bridging hemostasis, inflammation, and immunity - Wiley Online Library

International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology

REVIEW

INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Platelets: bridging hemostasis, inflammation, and immunity C. N. JENNE* , † , 1 , R. URRUTIA* , ‡ , 1 , P. KUBES* ,‡

*Calvin Phoebe & Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada † Department of Critical Care Medicine, University of Calgary, Calgary, AB, Canada ‡ Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada Correspondence: Dr Paul Kubes, Department of Physiology and Pharmacology, Calvin, Phoebe & Joan Snyder Institute for Chronic Diseases, University of Calgary, HRIC 4A26A, 3280 Hospital Drive N.W., Calgary, AB T2N 4N1, Canada. Tel.: +1 403 220 8558; Fax: +1 403 270 7516; E-mail: [email protected] 1

These authors contributed equally to this manuscript.

doi:10.1111/ijlh.12084

Received 1 January 2013; accepted for publication 6 February 2013

S U M M A RY

Although the function of platelets in the maintenance of hemostasis has been studied in great detail, more recent evidence has highlighted a central role for platelets in the host inflammatory and immune responses. Platelets by virtue of their large numbers and their ability to rapidly release a broad spectrum of immunomodulatory cytokines, chemokines, and other mediators act as circulating sentinels. Upon detection of a pathogen, platelets quickly activate and begin to drive the ensuing inflammatory response. Platelets have the ability to directly modulate the activity of neutrophils (phagocytosis, oxidative burst), endothelium (adhesion molecule and chemokine expression), and lymphocytes. Due to their diverse array of adhesion molecules and preformed chemokines, platelets are able to adhere to leukocytes and facilitate their recruitment to sites of tissue damage or infection. Furthermore, platelets directly participate in the capture and sequestration of pathogens within the vasculature. Platelet–neutrophil interactions are known to induce the release of neutrophil extracellular traps (NETs) in response to either bacterial or viral infection, and platelets have been shown to internalize pathogens, sequestering them in engulfment vacuoles. Finally, emerging data indicate that platelets also participate in the host immune response by directly killing infected cells. This review will highlight the central role platelets play in the initiation and modulation of the host inflammatory and immune responses.

Keywords Platelets, inflammation, neutrophil extracellular traps

HEMOCYTES Classically thought to be separate systems in higher organisms (vertebrates), in lower organisms, immunity and hemostasis are mediated by the same cell, the hemocyte. It is in these organisms that these 254

physiological systems first developed, and it is in these same organisms from which we can get a true sense of the overlap between hemostasis and immunity. Hemocytes are the primordial immune cell, possessing the ability to initiate both humoral (antimicrobial peptide) and cellular (phagocytosis) immune © 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2013, 35, 254–261

C. N. JENNE, R. URRUTIA AND P. KUBES | PLATELETS, INFLAMMATION AND IMMUNITY

responses. Hemocytes recognize foreign invaders through a number of pattern recognition and scavenger receptors [1, 2]. Upon activation, subsets of hemocytes rapidly degranulate, releasing antimicrobial peptides such as big defensin, tachyplesins, and antiLPS factor into the surrounding environment [3, 4]. These molecules bind to, sequester, and kill invading pathogens. Hemocytes are also efficient phagocytes that, upon binding to a foreign particle, rapidly internalize the target. Furthermore, upon activation, subsets of hemocytes aggregate to wall off the foreign entity and shield the host from the invader. In fact, some hemocytes have taken this strategy even further, producing compounds such as melanin to encapsulate and eventually kill the pathogen [3, 4]. In addition to these immune functions, hemocytes, through the release of numerous procoagulation factors such as factor C and factor G, also initiate and participate in the coagulation of hemolymph in response to tissue injury. These factors interact with soluble hemolymph proteins and, in the presence of Ca2+, initiate coagulation [1]. Coagulation is mediated by cross-linking of soluble proteins with receptors on the surface of hemocytes, generating large cellular aggregates. Interestingly, it is not only tissue damage that is able to initiate coagulation. The presence of a number of pathogen-associated molecules, such as LPS, is directly detected by soluble components of the hemolymph and is able to initiate coagulation [1, 4]. The resulting clots trap and kill bacteria, again reinforcing the physiological and functional overlap between hemostasis and immunity [1].

P L AT E L E T S In contrast to lower organisms, where hemocytes facilitate both immunity and hemostasis, in vertebrates, multiple cell types have evolved to perform these functions. Whereas the lion’s share of immunity falls in the domain of the leukocyte, platelets have evolved to be central mediators of hemostasis. Platelets are small, specialized, anucleated cytoplasmic bodies present in the circulation. These cell fragments are derived from megakaryocytes in the bone marrow and are essential for hemostasis and thrombosis. While it is tidy and convenient to separate immunity and hemostasis according to cell types, there is growing evidence this may not be entirely correct. Platelets express a © 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2013, 35, 254–261

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wide array of cell surface immune receptors and adhesion molecules and contain numerous granules loaded with a multitude of immune mediators. Because of these characteristics, and their high numbers in the circulation (150 – 400 9 109 platelets/L), platelets are thought to be circulating sentinels [5], able to activate, modulate, and participate in the host immune response. Moreover, the instantaneous presence of platelets at sites of injury and infection makes them, at the very least, guilty by association [6].

P L AT E L E T I M M U N E R E C E P TO R S Platelets express a number of innate immune receptors including an assortment of pattern recognition receptors (PRRs) [including Toll-like receptors (TLR1-9)], receptors for products of the humoral immune response (complement receptors), and receptors for immunoglobulins (FcR) [2, 7, 8]. Platelets have also been shown to express the coreceptors and signaling molecules associated with these PRRs [7]. Subsequent studies have demonstrated a number of these receptors are functional and, upon ligand binding, induce platelet activation [8], clearly illustrating the ability of platelets to detect, and respond to, pathogens.

P L AT E L E T- D E R I V E D I N F L A M M ATO RY A N D I M M U N E M E D I ATO R S Many of the functions of platelets result from the diverse array of preformed cell surface and soluble mediators contained with platelet granules. Platelets contain three types of granules: a-granules, dense bodies, and lysosomes [9]. Granule-stored mediators include coagulation and angiogenic factors, adhesion molecules, cytokines, and chemokines. A list of important platelet-derived inflammatory and immune mediators can be found in Table 1. Although preformed mediators allow for rapid release following platelet activation, it is important to note that platelets also posses the ability to synthesize additional mediators such as IL-1b [10]. a-Granules are the most abundant granules in platelets and are rapidly exocytosed upon activation to enhance hemostasis and inflammation. Many a-granule-derived mediators (i.e., P-selectin) promote interaction between platelets, leukocytes, plasma proteins, and the vessel wall [9], whereas others [platelet

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Table 1. Platelet granule contents, surface molecules, and platelet-derived mediators involved in inflammation and immunity a-Granules

Dense granules

Lysosome granules

Other soluble mediators Plasma membrane

Adhesive glycoproteins P-selectin, fibrinogen, vWF (vWF), fibronectin, thrombospondin Coagulation factors Factor V, protein S, factor XI, factor XIII Mitogenic factors PDGF, TGF-b, EGF Angiogenic factors VEGF, PF4 inhibitor Fibrinolytic inhibitors a2-plasmin inhibitor, PAI-1 Immunoglobulins Granule membrane-specific proteins P-selectin, CD63, GMP 33 Chemokines CXCL7, CXCL4 (PF4), CXCL1 (GROa), CXCL5, CCL5 (RANTES), CCL3 (MIP1a) Amines Serotonin (5-HT), histamine Bivalent cations Ca2+, Mg2+ Nucleotides ATP, ADP, GTP, GDP Acid proteases Carboxypeptidases (A, B), cathepsins D, E, acid phosphatase, collagenase Glycohydrolases Heparinase, b-N-acetyl-glucosaminidase, b-glucuronidase, b-glycerophosphatase, b-galactosidase, a-D-glucosidase, a-L-fucosidase, b-D-fucosidase Other molecules with immune functions: CCL7(MCP3), IL1b, HMGB1, Defensins, thromboxane A2, PAF, sCD40L TLR1, TLR2, TLR5, TLR4 TLR6, CD40, CD40L, TREM-1 ligand

VWF, von Willebrand factor; PDGF, platelet-derived growth factor; TGF-b, transforming growth factor b; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor; PAI-1, plasminogen activator inhibitor 1; PAF, platelet activating factor; TLR, Toll-like receptor.

factor 4 (PF4) and b-thromboglobulin-F (NAP2)] are able to activate and recruit cells to sites of inflammation. It is also important to note that a-granules take up proteins from the plasma, such as fibrinogen and immunoglobulins, serving as circulating reservoirs for these molecules, ready to rapidly release them at local sites following platelet activation.

In addition to a-granules, platelets also contain electron opaque structures called dense bodies (d-granules) [9]. These dense bodies contain adenine nucleotides (ADP and ATP) and serotonin, mediators that induce platelet aggregation, vasoconstriction, and pro-inflammatory cytokine production, potent modulators of inflammation. The third type of granule, lysosomes, contain glycosidases, proteases, and cationic proteins with bactericidal activity, such as b-glucuronidase, elastase, and collagenase [9]. These enzymes can aid in pathogen clearance, breakdown of extracellular matrix, and may contribute to the clearance of platelet thrombi and inactivation or degradation of heparin. Importantly, recent work has suggested that there are additional molecules, such as defensins, which are not located in any of the three aforementioned granules but rather exist as either cytosolic molecules or as constituents of an as yet unidentified granule [11].

P L AT E L E T A D H E S I O N M O L E C U L E S Platelets express numerous adhesion molecules and ligands that facilitate interactions between platelets, leukocytes, and endothelium. Platelets express large quantities of P-selection that, upon activation, is rapidly mobilized from a-granules to the platelet surface where it can mediate adhesion to cells expressing PSGL-1, primarily neutrophils, monocytes, and other leukocytes, but also endothelial cells and other platelets [2, 5]. This rapid P-selectin–PSGL-1 interaction results in tethering and rolling, slowing the platelet enough for firm adhesion to take place. Arguably the most important adhesion molecules found on platelets are integrins, heterodimeric transmembrane proteins that mediate interactions with extracellular matrix molecules and adhesion molecules on other cells. Integrins also play an important role in cell signaling and most require activation before they are able to bind with high affinity to their ligands. Platelets express a number of b1 and b3 integrins, including a5b1 (VLA-5), a6b1 (VLA-6), a2b1 (GPIa/IIa, VLA-2, or CD49b/ CD29), and GPIIb/IIIa (aIIbb3) [12, 13]. These molecules mediate platelet adhesion to ICAM and JAMs on leukocytes and endothelium and to extracellular matrix proteins, such as fibronectin, laminin, and collagen [12, 13]. © 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2013, 35, 254–261


In addition to selectins and integrins, platelets express other molecules that can facilitate their interaction with leukocytes and endothelial cells including a number of Ig superfamily cellular adhesion molecules such as the intercellular adhesion molecule-2 (ICAM-2), the junctional adhesion molecules (JAM-A, JAM-C), and the platelet endothelial cell adhesion molecule-1 (PECAM-1) [12]. These molecules interact with other cells through a mix of homophilic (PECAM-1, JAM-A) and heterophilic interactions (ICAM-2, JAM-A, JAM-C) whereby these adhesion molecules serve as ligands for integrins. Furthermore, platelets also express the glycoprotein (GP) Ib-V-IX complex that mediates initial platelet contact with exposed subendothelium through binding to von Willebrand factor (vWF), and GPVI, which mediates platelet binding to collagen [13]. This complex array of adhesion molecules and ligands allow platelets to bind to and to facilitate the binding of a number of diverse cellular and structural targets. It is also worth mentioning that a number of these molecules must perform their adhesive functions under shear conditions, a property not characteristic of most proteins.

P L AT E L E T S A S I M M U N E C E L L S Given the immense number of platelets within the circulation, their extensive battery of immune receptors, immunomodulatory mediators, and adhesion molecules, platelets have great potential to initiate, shape, and participate in the host inflammatory response. In fact, platelets have been reported to utilize a number of mechanisms to help drive inflammation and pathogen clearance (Figure 1), some of which will be discussed below. Substantial evidence exists to support this central role for platelets in innate immunity and inflammation. Clinical data indicate that many patients with severe bacterial or viral infections are thrombocytopenic, suggesting platelets are actively recruited to participate in the inflammatory response [14]. Furthermore, circulating platelet–leukocyte aggregates (PLAs) are a hallmark feature of sepsis, clearly illustrating the close interaction between platelets and the immune system during inflammatory responses [15]. PLAs are also associated with a number of other disease states including cardiovascular disease, inflammatory bowel © 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2013, 35, 254–261

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disease, and cystic fibrosis. The diagnostic assessment of PLAs using flow cytometry is a useful, and very sensitive measure of platelet activation status within a patient and, as such, can provide some insight into disease mechanism and progression [16].

M O D U L AT I O N O F I M M U N I T Y Platelets have been reported to directly modulate leukocyte activation. Examination of chemokineinduced platelet–neutrophil aggregates reveals an upregulation of the amb2 (Mac-1) integrin on the neutrophil following platelet binding. Additional studies have demonstrated neutrophil degranulation following association with LPS-activated platelets [17] and enhanced phagocytosis following neutrophil–platelet interactions [18]. The platelet is an active participant in these responses, and studies have demonstrated that the platelet itself must be activated to modulate the leukocyte response. Incubation of activated neutrophils with unactivated platelets or platelets unable to detect the pathogen (i.e., TLR2 deficient) fails to enhance the neutrophil activity [17, 18]. Furthermore, platelets have been identified as the single largest source of soluble CD40L (sCD40L), a molecule that has been shown to induce ROS production and upregulate adhesion molecule expression by neutrophils, activate macrophages, and induce optimal cytotoxic T cell and B cell activation in response to infection [19]. These immune-modulator functions put platelets in a key position as a central regulator of the host innate immunity.

CELLULAR RECRUITMENT The wide variety of expressed adhesion molecules, cytokines and chemokines, places the platelet in a pivotal role regarding leukocyte recruitment to sites of tissue damage and inflammation. Following vascular damage, platelets rapidly adhere to the exposed subendothelium, forming a thrombus and preventing bleeding. This interaction involves an initial tethering step mediated between the GP Ib-IX-V complex on platelets and immobilized vWF followed by GPVI-mediated firm adhesion to collagen [13]. Alternatively, platelets can directly adhere to the vascular endothelium. During endotoxemia, tethering and rolling of platelets are mediated by GPIba and

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Figure 1. Platelet-mediated inflammation and immunity. Platelets play a central role in driving and modulating the host inflammatory and immune responses. (a) Platelets are able to directly modulate the function of other cells such as endothelium, neutrophils, and lymphocytes. (b) Platelets facilitate the adhesion of neutrophils to the subendothelium, release chemokines and cytokines that attract and activate leukocytes, and induce the expression of adhesion molecules by the endothelium. (c) Platelets play an active role in pathogen capture and sequestration through the induction of neutrophil extracellular traps (NETs), encasing pathogens within platelet aggregates, and direct internalization of pathogens. (d) Through the release of soluble mediators, platelets can directly induce the killing of infected cellular targets.

P-selectin followed by integrin-mediated (aIIbb3) firm arrest of the platelet on the inflamed vascular endothelium [12, 13]. Once adherent, platelets rapidly express large quantities of P-selectin generating a surface on which neutrophils can tether and roll via their expression of PSGL-1 [12, 13]. This initial rolling is followed by firm adhesion of the neutrophil to adherent platelets via Mac-1 binding to fibrinogen bound to platelet GPIIbIIIa or to platelet GPIba [12, 13]. In this way, platelets can amplify neutrophil recruitment signals or can serve to recruit neutrophils to areas of the vasculature devoid of, or with low levels of, classic neutrophil adhesion molecules. This ability of platelets to mediate adhesion of neutrophils has been clearly demonstrated in a mouse model of autoimmune

encephalomyelitis in which blockade of platelet– endothelium interactions was able to dramatically reduce neutrophil adherence within the brain microvasculature [20]. In addition to serving as a platform to which leukocytes can adhere, platelets also have the capacity to directly modulate the expression and activation of adhesion molecules on other cell types. Platelet CD40L has been demonstrated to upregulate expression of E-selectin, ICAM-1, and VCAM-1 and the secretion of chemokines by endothelial cells [21]. Furthermore, platelets themselves release a number of cytokines and chemokines upon activation, mediators that modulate adhesion molecule expression on leukocytes and induce the generation of the highaffinity conformation of integrins. © 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2013, 35, 254–261


PAT H O G E N C A P T U R E / S E Q U E S T R AT I O N Platelets not only modulate the immune response but also participate in pathogen capture and sequestration. During endotoxemia, LPS-activated platelets bind to neutrophils and induce the production of neutrophil extracellular traps (NETs) [17]. These NETs are comprised of decondensed chromatin that has been released into the extracellular environment forming a sticky mesh, decorated with numerous antimicrobial granular and nuclear proteins [22]. Subsequent studies have confirmed this critical role for platelets in the generation of NETs using in vivo models of bacterial and viral infection [23, 24]. Functionally, it has been demonstrated that NETs can ensnare pathogens within the vasculature under flow conditions, and the presence of NETs within the vasculature limits dissemination of infectious agents [17, 22, 23]. Importantly, NET production is dependent on direct neutrophil–platelet interaction as inhibition of these interactions completely blocks NET release, illustrating a critical role for platelets in this novel mechanism of pathogen capture [23]. This association between leukocytes and platelets leading to the release of intravascular NETS provides new opportunities for the development of diagnostic assays in severe inflammatory disease. Circulating, cell-free DNA (host-derived presumably from both NETs and damaged tissue) has been associated with a number of inflammatory conditions and is currently being explored as a prognostic indicator for severe sepsis [25]. In addition to NET-mediated pathogen capture, platelets have been reported to bind and sequester bacteria directly. Recent in vitro studies of platelets have demonstrated that upon detection of clusters of certain bacteria, platelets rapidly bind to the pathogen and initiate aggregation [26]. This aggregation progresses until the bacteria are completely encased within the platelet aggregate, a situation that is reminiscent of the response of hemocytes to foreign particles [3, 4]. Studies have shown that encapsulation by platelets actively attenuates bacterial growth, possibly through the activity of platelet-derived mediators such as b-defensin [11]. Platelets are also known to directly internalize pathogens, binding, and internalizing both bacteria and viruses into discrete, membrane-lined engulfing vacuoles [27]. Although analogous to phagocytosis by © 2013 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2013, 35, 254–261

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neutrophils, platelet-mediated pathogen internalization occurs by a fundamentally different mechanism. Engulfment vacuoles fuse with a-granules [27]; however, platelets do not appear able to kill the internalized organisms, possibly due to the lack of sustained oxidative burst and the failure to lower the pH within the engulfment vacuoles.

D I R E C T PAT H O G E N K I L L I N G Platelets are not restricted to simply trapping or sequestering pathogens. Recent work on the causative parasite of malaria, Plasmodium falciparum, has identified a unique mechanism whereby platelets can contribute directly to pathogen killing. Upon detection of infection, activated platelets bind to infected red blood cells and induce directed a-granule release [28]. One of the soluble mediators released in this response is platelet factor 4 (PF4), a CXC-type chemokine that inhibits parasite growth and kills the pathogen [28]. Interestingly, PF4 has also been shown to bind to the Duffy-antigen receptor (Fy), resulting in the death of infected red blood cells [28]. Moreover, human platelets, upon activation, release b-defensins that have direct and potent antimicrobial effects on Staphylococcus aureus [11]. Furthermore, peptides derived from a number of other platelet mediators (NAP-2, CTAP-III, IL-8) have also been demonstrated to have antimicrobial activity [5].

C O L L AT E R A L DA M AG E Although platelets play an important role in modulating the inflammatory response, it is not yet clear whether this participation in immunity is entirely beneficial to the host. In models of highly pathogenic influenza, excessive inflammation in tissues such as the liver and lungs has been associated with increased cell death and fibrin deposition [29]. Moreover, numerous studies have suggested that ischemia caused by platelet–leukocyte aggregates, frequently associated with sepsis, within the microvasculature contributes to pronounced organ dysfunction and tissue damage [15, 30]. Furthermore, increasing evidence indicates that the excessive production of NETs and the deposition of their associated cytotoxic molecules on endothelium can lead to cellular death and tissue damage [17, 23]. Finally, studies showing a role for platelets in pathogenesis in models of multiple

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sclerosis, rheumatoid arthritis, and other inflammatory diseases are emerging [6, 20]. Given the critical role platelets play in driving and modulating inflammation and in the induction of NETs, it may be necessary to reexamine platelet activation in the context of inflammation-associated tissue damage.

CONCLUSION With their diverse array of immune receptors, adhesion molecules, and immune mediators, it is not entirely surprising that platelets play a critical role in inflamma-

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