http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, 2015; 26(4): 302–308 ! 2015 Informa UK Ltd. DOI: 10.3109/09537104.2015.1014471

REVIEW ARTICLE

Platelet interaction with bacterial toxins and secreted products Oonagh Shannon Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden

Abstract

Keywords

Bacteria that enter the bloodstream will encounter components of the cellular and soluble immune response. Platelets contribute to this response and have emerged as an important target for bacterial pathogens. Bacteria produce diverse extracellular proteins and toxins that have been reported to modulate platelet function. These interactions can result in complete or incomplete platelet activation or inhibition of platelet activation, depending on the bacteria and bacterial product. The nature of the platelet response may be highly relevant to disease pathogenesis.

Bacteria, LPS, platelets, sepsis, toxins

Introduction Bacterial infection and complications of infection remain a significant global health problem. The increasing incidence of antibiotic resistance among bacterial pathogens has intensified this problem. The pathogenesis of infection typically involves multifactorial host–bacteria interactions and improved understanding of these interactions is essential for the development of novel treatment strategies. Platelets have recently emerged as important mediators of the host response to infection [1, 2]. Platelets patrol the vasculature at levels ranging from 150 to 400  106 per ml of blood. As such, they represent important blood sentinel cells. The primary role of platelets is maintenance of haemostasis by adhesion and aggregation at sites of tissue injury and release of procoagulants. Platelets also participate in inflammation. Upon activation, platelets release their preformed granules, which contain diverse active substances, including proinflammatory molecules: chemokines, co-stimulatory molecules, and anti-microbial peptides [1]. Furthermore, activated platelets can bind to leukocytes and stimulate leukocyte function [3]. In response to activation, platelets can also synthesise proteins, including the pro-inflammatory cytokine IL-1beta [4]. The interaction between platelets and bacteria may have important consequences for the pathophysiological response to bacterial infection.

Platelet function during bacterial infection When bacteria gain access to the bloodstream, clinical infection may occur. For example, sepsis can occur when an initial controlled response to infection becomes augmented and deregulated. Sepsis is a multifactorial clinical syndrome that may escalate to septic shock. The complexity of this syndrome is illustrated by the findings that sepsis is not merely an overwhelming pro-inflammatory response to infection but patients also exhibit features of immunosuppression [5]. Leading causative Correspondence: Oonagh Shannon, Department of Clinical Sciences, Biomedical Centre (BMC), B14, Lund University, SE – 22184 Lund, Sweden. Tel: +46 46 2224488. Fax: +46 46 157756. E-mail: [email protected]

History Received 17 October 2014 Revised 6 December 2014 Accepted 28 January 2015 Published online 20 March 2015

agents of sepsis include Staphylococcus aureus, Streptococcus spp, and Escherichia coli [6]. Multiple host defence cells and mediators contribute to the pathogenesis of sepsis. Coagulation disorders are common and range from subclinical activation of coagulation to systemic coagulation activation, consumption of coagulation factors and platelets, inhibition of anti-coagulation systems, and associated bleeding risk [7]. The most severe manifestation of this coagulation dysfunction is disseminated intravascular coagulation, which has been reported to be an independent predictor of organ damage and mortality in sepsis [7]. The platelet response in sepsis has not been completely elucidated, however important evidence exists for platelets responding to the bacteria. Thrombocytopenia is a frequent occurrence in sepsis and is reported to be an independent marker of mortality in response to diverse pathogens [8–10]. A recent systematic review of the literature on thrombocytopenia in critically ill patients confirmed that thrombocytopenia is a risk factor for mortality and in particular sepsis and organ dysfunction were correlated to the level of thrombocytopenia [11]. Thrombocytopenia can arise due to decreased production or increased destruction of platelets. The coagulation system is activated and endothelial dysfunction occurs during sepsis [12]. Increased destruction of platelets may reflect platelet activation in vivo in response to procoagulant or proinflammatory mediators, and consumption of activated platelets on the endothelium, however a limited number of patient studies of platelet activation during sepsis have been performed. Platelet activation has been detected in patients suffering from sepsis and septic shock [13–16]. In particular, platelet–neutrophil complex (PNC) formation has been reported to be associated with organ dysfunction. PNCs are increased in the bloodstream at early stages but decrease when septic shock and multiple organ dysfunction occurs, suggesting that PNCs have migrated to the damaged organs [14, 17, 18]. Sepsis patients exhibit decreased ex vivo platelet aggregation and the level of the defect correlates with the severity of symptoms [19]. This ex vivo platelet defect may reflect a platelet population that have already been activated in vivo and respond weakly to further stimulation. Platelet activation could involve a direct platelet response to the bacteria [2] or bacterial products. Herein, we will discuss bacterial toxins

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Table I. Bacterial toxins or released products that modulate platelet function. Bacterial product

Bacteria

Platelet response

Lipolysaccharide (LPS)

Gram-negative bacteria

Lipoteichoic acid (LTA) a Toxin

Gram-positive bacteria Staphylococcus aureus

a Toxin

Escherichia coli

Streptolysin O (SLO) Phospholipase C (PLC) Shiga toxin Lethal toxin Pertusis toxin Staphylococcal superantigen-like protein 5 (SSL-5) Gingipains Streptococcal pyrogenic enterotoxin B (SpeB) Extracellular fibrinogen-binding protein (Efb) Extracellular adherence protein (Eap)

Streptococcus pyogenes Clostridium perfringens E. coli Bacillus anthracis Bordetella pertussis Staphylococcus aureus

Release of pro-inflammatory substances Induction of protein synthesis Formation of platelet–neutrophil complexes Inhibition of aggregation Release of procoagulant factors Stimulation of aggregation Induction of apoptosis Induction of protein synthesis Induction of apoptosis Induction of protein synthesis Formation of platelet–neutrophil complexes Formation of platelet–leukocyte complexes Stimulation of aggregation and adhesion to the endotheliuma Inhibition of aggregationa Stimulation of aggregation Stimulation of aggregation and adhesion to the endothelium

Porphyromonas gingivalis Streptococcus pyogenes

Cleavage and activation of PARs Cleavage of PARs

Staphylococcus aureus

Inhibition of aggregation

Staphylococcus aureus

M1 protein

Streptococcus pyogenes

Integrin activation Granule release Stimulation of aggregation Integrin activation Granule release Stimulation of aggregation

a

Conflicting reports exist that demonstrate no effect of the toxin on platelet function.

and other secreted bacterial products that target the platelet and modulate platelet function (summarized in Table I).

Platelet responses to endotoxin and bacterial PAMPS A fundamental component of the innate immune response to infection is the ability to recognise and directly respond to a pathogen. In order to achieve this, innate immune cells recognise conserved bacterial components belonging to the class of pathogen-associated molecular patterns (PAMPS). These PAMPs are recognised by pathogen recognition receptors (PRRs) on distinct human cells. The Toll-like receptor (TLR) family is the most well characterised [20]. PAMPS are associated with the bacterial cell wall, a complex polysaccharide structure that is essential to the integrity of the majority of bacterial pathogens. The Gram-negative cell wall consists of a double membrane structure with an outer membrane rich in lipopolysaccharide (LPS). LPS is lacking in the Gram-positive cell wall, which is characterised by a thick peptidoglycan layer containing lipoteichoic acid (LTA). These cell wall components are released on bacterial cell lysis and are detected by TLRs. There has been some inconsistency among reports on the presence of TLRs on platelets. Animal models of LPS toxicity demonstrate that platelets accumulate in the lungs and liver post LPS treatment and this contributes to the pathological response, [21–24]; however, these studies did not investigate the direct effect of LPS on platelets. Platelets were initially reported to lack the receptor for LPS, TLR4, and failed to respond to LPS [25]. TLR1 and TLR2 were detected at mRNA level, while other TLRs were not detected, however functional engagement of these TLRs with bacterial lipoproteins was not investigated [26]. TLR2 and TLR4 were detected at the surface of resting platelets and within intracellular stores [27]. Ward et al. confirmed that TLR2 and TLR4 were present on platelets, but treatment of platelets with

known ligands did not stimulate platelet activation or aggregation [28]. This led to speculation that platelets contained nonfunctional TLRs. In 2005, TLR4-dependent activation and adhesion of platelets in response to LPS was demonstrated for the first time. Importantly, the authors also demonstrated that platelet accumulation in the lungs of LPS-treated mice was dependent on platelet TLR4 [29]. This provided the first evidence of a link between direct activation of platelets by LPS and toxicity of LPS in an animal model. The presence of functional TLR4- and LPS-mediated activation of platelets was subsequently confirmed by other groups [30–32]. The discrepancies with regard to LPS stimulation of platelets may be explained by the findings that LPS does not generate conventional platelet activation, therefore the choice of platelet activation test is crucial when investigating the effects of LPS on platelet function. Studies that have confirmed activation in some platelet tests have also failed to demonstrate release of CD62P from the alpha granules or platelet aggregation [29, 33, 34], while others have reported release of only certain pro-inflammatory substances in response to LPS [34, 35, 36]. LPS can also initiate protein synthesis and cytokine production in platelets [37]. The differential activation of platelets after LPS engagement may reflect an immunomodulatory response as opposed to the prothrombotic response generated in response to classical platelet agonists. The signalling pathways downstream of platelet activation by LPS have not been clarified and this information will be essential to enhanced understanding of platelets as innate immune cells. Immune cells require CD14 to respond to LPS. Platelets do not express membrane CD14, however they can acquire sCD14 from plasma [37] and have been reported to have intracellular stores of CD14 [36]. Regardless of how the response is mediated, an important role for platelets in amplifying the immune response to LPS in vivo is emerging. Platelet depletion prior to administration of LPS results in diminished TNFa production, which is restored on platelet

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transfusion [30]. LPS-activated platelets have been shown to stimulate neutrophil activation and neutrophil extracellular trap (NET) formation [33], and this may be an important effector mechanism to remove bacteria from the bloodstream [38]. NET formation is an innate immune response to contain the infection, but the powerful activation of these granulated cells will also result in inadvertent tissue injury. Microvascular dysfunction is an important feature of LPS toxicity, and platelet TLR4 has been reported to contribute to vascular dysfunction in LPS-treated mice [39]. Platelet responses to Gram-positive cell wall components have not been extensively studied. There are studies that report engagement of platelet TLR2 with a synthetic ligand stimulates platelet activation and aggregation [40], while others have reported that the same synthetic ligand for TLR2 fails to mediate platelet activation [28]. Purified streptococcal LTA binds to platelets and decreases collagen-mediated platelet aggregation but not aggregation to other agonists [41]. Purified LTA from S. aureus has been demonstrated to decrease collagen-, ADP-, and thrombin-mediated platelet aggregation [42], as well as the platelet release reaction and platelet–monocyte complex formation [43]. Waller et al. recently confirmed that S. aureus LTA inhibits platelet aggregation in response to diverse agonists, including the bacteria itself [44]. The mechanism of action was dependent on PafR and increased generation of cAMP in LTAtreated platelets. Importantly, the authors also demonstrated that LTA administration resulted in defective platelet function in vivo and prolonged tail bleeding time in mice [44].

Bacterial exotoxins that modulate platelet function Bacteria produce and secrete diverse toxins that contribute to the pathogenesis of certain infections by inhibition or over stimulation of fundamental host functions. Important families of toxins include the pore-forming toxins, AB toxins, superantigens, and proteases. There are no reports of toxins that solely target platelet function during disease, but representative toxins within each family have been reported to target platelets, among other cells. Pore-forming toxins This family of toxins target the cell membrane and disrupt membrane function. The primary result is cell lysis and death. Staphylococcus aureus a-toxin is a pore-forming toxin with a broad range of tissue specificity. The toxin disrupts barrier function in the skin, lungs and vasculature, thereby contributing to the pathogenesis of diverse S. aureus infections [45]. As early as 1964 it was reported that a-toxin stimulated platelet aggregation [46]. Platelet activation in response to a-toxin led to enhanced platelet procoagulant response, in the absence of platelet lysis [47, 48]. Kraemer et al. have reported a direct toxic effect of S. aureus a-toxin and E. coli a-toxin on platelets by the induction of apoptosis [49]. Recent reports have confirmed that the treatment of platelets with a-toxin generates immediate responses in the form of platelet activation, aggregation and PNC formation, and also prolonged effects through initiation of protein synthesis [50–52]. The mechanism involved in generating platelet activation in response to this membrane-disrupting toxin has not been clarified. Pneumolysin is a pore-forming toxin and important virulence factor for Streptococcus pneumoniae. The toxin has been reported to mediate human platelet lysis [53]. Recently, Keane et al. reported that S. pneumonia bacteria stimulate platelet aggregation; however, this was not dependent on pneumolysin production by the bacteria [54]. Streptococcus pyogenes produce a pore-forming toxin, Streptolysin O (SLO) that can mediate platelet activation and CD62P-dependent PNC formation [55]. Importantly, the authors

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demonstrated that SLO decreases local blood flow in an animal model and speculated that PNCs induced by SLO occlude the vasculature. The same authors have also demonstrated that phospholipase C (PLC) produced by Clostridium perfringens stimulates platelet aggregation and platelet–leukocyte complex formation [56]. Furthermore, local administration of PLC decreased blood flow in an animal model and platelet–platelet and platelet–leukocyte aggregates were evident in tissue sections of occluded vessels from the animals [57]. The ability of both SLO and PLC to induce platelet-dependent vessel occlusion may be highly relevant to the pathogenesis of necrotising local tissue infection caused by the toxin-producing organisms. AB toxins This family of toxins are structurally related to one another. The toxins contain two domains, the A domain has toxic activity within the cell and the B domain binds to a host cell receptor to translocate the A domain into the cell [58]. Shiga toxin Shiga toxin (Stx)-producing E. coli can cause haemolytic uremic syndrome (HUS) in a susceptible host. Thrombocytopenia occurs during HUS and thrombi contribute to renal damage [59]. The contribution of Stx-mediated platelet activation to coagulation defects in HUS has been somewhat controversial. Some groups have reported binding and activation of Stx-treated platelets [60, 61], while others have failed to reproduce this [62–64]. These discrepancies may be explained by methodological differences between the studies, whereby determination of aggregation alone may not give a complete assessment of platelet function in response to the toxin and a panel of platelet activation tests should be utilised. Since Stx is an AB toxin, which can be internalised within the cell binding studies should determine both extracellular and intracellular Stx. Karpman et al. have demonstrated intracellular Stx in platelets [61]. Bacillus anthracis LT toxin Bacillus anthracis is the causative agent of anthrax, a toxinmediated disease with a high-mortality rate in a septic shock-like syndrome [65]. Haemostasis abnormalities and haemorrhage occur in animal models of anthrax, however only a limited number of studies have investigated platelet function and the results are not in agreement with one another. The purified LT toxin has been reported to block platelet aggregation and prolong the whole blood clotting time [66]; however, the same toxin has also been reported to have no effect on platelet activation [67]. Chauncey et al. also demonstrated that platelets lack the receptor for LT toxin and platelets failed to internalise the toxin [67]. These discrepancies remain to be clarified. Since anthrax is a clinical syndrome with an apparent haemostatic dysfunction, it would be very interesting to determine B. anthracis–platelet interactions at the molecular level. Pertussis toxin Bordetella pertussis produces pertussis toxin, a critical virulence factor for respiratory disease caused by this pathogen. The B subunit binds to a surface receptor on target cells and the A subunit is translocated into the cell where ADP-ribosylation of a target protein occurs and the toxic outcome depends on the cell targeted [68]. Pertusis toxin stimulates activation of platelets independently of ADP ribosylation [69], and the mechanism of activation is dependent on oligomerisation of receptors on the platelet surface by the B subunit of the toxin [70]. This is an extremely interesting mechanism of platelet activation, however

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evidence of a link to the pathogenesis of this local respiratory infection is lacking. Superantigens This family of toxins are mainly associated with Gram-positive pathogens and are considered to be critical virulence factors in the pathogenesis of sepsis. Superantigen binds to the MHC Class II receptor on antigen presenting cells and also to the T cell receptor on T helper cells. This triggers a non-specific activation of both cells and a massive pro-inflammatory immune response [71]. Interactions between classical superantigens and platelets have not been reported; however, the structurally related staphylococcal superantigen-like protein 5 (SSL5) can activate and aggregate washed platelets in a GPIb-dependent fashion, but in whole blood the effect is not as pronounced [72]. Proteases Many pathogenic bacteria elaborate proteases of broad or narrow specificity that contribute to multiple aspects of bacterial virulence. There is a paucity of information on the interactions between platelets and bacterial proteases, which is surprising when one considers that platelets express protease-activated receptors (PARs) that are critical for activation in response to thrombin. Porphyromonas gingivalis is a leading cause of adult periodontitis. Inflammation of the periodontal tissue can facilitate invasion of the bloodstream by bacteria [73]. Platelet activation occurs in patients with periodontitis and the level correlates with severity of disease [74, 75]. Proteases produced by the bacteria can stimulate platelet aggregation and granule release [76]. The proteases cleave PAR-1 and PAR-4 on the platelet surface resulting in autoactivation of the receptor [77, 78]. Streptococcus pyogenes produces a broad spectrum cysteine protease, SpeB that has recently been reported to cleave PAR1 and block subsequent thrombin-mediated platelet activation [79], however autoactivation of the receptor or direct platelet activation by SpeB was not observed. Secreted fibrinogen binding proteins Fibrinogen is an abundant plasma protein with important functions in coagulation and platelet aggregation. Staphylococcus aureus secretes three extracellular fibrinogen-binding proteins: coagulase, extracellular fibrinogen-binding protein (Efb) and extracellular adherence protein (Eap) [80]. Both Efb and Eap can interact with human platelets and generate opposing effects on platelet function. Efb inhibits platelet activation and aggregation [81, 82]. Efb exhibits powerful anti-platelet actions in vivo in experimental animal models [83]. Efb may contribute to wound healing defects during S. aureus wound infection and immunisation against Efb prior to infection improves wound healing in an experimental animal model [84]. Eap enhances thiol isomerase activity at the platelet surface resulting in GPIIb/IIIa integrin activation, granule secretion and aggregation [85]. Streptococcus pyogenes sepsis is associated with haemostasis dysfunction [86]. M protein is a cell wall-anchored protein of S. pyogenes that participates in multiple aspects of virulence [87]. The M protein of S. pyogenes emm1 serotype can also be released into the extracellular medium through the action of bacterial and host proteases [88, 89]. This soluble M1-protein forms a complex with plasma fibrinogen and this complex has been detected in abscess material from a patient suffering from S. pyogenes sepsis [90]. The M1 protein is a powerful platelet agonist. The M protein–fibrinogen complex binds to the surface of resting platelets and stimulates platelet activation [91, 92]. An interindividual variation in the ability of platelets to become activated

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in response to M protein has been observed and this is correlated to the level of anti-M protein IgG present in the individual’s plasma. This may represent an important strategy to cause thrombi at a distance from the bacteria and avoid entrapment of the bacteria in platelet aggregates. Furthermore, platelet thrombi may occlude the vessels and contribute to organ damage in sepsis.

Therapeutic targets based on platelet function It is apparent that bacterial pathogens have multiple mechanisms to modulate platelet function. This implies that the platelet is an important target for the bacteria. Interventions at the level of platelet function may therefore be beneficial during severe bacterial infection. Anti-platelet agents have improved the outcome in animal models of sepsis, and several restrospective clinical studies suggest that low-dose anti-platelet treatment may reduce the risk of multi-organ failure during sepsis, recently reviewed by Lo¨sche et al. [93] and Akinosoglou and Alexopoulos [94]. Sepsis is a complex clinical syndrome and interventions at the level of platelet function could also be associated with negative effects, such as increased bleeding risk. Anti-platelet therapy may only be relevant at a particular stage of sepsis or for sepsis caused by specific pathogens, therefore robust prospective clinical studies are required to assess this possibility. A deeper understanding of bacteria–platelet interactions may lead to the identification of alternative treatment strategies. The goal would be to identify a target that inhibits bacteria–platelet interactions, while maintaining normal platelet function and thereby avoiding the potential side effect of bleeding.

Declaration of interest The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article.

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