Published online before print December 6, 2007, doi: 10.1161/ATVBAHA.107.152058
© 2008 American Heart Association, Inc.
Cell Biology/Signaling |
Molecular Basis for Staphylococcus aureus–Mediated Platelet Aggregate Formation Under Arterial Shear In Vitro
From Molecular and Cellular Therapeutics (S.W.K., N.C., G.M., D.C.), Royal College of Surgeons in Ireland, Dublin; and the Microbiology Department (A.L., T.J.F.), Moyne Institute of Preventive Medicine, Trinity College, Dublin, Ireland.
Correspondence to Dr Dermot Cox, Cardiovascular Infection Group, Molecular and Cellular Therapeutics, 123 St. Stephens Green, Dublin 2, Ireland. E-mail dcox{at}rcsi.ie
Abstract
Objective— Staphylococcus aureus is the most frequent causative organism of infective endocarditis (IE) and is characterized by thrombus formation on a cardiac valve that can embolize to a distant site. Previously, we showed that S aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) can stimulate rapid platelet aggregation.
Methods and Results— In this study we investigate their relative roles in mediating aggregate formation under physiological shear conditions. Platelets failed to interact with immobilized wild-type S aureus (Newman) at shear rates <500 s–1 but rapidly formed an aggregate at shear rates >800 s–1. Inactivation of the ClfA gene eliminated aggregate formation at any shear rate. Using surrogate hosts that do not interact with platelets bacteria overexpressing ClfA supported rapid aggregate formation under high shear with a similar profile to Newman whereas bacteria overexpressing FnBPA did not. Fibrinogen binding to ClfA was found to be essential for aggregate formation although fibrinogen-coated surfaces only allowed single-platelets to adhere under all shear conditions. Blockade of the platelet immunoglobulin receptor Fc
RIIa inhibited aggregate formation.
Conclusions— Thus, fibrinogen and IgG binding to ClfA is essential for aggregate formation under arterial shear conditions and may explain why S aureus is the major cause of IE.
Both Staphylococcus aureus clumping factor A (Clf A) and fibronectin-binding protein A (Fnbp A) mediate rapid platelet aggregation. However, only Clf A can induce rapid aggregate formation under arterial shear conditions that is dependent on antibody and fibrinogen binding. The corresponding platelet receptors are GPIIb/IIIa and FcRIIa.
Key Words: Staphylococcus aureus • clumping factor A • aggregate formation • fibrinogen • IgG
Infective endocarditis (IE) is characterized by the formation of platelet-bacteria thrombi on a heart valve which, if untreated, can lead to valve failure or the formation of infected emboli.1 Infection of damaged or replacement valves is usually attributable to Streptococcus spp whereas infection of native valves is usually attributable to Staphylococcus aureus.2 Although many species of bacteria have been reported to cause endocarditis the majority of cases are attributable to S aureus.3 The reason for the dominance of S aureus–mediated IE is not known.
The ability of bacteria to interact with platelets has been shown to be important in the pathogenesis of IE.1 Bacteria can adhere to platelets and induce platelet aggregation. However, these are distinct properties and are often mediated by distinct surface proteins.4 Animal studies have shown that the ability of S aureus to adhere to platelets and the ability of S sanguis to induce platelet aggregation are important in infective endocarditis.5–8
The complex interplay between platelets and bacteria is still not completely understood.9 Successful colonization is likely to be the defining event leading to initiation of an infection.1 Several different surface adhesins called microbial surface component reacting with adhesive matrix molecules (MSCRAMMs)10 promote adhesion to and activation of platelets including clumping factor (Clf) A and fibronectin-binding proteins (FnBP) A and B.12 Both ClfA and FnBP bind fibrinogen allowing an interaction with platelet GPIIb/IIIa that supports bacterial adhesion to platelets. In the presence of specific antibodies to the MSCRAMM, engagement of the platelet Fc receptor FcRIIa occurs which stimulates platelet activation and subsequent aggregation.11,12
There are several major concerns with the studies of S aureus–platelet interactions. Firstly, most previous studies have been carried out under static (adhesion) or nonphysiological stirring conditions (aggregation). Therefore, it is difficult to relate these studies to the disease process as cells in the vasculature experience a range of shear conditions. Some studies have been performed in vitro under shear to better characterize platelet-bacteria interactions under more physiological conditions. Studies using a cone and plate viscometer have shown that both protein A and ClfA are important in thrombus formation. However, extremely high shear levels were used.13 A recent study showed a role for ClfA, SdrC, SdrD, SdrE, and protein A in a parallel plate flow chamber.14 An earlier study showed a role for antibody in thrombus formation under shear.15
Secondly, it may not be possible to investigate the interaction between S aureus and platelets using an animal model. Several early studies suggest that platelets isolated from rabbits interact with S aureus differently to platelets isolated from humans. For example, S aureus–induced aggregation of rabbit platelets was biphasic and was not inhibited by an RGD peptide that binds to GPIIb/IIIa.16,17 This is in direct contrast to S aureus–induced aggregation of human platelets which is monophasic and is inhibited by RGD peptides. Moreover, several studies have now highlighted the importance of the platelet Fc receptor, FcRIIa, in platelet activation by S aureus.11,12 Murine platelets do not express FcRIIa,18 suggesting that the mechanism of S aureus–induced platelet aggregation differs between human platelet and murine platelets.
To address this problem we investigated the roles of clumping factor A and fibronectin-binding protein A and serum proteins on S aureus–induced human platelet aggregate formation under venous and arterial shear, using a parallel flow chamber. We show that ClfA mediates rapid aggregate formation under high shear in a process that requires both fibrinogen and anti-ClfA antibodies, whereas FnBPA does not play a role in thrombus formation.
Methods
Materials
Fibrinogen and bovine serum albumin (BSA) were purchased from Calbiochem, Nottingham, UK. Human IgG was obtained from Baxter, UK. Platelet 
IIbβ3 antagonist, tirofiban, was purchased from Merck, UK. The anti-FcRIIa antibody, IV.3 was a kind gift from Dr Kimkowsky, Medarex Inc, Princeton, NJ. Lipophilic dye, 3,3'-dihexyloxacarbocyanine iodide (DiOC6) was purchased from Biosciences, Franklin Lakes, NJ. Flow chambers were purchased from GlycoTech. Glass cover slides were purchased from BioWorld. Bacterial growth media were purchased from Oxoid Ltd. All other reagents were purchased from Sigma.
Bacterial Strains and Growth Conditions
S aureus was grown in brain heart infusion (BHI) broth at 37°C with shaking (200 rpm) for 18 hours (stationary phase). S aureus Newman defective in clumping factor A has been described elsewhere.19 Lactococcus lactis was used to carry empty vector (pKS80) or for heterologous expression of the S aureus surface protein ClfA (pKS80clfA+).20 L lactis was routinely grown in M17 agar or statically in M17 broth incorporating 0.5% (wt/vol) glucose at 30°C for 18 hours.21 S aureus strain 8325-4 which lacks FnBPA and the plasmid containing the entire fnBPA gene have been described elsewhere.22 Bacteria were harvested and washed twice by centrifugation at 15 000g for 5 minutes. Washed bacteria were finally resuspended in phosphate buffered saline (PBS) and adjusted to an optical density of 1.4.
Platelet Preparation
Nine volumes of whole blood from healthy human adult volunteers were collected into 1 volume of 3.8% sodium citrate. Platelet-rich plasma (PRP) was prepared as previously described.4 To prepare platelets free of plasma proteins, prostaglandin (PG) E1 (1 µmol/L) and apyrase (1U/mL) were added to PRP and centrifuged at 650g for 10 minutes. The plasma was removed and the platelet pellet resuspended in the same volume of JNL buffer (6 mmol/L dextrose, 130 mmol/L NaCl, 9 mmol/L NaCl2, 10 mmol/L Na citrate, 10 mmol/L Tris base, 3 mmol/L KCL, 0.8 mmol/L KH2PO4, and 0.9 mmol/L MgCl2).
Preparation of Flow Chamber Slides
A 1 mL solution of either fibrinogen (20 µg/mL) or S aureus (109 bacterial cells) was applied to glass slides (75x25 mm) and allowed to attach overnight at 4°C or for 2 hours at room temperature in a humidity chamber. Uniform bacterial coverage on glass slides was verified by crystal violet staining. Slides were washed 3 times in PBS to remove any unbound bacteria or fibrinogen. Finally, the slides were blocked with 1% BSA for a further 1 hour at 37°C.
L lactis failed to adhere to the glass slides (probably because of its surface charge) therefore it was necessary to generate appropriate charges on both the glass slides and bacterial surface. Glass slides were submersed in 1% Triton-X 100 for 30 minutes and washed in running tap water for a further 30 minutes. The slides were then sterilized using 95% EtOH and allowed air dry. To produce a covalently bound amino group on the glass surface the slides were submersed in freshly prepared 2% solution of 3-aminopropyltriethoxysilane in dry acetone for 5 seconds. Finally, glass slides were washed in distilled water (5 minutes x 2) and dried overnight at 42°C. This method produces a surface covered in amino groups which will covalently bind to free aldehydes. Bacterial cells were centrifuged at 15 000g. Pellets were resuspended in 5 ml 0.5% formaldehyde for 1 hour at room temperature with occasional mixing. The suspension of bacterial cells was finally centrifuged at 15 000g and the resultant pellet resuspended and adjusted to a final OD of 1.4 using PBS. This method creates a bacterial surface covered in free aldehydes capable of covalently binding to the amino groups on the silane treated slides.
Epi-Fluorescence Digital Microscopy
Platelet interaction with immobilized protein or bacteria under various flow conditions was studied using a parallel flow chamber. Platelets were labeled in whole blood by incubation with DiOC6. A syringe pump (Harvard Biosciences) was used to aspirate blood through the flow chamber. Platelet adhesion and aggregate formation was visualized using phase contrast and fluorescent imaging microscopy (63X oil immersion lens-Achroplan objective) through the flow chamber mounted on a Zeiss Axiovert-200 epi-fluorescence microscope (Carl Zeiss). Images were taken at the same downstream location (from flow chamber entrance) in all experiments. Images were captured every second up to 300 s by a liquid chilled Quantix-57 charge-coupled device (CCD) camera (Photometrics Ltd). Platelet adhesion and aggregate formation was analyzed using MetaMorph software (Universal Imaging Corp).
Statistical Analysis
Data shown are the means plus or minus standard error of the mean, and comparisons between mean values were performed using the Student paired or unpaired t test.
Results
Aggregate Formation on Immobilized S aureus Strain Newman Under Shear Conditions
Using bright-field-imaging microscopy platelets (visualized as black cells) perfused under low shear conditions (50 s–1) in plasma failed to interact with immobilized S aureus Newman after 300 seconds (Figure 1A). At higher shear rates (>100 s–1) cells are concentrated in the center of the flow stream making interaction with the surface difficult, however if whole blood is used the larger cells (red and white blood cells) are carried to the center of the main stream of flow, leaving the smaller cells (platelets) in a better position to interact with immobilized bacteria on the glass slides. Platelets in whole blood stained with a lipophilic dye also failed to interact with immobilized S aureus Newman when the shear rate was increased to 200 s–1 or 500 s–1 (data not shown).
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Increasing the shear rate to 800 s–1 led to adhesion of platelets to immobilized S aureus strain Newman. Platelet aggregates began to form (white areas) within 100 seconds and became progressively larger as perfusion continued (Figure 1B). After 300 seconds aggregate formation covered almost the entire visible field of view (1±0.2% coverage at 50 s–1 and 90±5% coverage at 800 s–1, n=3, P<0.0001). Perfusing whole blood at 1500 s–1 led to more rapid platelet deposition and aggregate formation than at 800 s–1. Large platelet aggregates began to form at 100 seconds leading to complete occlusion of the field of view by 200 seconds. After 250 seconds the aggregate fragmented leaving behind the immobilized S aureus Newman. With further perfusion of platelets the process of aggregate formation began again on the remaining immobilized S aureus Newman. Platelets perfused at 800 s–1 failed to interact with S aureus strain 8325-4 (Figure 1C, 90±5% coverage on Newman and 0±0.0% coverage on 8325-4, n=3, P<0.0001).
Aggregate Formation on Immobilized S aureus Strain Newman Lacking ClfA Expression
The ability of the ClfA-containing strain Newman to induce platelet aggregation is in contrast to the lack of activity of strain 8325-4 which does not contain ClfA and suggests a role for ClfA in the interaction with platelets. It also suggests that this protein may play a more dominant role in aggregate formation than FnBPA. To address this question we studied a ClfA mutant of S aureus Newman (ClfA–). Platelets in whole blood perfused at 800 s–1 failed to interact with Newman ClfA– after 300 seconds (Figure 2B; 90±5% coverage on Newman ClfA+ and 2±1% coverage on Newman ClfA–, n=3, P<0.0001) or at the higher shear rate of 1500 s–1 (data not shown).
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Aggregate Formation on Immobilized Surrogate Hosts Overexpressing ClfA or FnBPA
To confirm the relative importance of ClfA in mediating aggregate formation under high shear both ClfA and FnBP A were separately overexpressed in surrogate hosts. Platelets perfused in whole blood failed to interact with fixed L lactis at any shear rate tested (Figure 3A). However, platelets perfused at 800 s–1 time-dependently adhered to fixed L lactis expressing ClfA. After 100 seconds large platelet aggregates formed with eventual complete occlusion at 300 seconds (Figure 3B) in a similar fashion to S aureus Newman (0.5±0.1% coverage on L lactis pKS80 and 89±4% coverage on L lactis ClfA+, n=3, P<0.0001). In contrast, platelets perfused in whole blood at 800 s–1 failed to interact with either S aureus strain 8325-4 (Figure 3C) or S aureus 8325-4 overexpressing FnBPA (0±0.0% coverage on 8325-4 FnBPA– and 1±0.2% coverage on FnBP+, n=3, NS). Comparative analysis by flow cytometry and Western blot of S aureus Newman and L lactis cells expressing ClfA showed that L lactis expressed similar levels of protein on their surface compared with S aureus. Furthermore, Western blot data demonstrated high levels of FnBPA in S aureus strain 8325-4 (data not shown).
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Platelet Adhesion to Immobilized Fibrinogen at High and Low Shear
We next investigated the molecular mechanisms of ClfA-induced aggregate formation. Fibrinogen plays a major role in platelet aggregate formation, an important step in thrombus formation. To investigate whether the platelet interaction with S aureus was simply due to ClfA binding fibrinogen and presenting passing platelets with a thrombogenic surface, we immobilized purified fibrinogen and perfused platelets at both high and low shear. Under low shear conditions (150 s–1) single platelets adhered to the immobilized fibrinogen in a time-dependent manner (Figure 4A). Increasing the shear rate to 800 s–1 also led to the development of a monolayer of single platelets but no aggregate was formed even at 300 seconds (Figure 4B). These results suggest that fibrinogen binding to ClfA is not sufficient to induce aggregate formation alone and that other plasma factors may play a role.
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L. Lactis ClfA-Induced Platelet Activation
To dissect the mechanisms leading to aggregate formation, whole blood was treated with aspirin (10 µmol/L) and perfused over immobilized L lactis ClfA at 800 s–1. Aggregate formation was strongly inhibited but single platelet adhesion occurred (Figure 5C; 89±4% coverage with L lactis ClfA and 7±1% coverage when treated with aspirin, n=3, P<0.0001). We next assessed the importance of fibrinogen binding to platelets in initiating aggregate formation. Addition of the GPIIb/IIIa antagonist, tirofiban (0.5 µmol/L) to blood completely inhibited both aggregate formation and single platelet adhesion (Figure 5D; 89±4% coverage over L lactis ClfA and 4±0.1% coverage in the presence of tirofiban, n=3, P<0.0001). These results suggest that GPIIb/IIIa and cyclooxygenase play an essential role in aggregate formation in this model system.
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Role of Plasma Proteins in S aureus ClfA–Induced Aggregate Formation
As platelets perfused under high shear (800 s–1) are capable of interacting with immobilized L lactis ClfA we set out to determine whether plasma factors are involved in aggregate formation. Single platelet adhesion was observed when gel-filtered platelets were combined with washed red blood cells and perfused over immobilized L lactis ClfA (Figure 6A; 13±2% coverage in plasma-free platelets perfused over L lactis ClfA and 89±4% coverage when plasma was included, n=3, P<0.001). Single platelet adhesion was also observed when antibody purified fibrinogen was also added (Figure 5B and 6
B, 89±4% coverage with plasma-rich platelets perfused over L lactis ClfA and 9±0.2% coverage with plasma-free platelets supplemented with fibrinogen, n=3, P<0.002). No interaction was observed when pooled human IgG was added (Figure 6C; 89±4% coverage with plasma-rich platelets perfused over L lactis ClfA and 2±0.5% coverage with plasma-free platelets supplemented with IgG, n=3, P<0.0001). However, when washed blood cells were combined with fibrinogen and pooled human IgG time-dependent aggregate formation (Figure 6D) similar to that with whole blood was observed (89±4% coverage with plasma-rich platelets perfused over L lactis ClfA and 74±3% coverage with plasma-free platelets supplemented with fibrinogen and IgG, n=3, P=NS). When gel-filtered platelets were treated with the monoclonal antibody IV-3 aggregate formation was reduced to single platelet adhesion (Figure 6E; 89±4% coverage with plasma-rich platelets perfused over L lactis ClfA and 8±2% coverage in the presence of IV.3, n=3, P<0.002).
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Discussion
S aureus is the major cause of infective endocarditis and its ability to interact with platelets is essential for infection of heart valves.23 Previous studies have shown that S aureus expresses a number of proteins capable of activating platelets including ClfA, FnBPA, and FnBPB.21,12 However, the relative importance of the individual proteins is not clear. This is complicated by the fact that the roles of these proteins have been characterized with in vitro assays such as platelet aggregation which may not reflect the situation in vivo.
Using a parallel plate chamber and a range of shear rates we investigated the ability of platelets to interact with immobilized S aureus. The S aureus strain Newman expressing ClfA induces platelet aggregation with a short lag time and strongly supports platelet adhesion under static conditions in the presence of fibrinogen. Under low shear conditions (venous shear) there is no evidence of platelet adhesion, presumably because of the weakness of the interaction which cannot support platelet attachment under shear stress. However, under high shear conditions (arterial shear) very strong adhesion occurred followed by rapid aggregate formation. This is a unique interaction in that other IE microorganisms such as S sanguis24 or S gordonii25 failed to interact at high shear however did interact at low shear. The aggregate formation is not solely attributable to platelets attaching to fibrinogen bound to the surface of immobilized bacteria, as immobilized fibrinogen only supported single platelet adhesion without aggregate formation. This phenomenon of only supporting platelet adhesion under high shear is similar to that seen with vWf, although it is not involved in the interaction described here. Fibrinogen bound to ClfA is subject to shear stress under flow conditions resulting in an alteration in its conformation. This altered conformation of fibrinogen may be more effective at interacting with GPIIb/IIIa under high shear. However, under low shear conditions fibrinogen can still interact with GPIIb/IIIa, but this interaction is not strong enough to resist the shear stress, preventing thrombus formation.
To characterize this interaction further, we examined the role of MSCRAMMs, the major platelet-interacting proteins of S aureus. A ClfA-defective mutant (ClfA–) of S aureus Newman could still induce platelet aggregation (albeit with a longer lag time) and support platelet adhesion (data not shown). However, the mutant failed to support strong adhesion or aggregate formation under any shear condition. The essential role of ClfA in the interaction was confirmed by expression in the surrogate host L lactis. L lactis ClfA+ was as effective as Newman at inducing aggregation (data not shown) and adhesion under shear. Thus, although there are a number of surface proteins on Newman capable of activating platelets, the only one that can trigger aggregate formation under shear is ClfA.
Pawar et al demonstrated that protein A has the capability of binding to vWf under high shear but not low shear conditions.13 This interaction acts as a bridging mechanism for crosslinking to platelet GPIb thereby initiating platelet adhesion. These experiments were carried out with S aureus cells in suspension mixed with PRP and subjected to shear rates only found in severe pathological conditions (5000 s–1). A more recent study demonstrated that L lactis expressing protein A adhered time-dependently to immobilized vWf at low shear rates (100 s–1).26 Together these results suggest that the protein A–vWf interaction may mediate the initial interaction leading to immobilization of bacteria at the site of injury, but it is unlikely that protein A alone causes thrombus formation. We have shown that platelets fail to interact with immobilized L lactis expressing protein A at any shear rate,26 so it is more likely to act as a costimulator rather than a primary activator.
Previous studies have shown that ClfA binds to fibrinogen which in turn binds to the platelet integrin GPIIb/IIIa. However, binding fibrinogen per se is not enough to induce platelet aggregation. When platelets were perfused over immobilized fibrinogen at any shear, single platelet adhesion was observed but not aggregate formation. S aureus is a common commensal of the human body and antibodies to surface proteins are present in the plasma of most if not all healthy individuals. We have shown previously that specific anti-ClfA antibodies are required for ClfA-induced platelet aggregation,11 therefore we investigated their role in platelet interactions with bacteria under shear stress. Antibody binding to ClfA is itself not sufficient to induce aggregate formation. However, when fibrinogen and specific antibody together bound to ClfA, aggregate formation occurred. The role of antibody is to interact with the platelet Fc receptor FcRIIa. A function-blocking monoclonal antibody directed against FcRIIa inhibited the rapid aggregate formation. A requirement for antibody is in agreement with the previous study of Sjobring and coworkers.15
Several studies have recently identified the binding capabilities of S aureus FnBPs to human blood platelets.12,27 Our results presented here do not disagree with these findings but have demonstrated that under high shear conditions FnBPs do not have the ability to induce aggregate formation. Similarly recent studies have suggested that FnBPs play a role in binding to healthy resting endothelial cells,28 however under shear this interaction does not take place.29 Collectively these results suggest that the fibronectin-binding proteins may have little or no role in aggregate formation, but as IE is a complex disease they may still play a role in the pathology of the disease. It is also important to note that although there is high shear in the coronary vessels the situation is much more complex at the valve surface where turbulence is also likely to play a role.
In summary, the results from this study demonstrate that the simultaneous binding of fibrinogen and antibody to ClfA, which interact with GPIIb/IIIa and FcRIIa on the platelet surface, is essential for aggregate formation. This rapid aggregate formation is unique to ClfA and may explain the high incidence of S aureus in infective endocarditis as it is a very powerful trigger of aggregate formation in the presence of anti–S aureus antibody. ClfA may be a good candidate for a vaccine for patients at high risk of IE. However, it is essential that the antibodies generated would block the ClfA-fibrinogen interaction, otherwise the presence of high levels of nonneutralizing antibody would likely exacerbate IE.
Acknowledgments
Sources of Funding
This work was funded by a postdoctoral research fellowship (to S.W.K.) and a program grant (to T.J.F. and D.C.) from the Health Research Board of Ireland.
Disclosures
None.
Footnotes
Original received July 18, 2007; final version accepted November 21, 2007.
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