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. Author manuscript; available in PMC: 2014 Jul 15.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2010 Nov 11;30(12):2408–2415. doi: 10.1161/ATVBAHA.110.216515

Mechanism of outside-in αIIbβ3-mediated activation of human platelets by the colonizing bacterium, Streptococcus gordonii

C Keane 1, H Petersen 2, K Reynolds 1, DK Newman 3,4,5, D Cox 1,7, PJ Newman 3,4,6,7, SW Kerrigan 1,8
PMCID: PMC4098661  NIHMSID: NIHMS252827  PMID: 21071690

Abstract

Objective

Infective endocarditis (IE) is a bacterial infection of the heart valves which carries a high risk of morbidity and mortality. The oral bacterium, Streptococcus gordonii, is amongst the most common pathogens isolated from IE patients, and has the property of being able to activate platelets, leading to thrombotic complications. The mechanism of platelet recruitment and activation by S. gordonii is poorly understood.

Methods and Results

Platelets interact with S. gordonii via GPIbα and αIIbβ3 recognising S. gordonii surface proteins Hsa and PadA, respectively. Inhibition of GPIbα or αIIbβ3 using blocking antibodies or deletion of S. gordonii Hsa or PadA significantly reduces platelet adhesion. ITAM containing proteins have recently been shown to play a role in transmitting activating signals into platelets. Platelet adhesion to immobilised S. gordonii resulted in tyrosine phosphorylation of the ITAM-bearing receptor, FcγRIIa, as well as phosphorylation of downstream effectors, Syk and PLCγ2. Tyrosine phosphorylation of FcγRIIa resulted in platelet dense granule secretion, filopodial and lamellipodial extension, and platelet spreading. Inhibition of FcγRIIa abalated both dense granule release and platelet spreading.

Conclusions

S. gordonii binding to the αIIbβ3/FcγRIIa integrin/ITAM signalling complex results in platelet activation that likely contributes to the thrombotic complications of IE.

Keywords: Infective Endocarditis, Streptococcus gordonii, platelet spreading, FcγRIIa, Syk, ITAM

INTRODUCTION

Infective endocarditis (IE) is considered to be the fourth leading cause of life threatening infectious disease, and is associated with significant morbidity and mortality1. It typically develops in individuals with an underlying cardiac defect, and usually occurs in close proximity to lesions subjected to hemodynamic disturbance2. These lesions have the ability to generate turbulent blood flow, which in turn can damage the endothelial surface, exposing the subendothelial matrix. This area becomes highly thrombogenic, leading to platelet deposition and the formation of a fibrin network3. This sterile platelet fibrin nidus, in turn, recruits bacteria from either a distal source or a from a focal infection resulting from transient bacteraemia4, 5. Finally secondary accumulation of platelets encase the bacteria forming a septic thrombus6. Thrombus formation may lead to aortic valve leaflet perforation that can manifest itself as acute congestive heart failure7. The American Heart Association has recently issued evidence-based guidelines for the treatment of IE which consists of aggressive antibiotic therapy1. This approach however, is often less than optimal, as even with treatment, the mortality rate can be as high as 36%8, perhaps due to the fact that significant levels of antibiotic fail to penetrate the growing thrombus.

The viridans group of streptococci is responsible for 44% IE cases9. Streptococcus gordonii is a member of the viridans group and an oral bacterium that resides primarily in dental plaque on the tooth surface10, 11. Chronic oral disease such as periodontitis or dental manipulation provides a route of entry of bacteria into the circulation, leading to transient bacteraemia12. S. gordonii is well known for its ability to interact with human blood platelets13 and this may be an important contributor for the initiation of IE.

A picture demonstrating the complexity of the interactions between S. gordonii and platelets is starting to unfold in the literature13. It is now well established that multiple strains of streptococci can induce platelet aggregation and support platelet adhesion14–16. Recently we demonstrated that under low shear conditions platelets are capable of rolling along immobilised S. gordonii in a manner similar to platelet rolling on immobilised von Willebrand factor at sites of vascular injury15. Platelet rolling on immobilised S. gordonii results in firm adhesion. S. gordonii express two large, highly glycosylated proteins, GspB and Hsa, that bind specifically to N-linked sialic acid residues on GPIbα17 interactions that are believed to mediate platelet rolling. Inactivation of the hsa gene in S. gordonii ablated platelet rolling15. More recently, our group identified another large protein expressed on S. gordonii, platelet adherence protein A (PadA, 397 kDa). This protein specifically binds to the major platelet integrin, αIIbβ3. Disruption of the padA gene significantly reduced platelet adhesion to S. gordonii16. Therefore platelet adhesion to S. gordonii is a multifactorial event, where Hsa binds to GPIbα mediating platelet rolling, and PadA binds to αIIbβ3, mediating firm adhesion. Together these proteins act synergistically to recruit and activate platelets on the bacterial surface.

Ligand binding to platelet surface receptors results in the generation of outside-in signals that initiate a series of cytosolic changes that promote spreading. This spreading is essential for the platelet to withstand the shear forces experienced in the vasculature. Recently, Boylan et al., demonstrated that the ITAM bearing receptor, FcγRIIa, functions as a key component of outside-in signal amplification mediated by αIIbβ318. The purpose of this study was to determine whether platelet interactions with S. gordonii result in similar outside-in amplification signals leading to platelet spreading. Our findings have important implications for understanding the molecular mechanisms through which the platelet response to bacteria contributes to the development of a thrombus in IE patients. Understanding these mechanisms may lead to the development of novel therapeutics to treat this disease.

MATERIALS AND METHODS

Antibodies and reagents

Anti-Syk (Clone D-3) and anti-PLCγ2 (Clone B-10) and polyclonal anti-FcγRIIa (Clone C-17) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies detecting tyrosine–phosphorylated Syk (tyr525/526) and PLCγ2 (tyr759) were purchased from Cell Signaling Technology (ISIS Ltd, Ireland). The antiphosphotyrosine monoclonal antibody, 4G10, was purchased from Millipore (Watford, UK). Monoclonal anti-FcγRIIa antibody, IV.3 was purchased from Stem Cell Technologies (Grenoble, France). Monvalent Fab fragments were prepared from mAb IV.3 using a Fab preparation kit from Pierce Biotechnology (Rockford, IL). The integrin pepidomimetic H-Arg-Gly-Asp-Ser-OH (RGDS) was purchased from Calbiochem (Nottingham, United Kingdom). Monoclonal antibody abciximab was obtained from Eli Lilly (Leiden, The Netherlands) and MB4519 was obtained from Serotec (Oxford, United Kingdom). Brain heart infusion and M17 media were purchased from Oxoid (Basingstoke, United Kingdom). All other laboratory reagents were purchased from Sigma-Aldrich (Poole, UK).

Bacterial strains and growth conditions

Sources and strains of S. gordonii used in this study are listed in Supplementary Table 1. Deletion of hsa or padA in S. gordonii DL1 by allelic exchange mutagenesis has been described previously16, 20. S. gordonii strains were maintained on blood agar and subsequently grown in BHI broth in sealed tubes incubated statically overnight (stationary phase) at 37°C for use in experiments. For all experiments S. gordonii suspensions were adjusted to 1×109 cells/ml for platelet adhesion/spreading studies.

Platelet preparation

Whole blood was drawn from the antecubital vein of healthy volunteers who had abstained from taking non-steroidal anti-inflammatory drugs in the previous 10 days. Informed consent was obtained from all subjects. Nine volumes of blood were added to 1 volume of acid-citrate-dextrose (ACD). Platelet rich plasma (PRP) and gel filtered platelets were prepared as previously described13.

Static platelet adhesion assay

Static platelet adhesion was measured as described previously15. Briefly, 96 well microtiter plates were coated with 100 µl bacteria (1×109 cells/ml), fibrinogen (20 µg/ml) or BSA (1%). Gel filtered platelets (2×108 platelets/ml) were added to each well and allowed adhere for 45 minutes at 37°C. Adherent platelets were then lysed with lysis buffer containing a substrate for acid phosphatase and incubated for 20 minutes at 37°C. The resultant color was read at 410 nm in a microtiter plate reader (Wallac Victor2, Perkin Elmer, Cambridge, United Kingdom). In some experiments gel filtered platelets were preincubated with specific monoclonal antibody for 15 minutes prior to addition to bacteria coated wells.

Platelet spreading on immobilised bacteria

Poly-L-lysine coated glass slides were coated with human fibrinogen (50 µg/ml) or S. gordonii (1×109 cells/ml) overnight at 4°C. Slides were then blocked with 1% BSA for 2 hrs at 37°C and finally washed with TBS to remove any unbound BSA. Gel filtered platelets (5×106 platelets/ml) were preincubated with inhibitors (abciximab 50 µg/ml, RGDS 10 mM, apyrase 2 U/ml or 1 µg/ml anti-FcγRIIa mAb IV.3) for 15 minutes at room temperature and then allowed to spread on either fibrinogen or S. gordonii for 60 minutes. After gently rinsing 3 times with modified HEPES-Tyrodes buffer, spread platelets were fixed with 3.7% paraformaldehyde for 10 minutes at room temperature and permeabilised in ice cold acetone for 5 minutes. Platelets were stained using Alexa 546 phalloidin for 20 minutes at room temperature in the dark. Samples were mounted in Vectashield mounting media (Vector Laboratories, CA), and images acquired (63×) using a Zeiss LSM 510 confocal microscope in DIC (differential interference contrast) or using an argon laser at 488 nm.

Dense granule secretion

Dense granule secretion was measured by luminometry using a luciferin/luciferase assay. Platelets (2×108/ml) were incubated with either; PBS (as control), RGDS (10 mM), abciximab (10 µg/ml), IV.3 (5 µg/ml) or apyrase (2 U/ml) for 10 minutes at room temperature. Chrono-lume luciferin/luciferase mix was added and luminescence was read in a microtiter plate reader.

Western blotting and immunoprecipitation

Gel filtered platelets (1×109/ml) adhered to either immobilised fibrinogen or S. gordonii, in the presence or absence of a Fab fragment of IV.3, were lysed using 2 × Nonidet-P40 buffer containing a 1× protease inhibitor cocktail and phosphatase inhibitors (sodium fluoride and sodium orthovanadate). A small aliquot was dissolved with SDS sample buffer for detection of total phosphorylation. An monoclonal antibody specific for FcγRIIa, IV.3, was added to the resultant supernatant and incubated overnight. FcγRIIa was immunoprecipitated by addition of protein G sepharose. Cleared lysates were separated by SDS-PAGE, transferred to PVDF, blocked with BSA in TRIS-buffered saline Tween (TBST; 0.5%) for 60 minutes. Immunoprecipitated immunoblots were stained with antibodies specific for tyrosine-phosphorylated Syk and tyrosine phosphorylated PLCγ2. The antiphosphotyrosine monoclonal antibody 4G10, was used to detect immunoprecipitated phosphorylated FcγRIIa. Equal loading was detected using antibodies against Syk, PLCγ2 and FcγRIIa. Protein bands were detected using species specific horseradish peroxidase-conjugated secondary antibody and chemiluminescence.

Statistical analysis

Statistics were performed using InStat statistical software (GraphPad software, SD, USA). Data shown are the means plus or minus standard error of the mean (SEM) and comparisons between mean values were performed using the Student paired or unpaired t-test.

RESULTS

Platelet adhesion to immobilised S. gordonii is mediated by platelet membrane receptors αIIbβ3 and GPIbα and S. gordonii surface proteins PadA and Hsa

Previous studies have shown that PadA and Hsa are important mediators of platelet adhesion to S. gordonii16. As shown in Figure 1A, deletion of the gene encoding either protein significantly reduced platelet adhesion, however, deletion of genes encoding both proteins had no additional effect. In contrast deletion of the sspA and sspB genes, the products of which are known to play an important role in S. gordonii-induced platelet aggregation, had no significant effect on platelet adhesion. Platelet adhesion to S. gordonii was significantly inhibited by the anti-αIIbβ3 antibody, abciximab, or by the anti-GPIbα antibody, MB45, but not by a control antibody (Figure 1B). These data demonstrate that neither αIIbβ3/PadA nor GPIbα/Hsa interactions by themselves are sufficient to support platelet adhesion, as residual adhesion can still be observed in the absence of both of these interactions. Therefore, suggesting that another, as yet uncharacterised interaction exists between S. gordonii and platelets.

Figure 1.

Figure 1

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Platelet adhesion to S. gordonii under static conditions. A. Plasma free platelets (2×108 platelets / ml) were allowed adhere to wildtype S. gordonii or S. gordonii deficient in PadA (ΔpadA), Hsa (Δhsa) or SspA/B (ΔsspAsspB) B. or in the presence of αIIbβ3 inhibitor, abciximab (20 µg/ml), GPIbα antibody, MB45 (20 µg/ml) or irrelevant antibody (20 µg/ml).

Platelets adhesion to immobilised S. gordonii leads to the generation of an outside-in signal that initiates full platelet spreading

When platelets adhere to fibrinogen, a ligand binding-induced, αIIbβ3-mediated signal is generated leading to platelet spreading that enables adherent platelets to resist the shear forces at the site of vascular injury3. To investigate whether platelets adherent to S. gordonii were capable of spreading in a similar manner, platelets were allowed adhere to BSA, fibrinogen, or S. gordonii, and spreading was evaluated by differential interference contrast and immunofluorescence microscopy. To analyse the percentage of platelets spread, we randomly selected five areas containing 200 or more total cells. The percentage of platelets spread was calculated as a fraction of total platelets. Consistent with previous results, platelets failed to spread on BSA, but spread on immobilised fibrinogen (Figure 2). Platelet spreading on wildtype S. gordonii, as defined by evidence of pseudopod and lamellipod formation, occurred within 15 minutes. The morphology of platelets spread on S. gordonii at 15 minutes resembled that of platelets spreading on fibrinogen at 15 minutes.

Figure 2.

Figure 2

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Platelet spreading on immobilised S. gordonii. A. Plasma free platelets were allowed adhere to BSA, fibrinogen or S. gordonii for 45 minutes. The percentage of platelets spread was randomly selected from five areas containing 200 or more total cells. B. Images were acquired (63×) in DIC or using an argon laser at 488nm.

As S. gordonii surface proteins PadA and Hsa are important in supporting platelet adhesion, we next investigated whether these proteins contributed to platelet spreading. Deletion of the αIIbβ3 ligand, PadA, from S. gordonii abolished platelet spreading, showing little or no signs of cytoskeletal rearrangements, however deletion of Hsa failed to have any affect on platelet spreading (Figure 2). Deletion of both PadA and Hsa together also ablated platelet spreading, similar to that seen with the ΔpadA alone. These data suggest that although both αIIbβ3/PadA and GPIbα/Hsa interactions contribute to platelet adhesion on immobilised S. gordonii, however αIIbβ3/PadA interactions are the major contributor to outside-in signalling leading to platelet activation and spreading.

To confirm the importance of αIIbβ3 in platelet spreading on S. gordonii., platelets were pre-treated with either an inhibitory peptide mimetic for the common integrin recognition motif, RGDS, or the αIIbβ3-specific monoclonal antibody, abciximab. As shown in Figure 3, each of these significantly inhibited platelet spreading on fibrinogen and S. gordonii. Platelet adhesion to fibrinogen or S. gordonii was unaffected when pretreated with ADP scavenger, apyrase, (2±1 % inhibition of platelet adhesion to fibrinogen in the presence of apyrase and 0% inhibition of platelet adhesion to S. gordonii in the presence of apyrase, P=NS, n=6). Platelets, however, failed to spread on fibrinogen or S. gordonii in the presence of apyrase suggesting that platelet adhesion to fibrinogen or S. gordonii stimulated the release of small potentiating amounts of ADP into the local platelet environment. Taken together, these data confirm the importance of αIIbβ3 in S. gordonii-induced platelet activation leading to cytoskeletal rearrangements, granule secretion, and platelet spreading.

Figure 3.

Figure 3

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Platelet spreading on immobilised S. gordonii. Plasma free platelets were allowed adhere to A. Fibrinogen or B. S. gordonii in the presence of various inhibitors. The percentage of platelets spread was randomly selected from five areas containing 200 or more total cells. C. Images were acquired (63×) in DIC or using an argon laser at 488nm.

Platelet spreading on S. gordonii requires an αIIbβ3/FcγRIIa integrin/ITAM cluster

The exact mechanism by which integrins promote outside-in signals is not fully understood, however several lines of evidence suggest that ITAM bearing receptors function to amplify integrin-mediated cellular activation21 including the recently-described αIIbβ3/FcγRIIa integrin/ITAM cluster that is expressed on platelets18. When incubated with a small Fab fragment derived from the anti-FcγRIIa specific monoclonal antibody, IV.3, platelets failed to spread on immobilised fibrinogen (Figure 3A) or S. gordonii (Figure 3B). Platelet adhesion to fibrinogen or S. gordonii was unaffected in the presence of mAb IV.3, (3±0.5 % inhibition of platelet adhesion to fibrinogen in the presence of IV.3 and 0% inhibition of platelet adhesion to S. gordonii in the presence of IV.3, P=NS, n=5) suggesting that platelet FcγRIIa does not mediate platelet S. gordonii interactions but rather amplifies the outside-in signalling following engagement of αIIbβ3 with PadA on S. gordonii. Moreover preincubating gel-filtered platelets with mAb IV.3, abolishes S. gordonii-induced platelet aggregation (56±1% untreated vs 5±2% IV.3 treated, P<0.0001), further demonstrating the role of FcγRIIa for relaying αIIbβ3-mediated signals into the platelet.

Platelet dense granule secretion is both induced by and contributes to platelet spreading on S. gordonii

The observation that apyrase had no effect on platelet adhesion, but rather inhibited subsequent platelet spreading (Figure 3) suggested that platelet/S. gordonii interactions initiated by PadA binding to αIIbβ3/FcγRIIa induces release of ADP from platelet dense granules. ATP also exists in the dense granules and is often used as a measure of dense granule release. Therefore, to determine whether S. gordonii induces platelet secretion, platelets were allowed adhere to immobilised bacteria, and ATP release measured in a luminescence assay. As shown in Figure 4, platelets bound to S. gordonii exhibited secretion similar to that induced by TRAP. Granule release was significantly reduced when platelets were preincubated with mAb, IV.3, consistent with the notion that αIIbβ3/FcγRIIa mediates platelet activation induced by S. gordonii.

Figure 4.

Figure 4

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S. gordonii induce dense granule secretion from platelets. Dense granule release was inhibited by preincubating platelets with either IV.3 (5 µg/ml) or apyrase (2 U/ml). Dense granule release was measured using a chrono-lume luciferin/luciferase mix and was read in the luminescence channel in a microtiter plate reader.

Platelet adhesion to immobilised S. gordonii triggers the αIIbβ3/FcγRIIa → Syk → PLCγ2 signal transduction pathway

Integrin-mediated platelet adhesion to immobilised ligands results in the generation of outside-in signals that lead to tyrosine phosphorylation of numerous signalling molecules22. To confirm biochemically that FcγRIIa acts as an amplifier of integrin signalling when platelets adhere to S. gordonii, we allowed platelets to spread on immobilised wildtype S. gordonii and assessed the tyrosine phosphorylation state of FcγRIIa and its key downstream regulators, Syk and PLCγ2. As shown in Figure 5A, platelet spreading on S. gordonii led to phosphorylation of FcγRIIa, Syk and PLCγ2. Furthermore, preincubation of platelets with Fab IV.3 prior to adhesion on either fibrinogen or S. gordonii abolished phosphorylation of FcγRIIa (Fig 5B). Taken together with the observation that mAb IV.3 prevents platelet spreading on S. gordonii these data demonstrate that following adhesion to S. gordonii, FcγRIIa plays an essential role in transducing signals that mediate platelet spreading.

Figure 5.

Figure 5

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S. gordonii induces an outside-in signal amplified by FcγRIIA. Plasma free platelets were allowed spread on fibrinogen or S. gordonii in the presence and absence of 10µg/ml IV.3 Fab. Platelets were lysed, immunoprecipitated with FcγRIIA antibody and immunopercipitates were immunoblot for either A. tyrosine phosphorylation of FcγRIIA and its key downstream regulators Syk or PLCγ2. B. or FcγRIIa in the presence of a Fab fragment of IV.3. Equal loading was determined by immunoblotting for antigen (Ag) in precleared lysates.

DISCUSSION

The viridans group of streptococci are major constituents of supra and subgingival dental plaque10, 23. They exist in the oral cavity as harmless commensals, however, when they gain entry to the circulation they behave as pathogens. Severe oral disease or dental manipulation provides a route of entry for these bacteria to the blood. Once in the blood they interact with circulating platelets to initiate inappropriate thrombus formation. Thrombus formation is thought to play a critical role in IE24. IE describes a family of persistent microbial infections that typically target previously damaged or diseased heart valves6. Steptococcus gordonii is a prominent member of the viridans group of streptococci and is commonly isolated from patients presenting with IE. The ability of S. gordonii to activate platelets is considered a primary event in the pathogenesis of IE and the severity of the clinical signs6.

Many attempts have been made to understand the mechanisms through which S. gordonii can bind to platelets and generate an intracellular signal leading to thrombus formation in vitro. Recently we proposed a model of the early stages of infection where platelets roll along immobilised S. gordonii using an interaction that involves platelet GPIbα interacting with S. gordonii surface protein Hsa15. This rolling interaction slows platelets sufficiently to allow a second interaction between platelet αIIbβ3 and S. gordonii PadA resulting in firm adhesion16. Even though S. gordonii is not a natural ligand for platelets, this proposed model suggests that bacterial deposition on damaged endothelium mimics the platelet response to vascular injury.

In the present study we demonstrated that upon firm adhesion to immobilised S. gordonii, platelets receive a signal that leads to extension of filopodia and lamellipodia with controlled orientation of stress fibres leading to full platelet spreading. Platelet spreading reflects the ability of αIIbβ3 to bind PadA on the surface of S. gordonii. Disruption of the padA gene abolishes platelet spreading, while disruption of the hsa gene has no effect (Figure 2). The function of platelet spreading is essential for the platelet to withstand the shear forces experienced in the vasculature. Spreading is a particularly important step in the development of thrombotic vegetations in IE as shear forces around the lesion on the cardiac valve are often turbulent2. Platelets therefore require the conversion from a discoid shape to a fully spread cell to withstand turbulent shear force.

Platelet spreading is mediated by a signal generated as a result of ligand binding through a process called outside-in signalling. The best characterised outside-in signal in platelets is that mediated by αIIbβ322, 25–27. Outside-in signalling through αIIbβ3 on a vascular matrix of fibrinogen or immobilised von Willebrand factor plays a critical role in mediating platelet spreading25, 28. αIIbβ3 can bind several macromolecular adhesive glycoproteins which contain the short amino acid sequence RGD29. There are several sites on fibrinogen that bind to αIIbβ3 These include the widely studied RGD site found twice on the Aα-chain30 and a carboxyterminal dodecapeptide (HHLGGAKQAGDV) found on the γ-chain31. Previously we demonstrated that an N-terminal PadA recombinant polypeptide bound to platelets in a αIIbβ3-dependent manner16. Moreover wild type S. gordonii expressing PadA but not S. gordonii defective in PadA expression could bind to purified αIIbβ3 and CHO cells stably transfected with αIIbβ3. Together these results suggest that PadA binding to the platelet generates an outside in signal through αIIbβ3 to mediate spreading. S. gordonii defective in PadA expression failed to spread after 60 minutes suggesting that kinetics is not a factor here as platelets were spread on wildtype S. gordonii after 15 minutes, smilar to the time to platelet spreading on immobilised fibrinogen. Platelet spreading on immobilised fibrinogen or S. gordonii is abolished when platelets are preincubated with abciximab or a short peptide containing peptide RGDS (Figure 3). These results suggest that αIIbβ3 might bind S. gordonii through an RGD-like sequence on PadA. Protein analysis identified two potential RGD-like regions (RGG and RGT) and a dodecapeptide-like region (AGD) that may act as binding sites for αIIbβ3 on the N-terminus fragment of PadA.

There is a considerable body of evidence that concomitant granular secretion of ADP from adherent platelets is necessary for full platelet spreading32, 33. Consistent with these results, we found that preincubation of platelets with the ADP scavenger apyrase prevented platelet spreading not only on fibrinogen, but also on immobilised S. gordonii (Figure 3). It is therefore likely that platelet adhesion and spreading on S. gordonii stimulates the release of potentiating amounts of ADP into the local platelet milieu, facilitating the cytoskeletal rearrangements necessary for platelet spreading.

The presence of an Fc receptor on platelets has attracted much attention recently as numerous reports suggest platelets may play a role in host defence by recognising antibody bound foreign invaders13. Youssefian et al, reported that platelets can actively phagocytose IgG-bound Staphylococcus aureus, however whether platelets have the ability to destroy the bacteria once phagocytosed is still in question34. Other reports suggest that antibody binding to bacteria is essential for triggering platelet activation. For example, Streptococcus sanguinis strain 2017–78 triggers platelet activation in an antibody-dependent manner. Inhibition of FcγRIIa, using mAb IV.3, prevented this platelet activation35. In addition, Staphylococcus aureus strain Newman surface protein, clumping factor A, requires binding of IgG and fibrinogen in order to trigger aggregate formation under conditions of high shear. Blocking FcγRIIa prevented aggregate formation however failed to prevent single platelet adhesion36. While there is little doubt that antibody binding to bacteria is essential for triggering platelet activation/aggregation, this appears be strain- or species-specific. For example, in this report we demonstrate that S. gordonii can induce platelet aggregation in an antibody-free environment, and that aggregation can be abolished by blocking FcγRIIa. We have also shown this to be true for several other strains of streptococci capable of inducing platelet aggregation including S. sanguinis strain SK34 and S. pneumoniae strain IO1196 (unpublished data SWK, DC, AK, HJF).

FcγRIIa is a member of the immunoglobulin gene superfamily, and is the most widely distributed Fcγ receptor in nature37. It is expressed on neutrophils, monocytes, macrophages and platelets38. It is capable of functioning as a low affinity IgG receptor with approximately 2000–3000 copies per platelet39–41. It consists of a single- transmembrane domain, a C-terminal that contains the binding site for IgG and a cytoplasmic domain. The cytoplasmic domain contains two YXXL sequences separated by twelve amino acids that together constitutes its ITAM domain42. Significant emphasis has been placed on the importance of ITAM-containing receptors as signal amplifiers in various cell types. Mocsai et al demonstrated that the β2 integrin in neutrophils and monocytes require tyrosine phosphorylation of the ITAM-containing adapter molecules, FcRγ or DAP12 in order to trigger an intracellular signal21, 43, and there are a growing number of papers in the literature describing the importance of ITAM bearing receptors in platelet signalling. Previous studies demonstrated that GPVI, a receptor for collagen, associates with FcRγ which triggers platelet activation through Src kinases and Syk44, 45. CLEC-2 is a newly discovered ITAM receptor on platelets. Upon binding the toxin rhodocytin, the ITAM region of CLEC-2 becomes phosphorylated triggering activation of downstream effectors such as Syk, Brutons tyrosine kinase (Btk), LAT, SLP-76 and PLCγ246–48. More recently, a paper by Boylan et al, demonstrated that FcγRIIa plays a key role in the outside-in signal mediated by αIIbβ3 upon binding to either soluble or immobilised fibrinogen18. In our experiments platelet spreading on S. gordonii leads to phosphorylation of the tyrosine residues on FcγRIIa. This in turn mediates the activation of downstream regulators Syk and PLCγ2. Inhibition of FcγRIIa prevents tyrosine phosphorylation which results in inhibition of platelet dense granule secretion, filopodial and lamellipodial extension, and platelet spreading.

The involvement of FcγRIIa in mediating both platelet activation and spreading in response to S. gordonii makes it a potential target for therapeutic intervention. Inhibition of this receptor while not treating the infection would prevent the potentially fatal thrombotic complications of S. gordonii infection without compromising normal platelet function. They could be used in conjunction with antibiotics to treat IE or on their own as prophylaxis for high-risk patients.

Supplementary Material

Supp1

Acknowledgments

Sources of Funding

This work was supported by the Health Research Board of Ireland grant (to SWK, #RP/2006/211), Wellcome Trust Clinical Training Fellowship (to HJF, #084979) and a Walton Fellowship (to PJN) from the Science Foundation of Ireland.

Footnotes

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“This work has previously been published in Abstract form at the 11th Annual meeting of the Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB) Council of the American Heart Association (4/15/2010), where it was selected as a top-scoring abstract and invited to be re-presented at the upcoming Scientific Sessions of the AHA (11/13–17/2010)”

Disclosures

None

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