimulus. [7][8][9] For example, platelet secretion is required to secure the endothelial barrier in the ischemic brain but not in the inflamed skin or lung. 26 Similarly, platelet GPVI is required to maintain vascular integrity in the inflamed skin while GPIbα is required in the inflamed lung. [7][8][9] Whether the difference is due to the nature and intensity of the inflammatory stimulus, endothelial cell heterogeneity, and/or the microenvironment is not known.
Following injury, restoration of vascular integrity, apoptosis, and recruitment of progenitor, stromal and immune cells are crucial for tissue restructuring, remodeling, and functionality. Platelets are involved in many of the stages of wound healing, and this is emphasized by the beneficial use of platelet-rich plasma and platelet releasate in wound repair. 27,28 They promote progenitor cell recruitment; promote cytokine, chemokine, proangiogenic, and growth factor release; and support fibrin generation and modulate immune and stromal cell recruitment and activation. 29 Platelets can also promote the migration of leukocytes and stromal cells through the secretion of metalloproteinase (MMP) and stimulation of MMP secretion from leukocytes and endothelial cells, promoting tissue remodeling and repair. 28,30 In addition, we have recently shown that impaired vascular integrity due to loss of platelet function can also be beneficial in skin tissue repair. Deletion of platelet C-type lectin-like receptor-2 (CLEC-2) and GPVI impairs vascular integrity during skin wound healing in mice and results in bleeding in the tissue. 31 The leakage of blood cells and plasma in the tissue accelerates fibrin generation, inhibits inflammatory immune cell recruitment, and promotes angiogenesis. It is likely that the cause of bleeding and the related pathology define the beneficial potential of safeguarding vascular integrity.

| PLATELETS PROMOTE LEUKOCYTE RECRUITMENT IN INFLAMMATION
Platelets and leukocytes use a multistep pathway to accrue at the site of inflammation, culminating in integrin activation and firm adhesion, with leukocytes undergoing further migration into the tissue. The recruitment of leukocytes to the inflamed endothelium is mainly observed in the postcapillary venules, probably due to the increased density of cell adhesion molecules and wall shear rate, with a key role for platelets in leukocyte recruitment in the brain venules. 32 The interaction of platelets with leukocytes in the blood and at the site of activated and injured endothelium involves several receptor-ligand pairs including P-selectin-P-selectin glycoprotein ligand-1 (PSGL-1), GPIbα-macrophage 1 antigen (MAC-1, α M β 2 ), GPIIbIIIa-MAC-1 through fibrinogen, and CD40-CD40L. 21,[32][33][34] In the postcapillary venules, platelet P-selectin is required for platelet and leukocyte recruitment, as shown in a large number of inflammatory models. 20 The initial engagement of P-selectin-PSGL-1 induces rearrangement of the cytoskeleton of leukocytes and activation of β1-and β2-integrins, leading to firm adhesion [35][36][37] (Figure 1). Platelets can also interact indirectly with leukocytes through fibrinogen or VWF, and these interactions F I G U R E 1 Platelets promote leukocyte recruitment, adhesion, and transmigration at the site of inflammation while maintaining vascular integrity. Under inflammatory challenge, platelets are the first cellular blood component adhering on the inflamed endothelium in small postcapillary venules. Adherent platelets recruit neutrophils and guide them in their adhesion and transmigration through cellcell interactions or release of chemokines on the endothelium. Activated platelets and neutrophils cooperate to recruit inflammatory monocytes with specific inflammatory stimuli such as MCP-1 secretion required for efficient monocyte recruitment. At the site of leukocyte transmigration, platelets secure endothelial cell integrity to limit inflammatory bleeding. During inflammation, platelets promote the differentiation of monocytes into macrophages. Platelet receptors or releasate can shift macrophages towards a pro-or anti-inflammatory phenotype. CLEC-2, C-type lectin-like receptor-2; GPVI, glycoprotein VI; IL-1, interleukin-1; GPIb, glycoprotein Ib; HMGB-1, high-mobility group box 1; MAC-1, macrophage 1 antigen; NAP-2, neutrophil activating peptide 2; PAF, platelet-activating factor; PF4, platelet factor 4; PSGL-1, P-selectin glycoprotein ligand-1; ; TREM-1, triggering receptor-expressed myeloid cells 1 differentially regulate leukocyte recruitment and activation. 38,39 Under specific inflammatory challenges, such as ischemia and reperfusion injury, or injection of inflammatory molecules, such as angiotensin II, P-selectin, and β2-dependent leukocyte adhesion, is also observed in arterioles. 40 During acute inflammation, monocyte release from the bone marrow is delayed compared to neutrophils, but they persist for longer and can infiltrate tissues and differentiate into macrophages. 60 Neutrophils and monocytes share common phagocytic and inflammatory features; however, they differ in their chemotactic stimuli and receptors, release kinetics from the bone marrow, metabolic burst activity, and their interaction with the hemostatic system. 61 Similar to neutrophils, monocyte recruitment to the vessel wall and their interaction with platelets involves P-selectin, GPIb-α, CD40L, and GPIIbIIIa. 21 Platelets and neutrophils cooperate to promote subsequent stable inflammatory monocyte recruitment. 25  survival rate. 66 This shows that similar mechanisms differentially regulate thrombo-inflammation and disease-specific targeting is required for successful treatment. NETs release-induced thrombosis(DVT, sepsis) (127,147,148) NETs release promotes bacterial clearance (sepsis) (143) human monocyte activation and their inflammatory phenotype (61,91,92,102) Apoptosis-induced organ damage (Sepsis) (66) Apotosis-induced tissue repair and remodelling (stroke) (65)

95)
lipoxin A4, release of anti-inflammatory mediators:PGE2 (88,89,94) ↑ ↑ ↑ ↑ ↑ ↑ ↑ of inflammation through the secretion of prorepair molecules. The protective role of platelet-neutrophil interaction was shown in murine models of acute respiratory distress syndrome and postischemia reperfusion injury in the mesenteric artery through the release of proresolution mediators such as maresin-1 and lipoxin A4, respectively. 88,89 These studies show that platelets exert both protective and detrimental roles in thrombo-inflammation, and it is likely that the balance between proinflammation and proresolution functions is highly dependent on the stage and the nature of the disease as well as the microenvironment (Figure 3). During acute and chronic inflammation, platelets promote monocyte survival and differentiation into macrophages, partly through the secretion of chemokines such as CXCL-12 and PF4. 96,97 In human monocyte-derived macrophages, activated platelets promote LPS-mediated inflammatory cytokine release (high-dose LPS), which is largely dependent on platelet secretion. 98 Using mice bone marrow-derived macrophages in vitro, activated platelets induces IL-10 secretion from noninflamed macrophages and decreases TNF-α release. 94 At low doses of LPS, platelets promote TNF-α secretion from macrophages but inhibit macrophage-dependent inflammation at a high dose of LPS and during experimental bacterial peritonitis. 99

| THE RELEVANCE OF PLATELET-LEUKOCYTE INTERACTIONS IN THROMBO-INFLAMMATORY DISEASES
The contribution of platelet-leukocyte interactions to thrombo-inflammation has been extensively studied in the last decade with recognition that the underlying mechanisms are tissue/organ specific.
Below, we illustrate some known roles of these interactions in different vascular beds and in response to different insults, and discuss the involvement of both common and disease-specific pathways in regulation of thrombo-inflammation.

| ATHEROSCLEROSIS
Atherosclerosis is a thrombo-inflammatory disorder involving inflammatory and immune responses to oxidized lipids, endothelial dysfunction, and the formation of an atherosclerotic plaque. At the site of atherosclerosis, leukocytes and platelets accumulate and promote plaque growth and progression and eventually destabilize the endothelial layer leading to plaque rupture. [105][106][107][108] Platelet and leukocyte recruitment promote atherosclerosis as depletion of platelets, neutrophils or monocytes reduces plaque size. [106][107][108] In severe atherosclerosis, platelet recruitment and adhesion preceded the development of atherosclerotic lesions followed by leukocyte recruitment to the arterial vasculature. 106 On the intact plaque, platelets are recruited through GPVI-laminin interaction promoting atheroprogression. 109 At the site of fissured lesions, plaque rupture triggers platelet recruitment through GPVI-collagen interaction. Inhibition of GPVI extracellular domain or downstream signaling inhibits thrombus formation on atherosclerotic plaque in vitro. 110,111 Platelet activation significantly contributes to the pathogen- DVT predominantly by providing tissue factor, which triggers blood coagulation, neutrophils promote thrombosis by releasing NETs. 127 The factor triggering NETosis in the sterile environment inside the blood vessel has been proposed to be platelet-derived HMGB-1. The effect of HMGB-1 is potentiated via the P-selectin-PSGL-1 axis, 128 with P-selectin-deficient mice being protected against DVT. 46,129,130 In addition to promoting NETosis, platelet-originated HMGB-1 also increases neutrophil and monocyte sequestration at the venous wall, increasing local inflammation. More recently, complement activation was shown to regulate the development of DVT in mice, with complement components displaying distinct roles in thrombus formation. C3 activation leads to platelet and fibrin deposition, whereas C5 increases tissue factor expression on monocytes and precipitates fibrin generation, independently of platelets, promoting thrombo-inflammation. 131 An increase in PLAs is observed in patients with venous thrombosis. 16 In particular, the increase in circulating platelet-neutrophil aggregates has been shown to correlate with platelet activation in individuals with DVT, 132 with the ROC revealing that the level of platelet-neutrophil aggregates represents a risk factor for venous thrombosis. Platelet-monocyte aggregates are also increased after surgery in patients with venous thromboembolism (VTE), and their count correlates with plasma levels of an important systemic proinflammatory marker, C-reactive protein. 133 Anticoagulants are the common mainstay of treatment of DVT, although thrombolysis, mechanical thrombectomy, and angioplasty are also used. Antiplatelet drugs, in particular aspirin, were shown to reduce the risk of primary thromboembolism and the recurrence of secondary VTE when used as a long-term secondary preventive strategy in patients with VTE following an initial anticoagulant treatment. [134][135][136] Thus, although the activation of the coagulation system drives venous thrombosis, the interaction of platelets with innate immune cells, both in the flowing blood and at the vessel wall, directly contribute to the initiation and progression of DVT and the associated inflammatory response.

| SEPSIS
Platelets express a wide range of complement receptors, pattern recognition receptors, in particular toll-like receptors and Fc receptors, providing the ability to sense and respond to endogenous and exogenous inflammatory and infectious signals and initiate an immune response. 137 Following activation, platelets secrete a large array of antimicrobial and immunomodulatory molecules that can directly kill pathogens and/or enhance immune cell differentiation and activation. Platelet depletion and thrombocytopenia are associated with worse outcome, suggesting their protective role in sepsis. 85,138 In mouse models of bacterial peritonitis, early platelet transfusion is protective through the regulation of macrophage activation. 99,101 Platelet transfusion attenuates thrombocytopenia, decreases plasma levels of inflammatory cytokines such as TNF-α and IL-6, and improves survival. 99 Platelet transfusion was also shown to increase inflammatory macrophage recruitment to the infected peritoneum and improve bacterial clearance through GPIb-CD11b interaction. 101 It is possible that platelet transfusion dampens the systemic inflammatory response partly through sequestration of cytokines released from activated immune cells and the regulation of immune cell activation. 83,85,86,101 During sepsis, platelets contribute to leukocyte recruitment and activation through both direct interaction and via secretion supporting immune cell recruitment and pathogen clearance, with an organ and pathogen role for platelet receptors and secretion. 139,140 Deficiency in these interactions reduces PLAs in bacterial sepsis and increases bacterial growth in an organ-and insult-dependent manner. 85,87,139,141,142 Interestingly, the protective role of platelet receptors depends on the pathogen and the site of infection. Following gram-negative bacterial infection in mice, LPS binding to TLR4 on platelets induces NET release from neutrophils and sequesters bacteria within the vasculature, in particular in pulmonary capillaries and liver sinusoid. 143 NET release is also induced by P-selectin, 128 , platelet release of HMGB-1, 144 β1-defensins, 145 and other interactions. Moreover, LPS binding to TLR4 on platelets induce the shedding of IL-1β rich microparticles increasing endothelial cell activation and propagating the inflammatory response. 146 Platelets and NETs also induce disseminated intravascular coagulation and alter organ functions. 147 Targeting NETs in sepsis must be finely tuned to limit organ damage but contain bacterial growth and spreading. This is evidenced by the beneficial role of the delayed injection of DNase in cecal ligation and puncture models compared to the detrimental effect observed at an earlier time in sepsis. 148 The liver has specialized macrophages, known as Kupffer cells, that line the walls of the sinusoids and are thus constantly exposed to the blood flow. Under physiological conditions, platelets transiently interact with intravascular Kupffer cells via GPIbα-VWF as part of the innate immune surveillance system of the liver. 149 The interaction of platelets with Kupffer cells is stabilized in the presence of bacteria such as Bacillus cereus and methicillin-resis-  84 These thrombi did not contain Salmonella and peaked when bacteria in the blood and tissues were declining. Deletion of platelet CLEC-2 abolished liver thrombosis without altering bacterial count. 142 In contrast, the CLEC-2-podoplanin axis inhibits inflammation in mouse models of LPS-induced acute respiratory distress syndrome and cecal ligation and puncture without altering thrombosis. 83,85,86 The inhibition of CLEC-2-podoplanin interaction exacerbates the cytokine storm and impairs macrophages recruitment to the infected peritoneum increasing bacterial load. Therefore, the nature and receptors involved in PLAs formation in sepsis depends on the organ involved, the time course of the infection, and the insult.
PLAs are also increased in septic patients and exhibit a reciprocal relationship to survival in patients developing multiple organ failure, probably due to an increase in sequestration. 151 Indeed, platelet-neutrophil aggregates are elevated during the early phases of sepsis but significantly decrease with sepsis progression. 151 An increase in platelet-monocyte aggregates is associated with increased mortality in older septic patients but not in younger patients. 152 Therefore, targeting platelet-immune cell interaction in sepsis plays either beneficial or detrimental roles according to the model, disease stage, and associated comorbidities. Similarly, the role of PLAs depends on the stage of the infection, the immune cells involved, and their contribution to pathogen clearance, and organ damage.

| CONCLUSION
There is compelling evidences for a functional relevance of platelet-leukocyte interactions in thrombo-inflammatory diseases.
Many antiplatelet drugs or inhibitors targeting selective interactions between platelets and leukocytes were shown to modulate clinical outcome in experimental models. The protective or deleterious effect of these interactions on the clinical outcome largely depends on the pathophysiological context and disease stage. 21