Platelets and the innate immune system: mechanisms of bacterial-induced platelet activation

Authors


Dermot Cox, Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin, Ireland.
Tel.: +353 1 4022152; fax: +353 1 4022453.
E-mail: dcox@rcsi.ie

Abstract

Summary.  It has become clear that platelets are not simply cell fragments that plug the leak in a damaged blood vessel; they are, in fact, also key components in the innate immune system, which is supported by the presence of Toll-like receptors (TLRs) on platelets. As the cells that respond first to a site of injury, they are well placed to direct the immune response to deal with any resulting exposure to pathogens. The response is triggered by bacteria binding to platelets, which usually triggers platelet activation and the secretion of antimicrobial peptides. The main platelet receptors that mediate these interactions are glycoprotein (GP)IIb–IIIa, GPIbα, FcγRIIa, complement receptors, and TLRs. This process may involve direct interactions between bacterial proteins and the receptors, or can be mediated by plasma proteins such as fibrinogen, von Willebrand factor, complement, and IgG. Here, we review the variety of interactions between platelets and bacteria, and look at the potential for inhibiting these interactions in diseases such as infective endocarditis and sepsis.

Introduction

Hemostasis is a critical process that acts to seal breaches in the vascular system. This serves two functions: prevention of further blood loss, and denial of access for pathogens to the vascular system. Platelets are key mediators of this response, and act to stop the leak and facilitate wound healing. In addition, platelets play a key role in preventing infection. When activated, platelets secrete the contents of their granules, which are known to contain over 300 proteins [1] as well as bioactive molecules such as ADP and serotonin. ADP acts to recruit more platelets into the growing thrombus, while serotonin causes vasoconstriction to reduce blood loss. Secreted cytokines and chemokines recruit leukocytes to deal with any potential infection, and secreted antimicrobial peptides act to kill pathogens.

Although thrombus formation at the site of a wound prevents blood loss, it can also occur in a diseased vessel, such as a coronary or cerebral artery, causing a potentially fatal myocardial infarction (MI) or stroke. Equally, activation of platelets by pathogens at locations other than a wound can lead to serious consequences, such as infective endocarditis (IE) or disseminated intravascular coagulation (DIC). However, although therapies have been developed to prevent thrombosis in stroke and MI, it is essential to develop therapies to prevent pathogen-induced platelet activation, which will, in all probability, be different from existing antiplatelet agents.

The activation of platelets leads to secretion of antimicrobial peptides, although many bacteria have become resistant to these peptides [2]. Bacteria have also developed the ability to interact with platelets without inducing platelet activation, allowing them to adhere to surfaces coated with platelets, such as a damaged cardiac valve. This ability to bind to platelets without activating them or to be resistant to their antimicrobial actions enables bacteria to survive in the circulation, either surrounded by or phagocytosed by platelets, and invisible to leukocytes.

Infection and thrombosis

As platelets are usually the first cells to respond to a wound, they have an important role in regulating the host response to infection, which takes place through platelet activation by bacteria [3,4]. However, this process can contribute to diseases such as IE, a serious infection of the heart valves that is usually caused by infection with either staphylococci (e.g. Staphylococcus aureus) or streptococci (e.g. Streptococcus sanguinis or Streptococcus gordonii). IE results from the formation of a bacteria–platelet thrombus on one of the heart valves, which, as it grows, leads either to valve failure requiring valve replacement or to the formation of a septic embolus, which can cause a stroke, heart attack, or pulmonary embolism. The major risk factors for IE are dental disease or manipulation and intravenous drug abuse, which allow entry of oral streptococci and S. aureus, respectively, into the bloodstream [5].

Another thrombotic disease associated with infection is septicemia [6]. Patients with septicemia typically develop DIC characterized by microthrombus formation, which can lead to blockage of the microvasculature and organ damage. Thrombus formation can also lead to consumption of coagulation factors and platelets, placing the patient at risk of a bleeding event [7]. Thrombocytopenia resulting from platelet activation in sepsis is a common occurrence, and its extent is related to outcome [8].

The studies of bacterial interactions with platelets have primarily been confined to Gram-positive bacteria, especially staphylococci (S. aureus and Staphylococcus epidermidis) and streptococci (S. sanguinis and S. gordonii), although the interaction with the Gram-negative Helicobacter pylori has also been characterized [4].

Mechanisms of interaction

There are three basic mechanisms for mediation of the interaction between pathogens and platelets: (i) binding to bacteria of a plasma protein that is a ligand for a platelet receptor; (ii) direct bacterial binding to a platelet receptor; and (iii) secretion of bacterial products, i.e. toxins, that interact with platelets. The presence of multiple mechanisms makes it difficult to identify the roles of the different proteins (both bacterial and platelet), and this is further complicated by interactions that are not only species-specific but also strain-specific. Some interactions lead to platelet activation, whereas others have no effect on the platelet. These non-activating interactions are usually of high affinity, and probably play a role in supporting platelet adhesion under the shear conditions found in the circulation. Typically, bacterial proteins that mediate adhesion are distinct from those that mediate aggregation. Thus, bacteria can support platelet adhesion and/or trigger platelet activation (see Table 1).

Table 1.   Platelet–bacteria interactions
Platelet receptorBacteriaBacterial proteinBridging protein
  1. ClfA, clumping factor A; Fnbp, fibronectin-binding protein; GspB, glycosylated streptococcal protein B; Hsa, hemagglutinin salivary antigen; IsdB, iron-regulated surface determinant B; LPS, lipopolysaccharide; PadA, platelet adhesion binding protein A; SdrG, serine–aspartate repeat G; SrpA, serine-rich protein A; TLR, Toll-like receptor; VWF, von Willebrand factor.

GPIIb–IIIaS. epidermidisSdrGFibrinogen
S. aureusFnbpA/BFibronectin
S. aureusFnbpA/BFibrinogen
S. aureusClfAFibronectin
S. aureusClfAFibrinogen
S. aureusIsdBDirect
S. pyogenesM1Fibrinogen
S. gordoniiPadADirect
S. lugdunensisFblFibrinogen
GPIbαS. sanguisSrpADirect
S. gordoniiGspB/HsaDirect
S. aureusProtein AVWF
Helicobacter pylori?VWF
FcγRIIaS. aureusFnbpA/BIgG
S. aureusClfAIgG
TLR2S. pneumoniae?Direct
?LipoproteinDirect
TLR4Escherichia coliLPSDirect
gC1q-RS. sanguinis?C1

Bacterial-induced platelet aggregation is often uniquely characterized by a distinct delay known as the lag time (Fig. 1). When a soluble agonist such as ADP is added to a platelet suspension, the aggregation response occurs within a few seconds. When bacteria are added to a suspension of platelets, there is a delay in the aggregation response that is concentration-dependent. Increasing the bacterial concentration shortens this lag time but never abolishes it. Bacteria such as S. aureus induce platelet aggregation with a lag time of approximately 2–3 min. However, a complement-dependent strain such as S. sanguinis NCTC 7863 usually takes between 10 and 15 min to induce aggregation. Unlike that caused by soluble agonists, bacterial-induced platelet aggregation is an all-or-nothing process.

Figure 1.

 Lag time to platelet aggregation. ADP induced platelet aggregation with a lag time of 15 s, whereas Streptococcus sanguinis induced platelet aggregation with a lag time of 4 min. Lag time is defined as the time from addition of agonist or bacteria to the first signs of platelet aggregation.

Platelet receptors

Although bacteria utilize many different proteins to interact with platelets, there are a limited number of platelet receptors that mediate adhesion and/or activation, notably glycoprotein (GP)IIb–IIIa, GPIb, and FcγRIIa (Figs 2 and 3). This limited number of platelet receptors makes it possible to develop antiplatelet agents that target a wide range of bacteria–platelet interactions.

Figure 2.

 Summary of indirect interactions between bacteria and platelets. Different species of bacteria bind different plasma proteins that act as bridges to their respective platelet receptors, thus triggering activation. ClfA, clumping factor A; C1q, complement 1q; FnbpA, fibronectin-binding protein A; GP, glycoprotein; PA, protein A; VWF, von Willebrand factor. Note that protein A does not require antibody, whereas Helicobacter pylori does.

Figure 3.

 Summary of direct interactions between bacteria and platelets. Different species of bacteria contain ligand mimetic motifs that act as agonists on platelet receptors. GP, glycoprotein; GspB, glycosylated streptococcal protein B; Hsa, hemagglutinin salivary antigen; IsdB, iron-regulated surface determinant B; LPS, lipopolysaccharide; PadA, platelet adhesion protein A; SdrG, serine–aspartate repeat G; SrpA, serine-rich protein A; TLR, Toll-like receptor.

Table 2.   Platelet–toxin interactions
Platelet receptorBacteriaBacterial toxin
  1. GP, glycoprotein; PAR1, protease-activated receptor 1; SSL, staphylo-coccal superantigen-like.

PAR1Porphyromonas gingivalisGingipains
GPIbαS. aureusSSL-5
GPVIS. aureusSSL-5
PhospholipidsS. pneumoniaePneumolysin
S. aureusα-Toxin
S. aureusLeukocidin
S. pyogensStreptolysin-O

GPIIb–IIIa

GPIIb–IIIa is the most abundant platelet surface membrane GP, and its expression is specific to platelets and megakaryocytes. GPIIb–IIIa is a member of the integrin family of heterodimeric receptors, which mediate cell adhesion and signaling. Resting platelets contain approximately 80 000 surface copies, and there are additional pools of GPIIb–IIIa in the membranes of α-granules and the open canicular system. Upon platelet activation, surface expression can increase by as much as 50%. As the platelet fibrinogen receptor, GPIIb–IIIa mediates crosslinking of platelets by fibrinogen, which is responsible for aggregate formation [9].

Fibrinogen-binding proteins

Staphylococci have a family of surface receptors that are members of the ‘microbial surface components recognizing adhesive matrix molecules’ (MSCRAMM) family of proteins, often characterized by the presence of a domain rich in serine–aspartate repeats (Sdrs) [10]. Examples of MSCRAMMs include S. aureus clumping factor (Clf)A [11], ClfB [12], fibronectin-binding protein (Fnbp) A, and Fnbp B [13], Staphylococcus lugdunensis Fbl [14], and S. epidermidis SdrG [15]. Most of the MSCRAMMs bind plasma proteins such as fibrinogen or fibronectin [16].

Although fibrinogen-binding MSCRAMMs are related proteins, they bind to different domains in fibrinogen. Both ClfA and Fbl bind to the C-terminal region of the fibrinogen γ-chain [17], as do the non-Sdr proteins FnbpA and FnbpB. ClfB binds to the C-terminus of the Aα-chain [18] and SdrG to the Bβ-chain [19]. Fibrinogen-bound bacteria mediate platelet activation in a similar manner to other fibrinogen-coated surfaces. As their name implies, Fnbps also bind fibronectin, and this can also bind to GPIIb–IIIa [20]. In all cases, the MSCRAMM-bound fibrinogen/fibronectin can interact with GPIIb–IIIa, generating an outside-in signal that is capable of triggering platelet activation.

Streptococci also contain fibrinogen-binding proteins such as Streptococcus pyogenes M1 protein, which triggers platelet aggregation [21]. Streptococcus mitis lysin binds to the α and β subunits of the fibrinogen D fragment, although it is not known whether this induces platelet aggregation [22]. While both proteins are shed/secreted from the bacteria, lysin probably remains associated with the bacterial surface, because of its choline-binding properties.

Direct binding to GPIIb–IIIa

More recently, reports have demonstrated that some bacteria express proteins that can directly bind to GPIIb–IIIa in the absence of a bridging molecule. S. epidermidis SdrG can bind directly to GPIIb–IIIa and can also crosslink GPIIb–IIIa and FcγRIIa [15]. More recently, a heme-binding protein on S. aureus, iron-regulated surface determinant B, has been shown to support platelet adhesion and induce platelet aggregation through a direct interaction with GPIIb–IIIa [23]. S. gordonii also expresses platelet adhesion binding protein A (PadA), a novel high molecular weight protein that binds directly to GPIIb–IIIa and is critical for supporting platelet adhesion but not platelet aggregation [24]. The site of interaction between iron-regulated surface determinant B or PadA and GPIIb–IIIa has not yet been mapped, although it is noteworthy that preincubation of platelets with the peptide mimetic RGD completely abolishes adhesion.

GPIbα

GPIbα is a member of the leucine-rich repeat family of proteins, which is exclusively expressed on platelets and megakaryocytes. It can bind several different ligands, but its crucial role in primary hemostasis relies on its ability to interact with von Willebrand factor (VWF). GPIbα exists in a complex with GPIbβ, GPIX and GPV in a ratio of 2 : 2 : 2 : 1. Platelets express approximately 25 000 copies of GPIbα, which both mediates platelet tethering to surface-exposed VWF and supports platelet activation under high-shear conditions [25].

Several species of streptococci have been shown to directly interact with GPIbα, the interaction being mediated by a family of serine-rich glycoproteins. This family includes the S. sanguinis protein serine-rich protein A [26] and the S. gordonii proteins GspB (glycosylated streptococcal protein B) [27–29] and hemagglutinin salivary antigen (Hsa) [30], which are all structurally related. These are large, highly glycosylated, serine-rich proteins that bind sialic acid residues on host receptors. GspB predominantly binds O-linked sialic acid residues, whereas Hsa predominantly binds to N-linked sialic acid residues [29]. The interactions with GPIbα trigger platelet aggregation and support platelet adhesion. S. aureus expresses SraP, which is a homolog of GspB and supports platelet adhesion [31], possibly through GPIbα.

Just as some bacterial proteins can bind fibrinogen, there are also VWF-binding proteins on bacteria. S. aureus protein A has been shown to bind VWF, which in turn can interact with GPIbα [32]. H. pylori has also been shown to bind plasma VWF through an unknown protein, which in turn enables it to interact with GPIbα and trigger platelet aggregation [33]. Unlike soluble or immobilized VWF, bacteria-bound VWF can interact with GPIbα in the absence of high shear. However, it is not clear whether the interaction with VWF is simply an adhesive interaction mediating the binding of bacteria to platelets, thereby facilitating an interaction with an activating receptor, or whether it also plays a role in platelet activation. Certainly, the protein A-mediated interaction does not lead to platelet activation, whereas the H. pylori-mediated interaction does, although only through engagement of FcγRIIa.

Toll-like receptors (TLRs)

TLRs are a family of receptors in the innate immune system that mediate the host response to infection. These receptors recognize conserved pathogen-associated molecular patterns that are found on different classes of infectious agents [34]. To date, at least 11 TLRs have been described in various immune and non-immune cells. Recently, platelets have been reported to express TLR2, TLR4, and TLR9, and to show very weak expression of TLR1, TLR6, and TLR8, reinforcing their role as primitive immune cells in host defense [35,36]. The discovery of TLRs on platelets led to a search for their role in platelet function, with most studies focusing on TLR2 and TLR4.

The ligand for TLR4 is lipopolysaccharide (LPS) from Gram-negative bacteria [37]. Some studies have shown that LPS can induce platelet aggregation [38–41], whereas others have shown no effect [42] or even inhibition of platelet aggregation [43]. Exposure to LPS has also been shown to reduce platelet adhesion to fibrinogen in a calcium-dependent process [44]. More recently, it has been shown that, rather than inducing platelet aggregation, LPS leads to enhanced formation of neutrophil–platelet complexes, resulting in the formation of neutrophil extracellular traps [45], and that LPS-induced thrombocytopenia in mice is neutrophil-dependent [46], owing, in part, to increased phagocytosis [47]. LPS also induces expression on endothelial cells and monocytes of tissue factor, which in turn serves as a binding site for platelet GPIIb–IIIa [48]. In addition, LPS stimulates the release from platelets of sCD40L, which is widely regarded as a predictive indicator of cardiovascular events such as stroke or MI [49], as well as tumor necrosis factor release [36]. Soluble CD40L release is significantly reduced using a blocking monoclonal antibody against TLR4 [50]. Escherichia coli O157 LPS has been shown to bind to platelet TLR4, leading to activation [51], although other studies have failed to show any effect of LPS on platelet aggregation [42].

The natural ligand for TLR2 is lipoteichoic acid [52], and this has been shown to have mixed effects on platelet aggregation. It has been shown to inhibit platelet aggregation and to support platelet adhesion to S. epidermidis [53]. Studies with Pam3CSK4, a synthetic TLR2 agonist, have shown no effect on platelet aggregation at concentrations that activate TLR2 in other cell types [42], although it did induce aggregation and formation of platelet–neutrophil aggregates at a 10-fold higher concentration in wild-type but not TLR2-deficient mice [54]. More recently, it was shown to induce platelet aggregation and secretion in an ADP receptor-dependent manner [55]. Streptococcus pneumoniae was shown to induce platelet aggregation in a TLR2-dependent manner, and also generated an intracellular signal that triggered dense granule release and activated the phosphoinositide-3-kinase–RAP1 pathway [56]. However, S. aureus-derived lipoteichoic acid has been shown to inhibit platelet aggregation [57].

Although the molecular basis of these effects is still unclear, it appears that the primary effect of TLR-mediated activation of platelets is the secretion of immunomodulatory agents and the activation of other cells such as neutrophils and endothelial cells, rather than the formation of a thrombus. In this context, platelets are acting as components of the innate immune system [58] rather than as components of hemostasis, as they act to detect the presence of an infectious agent and coordinate the response to the pathogen.

Complement receptors

When bacteria enter the blood, they frequently trigger complement generation, either in an antibody-dependent manner or in an antibody-independent manner (alternative pathway) [59]. Complement-coated bacteria have been shown to be capable of inducing platelet aggregation. Some strains of S. sanguinis have been shown to induce platelet aggregation in a process that involves complement but also requires antibody binding [60,61]. Human gC1q-R is a multiligand binding protein for the first component of complement, C1q [62]. Low levels of gC1q-R are expressed on platelets under resting conditions; however, upon activation, the receptor number increases [63], thus possibly leading to gC1q-R serving as a receptor for complement-coated S. sanguinis. S. aureus ClfA and ClfB can induce platelet aggregation in a complement-dependent and antibody-dependent process [64,65]. In all cases, complement-mediated aggregation is FcγRIIa-dependent. It would appear to be dependent on the presence of an unidentified complement receptor on platelets as well.

FcγRIIa

The Fc portion of antibodies mediates its effects through a family of receptors known as Fc receptors. Each antibody type has a subfamily of Fc receptors, with IgG interacting with the FcγR subfamily. FcγRIIa is the most widely distributed Fcγ receptor in nature. It is predominantly expressed on neutrophils, monocytes, macrophages, and platelets. FcγRIIa is a low-affinity IgG receptor, with approximately 2000–3000 copies per platelet. It consists of a single transmembrane domain, a C-terminal domain, which contains the binding site for IgG, and a cytoplasmic domain. The cytoplasmic domain contains two YXXL sequences separated by 12 amino acids that, together, constitute an immunoreceptor tyrosine-activation motif (ITAM) [66].

Evidence suggests that FcγRIIa plays a critical role in bacterial-induced platelet aggregation [33,61,65,67,68]. FcγRIIa not only acts as an IgG receptor, but also plays an important role in platelet function. FcγRIIa enhances GPIIb–IIIa-mediated platelet spreading on fibrinogen in an IgG-independent manner [69]. It has also been shown to be colocalized with GPIbα and to play a role in GPIbα-mediated signaling in an IgG-independent manner [70].

The interaction of fibrinogen-bound or fibronectin-bound S. aureus or S. pyogenes with platelet GPIIb–IIIa induces platelet aggregation in an antibody-dependent manner. Thus, S. aureus ClfA-mediated aggregation requires binding of fibrinogen and antibody to ClfA, and these in turn bind to GPIIb–IIIa and FcγRIIa, respectively [65]. Similarly, VWF-bound bacteria such as H. pylori induce platelet aggregation in an FcγRIIa-dependent manner [33]. In this case, the VWF binds to GPIbα, and the antibody binds to FcγRIIa. Complement-dependent platelet aggregation is also antibody-dependent and FcγRIIa-dependent [61].

In the case of S. sanguinis [68], S. gordonii, and S. pneumoniae [56], aggregation is also FcγRIIa-dependent, but there is no requirement for IgG to induce aggregation. This is analogous to the role of FcγRIIa in promoting cell signaling through GPIIb–IIIa [69] and GPIbα [70–72].

Bacterial toxins

As well as interacting with platelets through surface proteins, bacteria can secrete toxins that can activate platelets [73]. Porphyromonas gingivalis is an oral pathogen that secretes a family of cysteine proteases known as gingipains [74]. These toxins can induce platelet aggregation by cleaving protease-activated receptor 1 in a manner analogous to thrombin [75,76]. S. aureus secretes a 34-kDa pore toxin called α-toxin [77], which is produced by almost all strains of S. aureus. It binds to the lipid bilayer of platelets, creating a transmembrane pore and an influx of calcium [78,79], which in turn triggers platelet activation in a manner analogous to the calcium ionophore A23187 [80]. Other pore-forming toxins include streptolysin O [81] from S. pyogenes and pneumolysin [82] from S. pneumoniae, which activate platelets in a similar manner to α-toxin.

S. aureus and S. pyogenes can produce a superfamily of toxins known as superantigens and staphylococcal superantigen-like (SSL) toxins [83]. One of these (SSL5) has been shown to directly interact with GPIbα via the sLacNac residues that terminate its glycan chains [84]. SSL5 has also been reported in an abstract to bind directly to GPVI [85]. The binding of SSL5 to platelets triggered platelet activation and aggregation.

Effect of bacteria on platelet function

Although it is clear that many bacteria can adhere to platelets and induce platelet aggregation, it is important to confirm that this is not simply an in vitro artefact. Key elements here are evidence for signal generation in platelets in response to their interactions with bacteria, evidence of a response in models that better reflect in vivo conditions, or evidence of response in animal models of disease.

Role of shear stress

Platelet aggregation and static adhesion studies involve artificial systems that do not truly reflect the dynamic nature of the circulatory system. Platelets are routinely exposed to a range of shear stresses, reflecting both venous and arterial conditions. Platelet function is sensitive to shear stress; for example, the interaction between GPIbα and VWF occurs only under conditions of high shear stress.

S. sanguinis and S. gordonii both interact with GPIbα, and therefore it is not surprising that this interaction is shear-dependent. However, in contrast to the high-shear-dependent rolling of platelets over immobilized VWF, platelets roll over both streptococci under low-shear conditions [26,30]. Deletion of the serine-rich, highly glycosylated proteins serine-rich protein A (S. sanguinis) or GspB/Hsa (S. gordonii) completely abolished rolling. Under low-shear conditions, thrombus formation on S. pyogenes is antibody-dependent, FcγRIIa-dependent, fibrinogen-dependent, and GPIIb-IIIa-dependent, as is platelet aggregation [86]. However, platelet aggregation induced by S. aureus is more complex, with potential roles for ClfA, ClfB, FnbpA, and FnbpB. Studies employing shear conditions showed that thrombus formation occurred only under high-shear conditions (> 800 s−1), and that it was entirely dependent on ClfA, as none of the other proaggregatory proteins could support thrombus formation. As with aggregation, thrombus formation was antibody–FcγRIIa-dependent and fibrinogen–GPIIb–IIIa-dependent [67].

Platelet signaling in response to pathogens

The ability of bacteria to generate intracellular signals upon binding to platelets is important in establishing a biological relevance for the interaction. There is a paucity of data on this, in part because of the complex, multicomponent nature of the interactions.

Upon activation by S. sanguinis, platelets release their dense granules, which contain vasoactive substances, including the adenosine nucleotides ATP and ADP [87]. S. sanguinis also expresses an ectoATPase that hydrolyses the released ATP to ADP [88,89]. ADP binds to the platelet ADP receptors P2Y12 and P2Y1, to serve as an amplification step that is essential for stable aggregate formation. Further studies have characterized the signal induced by S. sanguinis and demonstrated that it is also cyclooxygenase-dependent and thromboxane A2-dependent [68]. More recently, Pampolina et al. demonstrated that, in the presence of IgG, S. sanguinis caused tyrosine phosphorylation of platelet FcγRIIa within 30 s, followed by phosphorylation of phospholipase (PL)Cγ2, Syk, and LAT. Subsequently, there was tyrosine phosphorylation of PECAM-1 and the tyrosine phosphatase SHP-1, leading to dephosphorylation of PLCγ2, Syk, and LAT. As aggregation progressed into the early phase, platelets released thromboxane and the contents of their dense granules, acting to amplify and stabilize the platelet aggregate [90].

Keane et al. also demonstrated that platelet adhesion to immobilized S. gordonii resulted in tyrosine phosphorylation of the ITAM-bearing receptor FcγRIIa, as well as phosphorylation of the downstream effectors Syk and PLCγ2. This signal resulted in platelet dense granule secretion, filopodia and lamellipodia extension, and platelet spreading. Inhibition of either GPIIb–IIIa or FcγRIIa completely abolished dense granule release and platelet spreading [91].

S. mitis has also been shown to bind to platelets in a GPIIb–IIIa-independent and GPIbα-independent manner; however, no platelet-activating signal was generated [92,93]. Relatively little is known about the signal generated in platelets upon binding to S. aureus, other than it is cyclooxygenase-dependent and thromboxane-dependent [94].

Phagocytosis

The presence of FcγRIIa on the platelet surface suggests that platelets may have the capacity to phagocytose, as this receptor is important in immune complex clearance. Platelets have been shown to phagocytose immune complexes in an FcγRIIa-dependent manner, and can also be phagocytosed themselves [95,96], also in an FcγRIIa-dependent manner [47]. Platelets have been shown to enhance the phagocytosis of periodontal pathogens by neutrophils [97]. Platelets can also directly phagocytose bacteria such as P. gingivalis [98,99] and S. aureus [100–102]. However, it is often the case that phagocytosis does not result in bacterial killing, and this has been suggested to be attributable to the structure of their vacuoles [100]. Bacteria can also become trapped in the space between platelets in an aggregate [99]. As a result, phagocytosis of bacteria by platelets can lead to the formation of a pool of viable bacteria, present either intracellularly or within a thrombus, that are protected from the immune system and play a role in the pathogenesis of diseases such as IE.

Bacteria–platelet interactions in vivo

Several studies have investigated the interaction of bacteria with platelets under in vivo conditions. Mice infected with S. aureus develop platelet-rich thrombi in a process that is dependent on ClfA, as administration of the fibrinogen-binding domain of ClfA prevented thrombus formation [103]. Dogs infected with S. aureus develop sepsis, with an associated drop in platelet count [104]. Resistance to platelet antimicrobial peptides was a virulence factor in S. aureus for IE [105], whereas hyperproduction of α-toxin reduced the extent of S. aureus-mediated endocarditis [106], presumably because of increased levels of antimicrobial peptides. S. aureus SraP is a virulence factor in IE [31], as is wall teichoic acid [107]. Lactococcus lactis expressing either ClfA or FnbpA was shown to be 100 times more infective than the wild-type L. lactis strain in an animal model of IE [108]. However, MSCRAMMs have been shown to have only a modest role to play in S. aureus-mediated endocarditis in animal models [109–111], which is probably attributable to the presence of multiple platelet-interacting proteins on the bacterial surface and the difficulty of generating a strain of S. aureus devoid of any interaction with platelets, especially as complement formation can occur in the absence of these proteins. Thus, blockade of the complement receptor gC1qR was shown to be beneficial in S. aureus-mediated endocarditis [112]. Deletion of the lysin gene from S. mitis significantly reduced endocarditis in a rat model [22]. There was evidence of increased rates of embolization in H. pylori-infected mice after laser-induced arterial damage [113].

Evidence from studies in mice suggests that, in the case of S. pneumoniae infection, thrombocytopenia and DIC result from a bacterial neuraminidase that removes sialic acid from platelet proteins, making them substrates for the Ashwell receptor in the liver. Binding to the Ashwell receptor leads to clearance of platelets from the circulation, resulting in thrombocytopenia [114].

H. pylori infection is associated with platelet activation in patients [115,116]. Clinical studies showed that H. pylori eradication therapy in patients with idiopathic thrombocytopenic purpura who were H. pylori-positive was effective at improving the platelet count [117–120]. This suggests that ongoing infection with H. pylori leads to platelet activation and subsequent thrombocytopenia.

There are several differences between human and rodent platelets, most notably the absence of FcγRIIa. As FcγRIIa has been shown to play a significant role in the interaction of bacteria with human platelets, the relevance of data from traditional mouse models of sepsis is questionable. As transgenic mice expressing FcγRIIa are now available, it will be possible to use these to better understand the interaction of bacteria with platelets in vivo.

There is a paucity data on the role of platelets in infection in humans, but a study of patients with S. pyogenes toxic shock syndrome showed evidence of microthrombi in biopsy specimens. These platelet aggregates formed in a process dependent on M1 protein, IgG, and FcγRIIa [21], similar to that seen in vitro.

Conclusions

There is no doubt that platelets play an important role in the innate immune system. As the first responders to injury, they are ideally placed to initiate an immune response to potential pathogens, through secretion of antimicrobial peptides to kill bacteria, and of chemokines to attract other immune cells. Both the hemostatic and immune functions of platelets require platelet activation to occur. There are many different mechanisms by which bacteria can interact with platelets, including direct interactions with platelet receptors and the secretion of bioactive agents such as LPS. However, there is a paucity of in vivo data to identify the key mechanisms in infectious diseases such as sepsis. Is there a primary interaction driving the response, or is it a combination of all of the interactions?

Some bacteria have developed resistance to the antimicrobial effects of platelets, and have the ability to recruit platelets into the infection process. By inducing platelet activation while being resistant to the antimicrobial peptides, they can become engulfed in a septic thrombus, as occurs in IE. They are then protected from the other cells of the immune system, allowing them to persist in the circulation. Even when bacteria are susceptible to antimicrobial peptides, rapid bacterial growth during sepsis leads to extensive platelet activation, which, in turn, leads to DIC and shock. Thus, in these cases, inhibition of platelet activation by bacteria may prevent some of the serious consequences of sepsis and IE.

As each species of bacterium, and even individual strains, have different mechanisms for interacting with platelets, it will, in all likelihood, prove impossible to target the bacteria as a mechanism to prevent platelet activation. However, there appear to be a limited number of platelet receptors involved, making the platelet a better target. GPIIb–IIIa is an obvious target, as it is important in S. aureus-induced platelet activation and there are approved inhibitors available. However, bleeding is a serious problem with these drugs, and in a patient who is already thrombocytopenic because of sepsis, they would further compromise the remaining platelets. GPIbα is another important target, as it is important in streptococcal sepsis; however, despite much effort, there are no approved GPIbα inhibitors. Aspirin could also be used to prevent platelet activation but, as it also compromises platelet function and as some species of bacteria can induce platelet activation in a cyclooxygenase-independent manner, it is of limited use. The most promising target is FcγRIIa, as it plays a critical role in platelet activation induced by most species of bacteria, either because they require bound IgG to induce aggregation, or because FcγRIIa is important in the activation process, even if antibody is not required. Another advantage of targeting FcγRIIa is that it has minimal effects on normal platelet function, thus allowing the preservation of platelet function and not causing bleeding. Although there are no inhibitors of FcγRIIa at present, the possibility of synthesizing such compounds has recently been demonstrated [121].

The conventional view of platelets has been that of anucleate cellular fragments that play a key role in hemostasis. The discovery of evidence for protein synthesis by platelets [122] suggested that they are more sophisticated than originally thought. We now have strong evidence that platelets are also key components of the innate immune system, where they play important roles in infection and inflammation. Although our understanding of the role of platelets in the immune system is far from complete, we do see the possibility and potential benefits of specifically targeting the immune function of platelets in both autoimmune and infectious diseases.

Acknowledgements

Work in the authors’ laboratories is supported by grants from the Science Foundation Ireland, the Health Research Board, Ireland, the British Heart Foundation, and the Wellcome Trust.

Disclosure of Conflict of Interest

The authors state that they have no conflict of interest.

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