A peptide from the staphylococcal protein Efb binds P‐selectin and inhibits the interaction of platelets with leukocytes

P‐selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N‐terminal domain of Staphylococcus aureus extracellular fibrinogen‐binding protein (Efb) binds to P‐selectin and interferes with platelet‐leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet‐leukocyte aggregates.


| INTRODUC TI ON
Our understanding of the role of platelets in vascular health has significantly progressed in recent years. In addition to platelets' established role in hemostasis and thrombosis, their participation in inflammatory responses has become evident. 1 The role of platelet pro-inflammatory activity for progression of cardiovascular disease has attracted considerable attention. 2 In addition to their ability to release pro-inflammatory cytokines 3 and modulate the release of cytokines by leukocytes, 4 platelets directly interact with leukocytes to form platelet-leukocyte aggregates (PLAs). 5 PLA formation has been shown to facilitate leukocyte homing and extravasation at the site of vascular injury, thus promoting inflammation. 6 Inflammation is a key factor in vascular complications and cardiovascular diseases. 7 PLAs are in fact increased in coronary syndromes, in the form of platelet-monocyte [8][9][10][11][12] and platelet-neutrophil complexes. 13 PLA formation also increases as a consequence of coronary surgical intervention. 14 Although CD40-CD40L interactions 15 and the binding of the leukocyte receptor CD11b/CD18 (Mac-1) with either platelet glycoprotein receptor GPIBα 16 or platelet integrin α IIb β3 17 participate in the formation of PLAs, the interaction of platelet P-selectin and its physiological ligand P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes is critical for the heterotypic aggregation of these cell types. 18 The interaction of platelets with neutrophils has particularly important pathophysiological consequences. Platelets can induce the formation of neutrophil extracellular traps (NETs). 19 While NETs have originally been described in host-defense processes, 20 their role in the onset and progression of venous and arterial thrombosis has been shown in animal models and clinical studies. [21][22][23] As for PLAs, the binding of P-selectin on platelets with PSGL-1 on leukocytes is a critical step in NET formation. 24 Because of its role in the interaction of platelets with leukocytes, P-selectin has attracted increasing interest as a drug discovery target to develop pharmacobiological agents able to control inflammation and vascular degeneration via disruption or reduced formation of PLAs and NETs. The clinical potential of P-selectin inhibitors requires further investigation. We have shown previously that extracellular fibrinogen binding protein (Efb), a protein secreted by Staphylococcus aureus (S. aureus), directly binds P-selectin and inhibits its interaction with PSGL-1. 25 In contrast to numerous bacterial proteins that have been reported to positively modulate platelet function, Efb inhibits platelet activation and thrombus formation, 26,27 facilitating bacterial survival in the blood and aggravating infection. 28 Efb comprises an N-terminal secretion signal, a N-terminal domain lacking structural organization (Efb-N, residues Ser30-Thr104), and a tri-helical bundle C-terminal domain (Efb-C, residues Ile105-Lys165) 29 ( Figure 1A).
Efb-N includes two repeated motifs (residues Asn46-Pro67 and Asn77-Ala98) that are homologous to S. aureus coagulase repeats and that are part of fibrinogen binding sites comprising residues Ser30-Pro67 and Lys68-Ala98. 30 Efb-C plays an immunosuppressive role by interfering with the complement system. 29,31 In combination, Efb-N and Efb-C facilitate S. aureus escape from phagocytosis and increase S. aureus pathogenicity. 32 In fact, S. aureus infections are significantly exacerbated in vivo in the presence of Efb. 33 In this study, we mapped the P-selectin-binding site in Efb-N using a peptide scanning approach. A 20 aa-peptide located within Efb-N between Lys68 and Glu87 bound P-selectin. We show here that this peptide selectively binds platelets, without affecting their hemostatic function, and inhibits platelets' ability both to complex with leukocytes to form PLAs and to induce NET formation.
The therapeutic potential of this Efb-derived peptide to control thromboinflammation in cardiovascular patients will require further investigation.
In order to fluorescently label lysine residues, synthetic peptides (or recombinant Efb-N 30-

| Blood collection and platelet isolation
Procedures using human blood conformed to the principles outlined in the Declaration of Helsinki. Human blood was collected at the Institute of Clinical Chemistry and Laboratory Medicine (University Medical Center Eppendorf -Hamburg) after informed volunteers' consent was given in written form. Sodium citrate (0.5% w/v) was used as an anticoagulant. Platelet-rich plasma (PRP) was separated from whole blood by centrifugation (250 g, 17 min) and platelets were separated from PRP by a second centrifugation step (500 g, 10 min) in the presence of prostaglandin E1 (PGE1, 40 ng/ml) and indomethacin (10 μM). All centrifugations were performed with soft deceleration settings. Platelets were resuspended in modified Tyrode's buffer at a density of 2 × 10 8 platelets/ml throughout the study.

| Flow cytometry (Efb binding)
For isolated platelet binding, 10 7 platelets/ml were incubated in the presence or absence of 3 mg/ml fibrinogen and 0.2 U/ml thrombin for 10 min at room temperature. 1 µM FITC-Efb peptide, FITCscrambled Efb peptide or FITC-BSA was then added to the platelets followed by another 10 min incubation. Platelet binding was assessed by flow cytometry using a FACSAria III (BD Biosciences).
Alternatively, 10 µM FITC-conjugated Efb peptide was incubated for 30 min at room temperature in heparin-anticoagulated (5 U/ml) human blood with CD42b/PE (#561854, BD Biosciences) and CD45/APC (#555745, BD Biosciences) antibodies. Cells were then fixed with 1% paraformaldehyde (PFA) for 15 min and binding was assessed by flow cytometry using a FACSCanto II (BD Biosciences). The signal peptide is shown in light blue, the first fibrinogen binding motif (Fg B1) in red, the second fibrinogen binding motif (Fg B2) in green. Efb peptide binding to resting (B), thrombin-stimulated (C) or fibrinogen-treated (D) platelets. 1 µM FITC-labelled Efb or scrambled control peptides were incubated with isolated human platelets and their binding was assessed by flow cytometry. In (C), thrombin stimulation was obtained with 0.1 μ/ml human thrombin for 10 min, while in (D) platelets were treated with 3 mg/ml fibrinogen for 10 min. Values are the median fluorescence intensity (MFI) from 10 000 events in five independent experiments (mean ± SEM). Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test; p <.05 (*), p <.01 (**), p <.001 (***)

| Isolated platelet fluorescence imaging
Coverslips were coated with 0.01% w/v poly-L-lysine and blocked with 0.1% w/v solution of BSA in PBS. Isolated platelets (resting, or activated in suspension with 10 µM TRAP6 peptide for 5 min at 37°C) were dispensed onto the prepared coverslips at 0.5 × 10 7 platelets/ml density and incubated for 1 h at RT. Platelets were then fixed for 15 min in 4% PFA and stained with 2 μM solution of FITC-labelled peptide (Efb 68-87 or Efb 68-87 (ctrl)) and, subsequently, with 1 U/ml phalloidin-rhodamine in PBS-Tween20 0.1% v/v. The coverslips were mounted onto the glass slides with Fluoromount mounting medium. Imaging was performed with a Leica TCS SP5 confocal microscope.

| Dot blots
200 ng of Fc-P-selectin (R&D Systems, # 137-PS-050) was dotted onto nitrocellulose membrane and air-dried. The membrane was then blocked with 5% w/v BSA in TBS-T for 30 minutes before being incubated with 1.0 μg/ml FITC-Efb peptides (labelling described above) for 45 minutes. Following three washes in TBS-T, imaging was performed with a Licor Odyssey CLx scanner.  Signaling Technology) and phospho-Src (R&D Systems, # AF2685).

| Pull-down experiments
Densitometry was performed using ImageJ 1.47v (Wayne Rasband, National Institute of Health, US) and presented as intensity ratio over actin staining (i.e. loading control).

| Platelet aggregation
Platelets resuspended in modified Tyrode's buffer at a density of 2 × 10 8 platelets/ml were stimulated using a Chrono-Log 490 4+4 aggregometer. Aggregation was induced with 0.1 U/ml human thrombin or 3 µg/ml Horm collagen. Absorbance was measured for 10 minutes and expressed as percentage change of absorbance.

| PLA quantification
Heparin-anticoagulated (5 U/ml) human blood was treated with PLAs were quantified as events positively stained for both markers (see Figure 5A) and their density was expressed as number of double-positive events over 10 000 events.

| NET visualization and quantification
Neutrophils were isolated from human peripheral blood as previously described. 34 5 × 10 4 neutrophils per well were seeded in 96-well F I G U R E 2 Efb 68-87 binding to platelet P-selectin. (A) Phalloidin-rhodamine (red, 1 U/ml) and FITC-labelled Efb 68-87 (green, 2 μM) were used to stain human platelets fixed either before or after stimulation in suspension with TRAP6. FITC-labelled scrambled Efb 68-87 was used as control.

| Statistical analysis
Data normality and homoscedasticity were tested with Shapiro-

| Efb 68-87 selectively binds platelets and captures them from blood under flow
Amongst blood cells, P-selectin is selectively expressed in platelets. Therefore, we assessed whether Efb 68-87 selectively binds platelets or might interact with other blood cells. Antibodies for plateletspecific (CD42b) and leukocyte-specific (CD45) markers were utilized in flow cytometry to identify platelets and leukocytes (white blood cells (WBCs) and red blood cells (RBCs)) ( Figure 3A). FITC-Efb 68-87 exclusively bound to platelets, while binding of WBCs and RBCs was negligible. We then used an adhesion assay under physiological flow to probe the stability of Efb 68-87 binding to platelets, F I G U R E 3 Efb 68-87 binds to platelets in whole blood without interfering with collagen-dependent thrombus formation. (A) CD42b/PE and CD45-APC antibodies were used to distinguish platelets (CD42b + ), leukocytes (CD42b − , CD45 + ) and red blood cells (CD42b − , CD45 − ) from human blood by flow cytometry. FITC-Efb 68-87 binding to each cell population was tested by flow cytometry. Data are mean ± SEM of the MFI values from 10 000 events in six independent experiments. Statistical significance was assessed using one-way ANOVA with Bonferroni post-hoc test: p <.05 (*), p <.01 (**), p <.001 (***). (B) Platelet adhesion to Efb 68-87 under physiological flow. Platelet adhesion to Efb 68-87 was tested at shear stress 200 sec −1 (venous) and 1000 sec −1 (arterial, not shown). In addition, (C) the effect of Efb 68-87 (5 μM) on platelet adhesion and thrombus formation on collagen was tested (1000 sec −1 ). Platelets in whole blood were fluorescently labelled with DiOC 6 and their surface coverage was assessed by fluorescence imaging/densitometry analysis. Data presented are mean ± SEM of the surface coverage from 6 independent experiments. Statistical significance was assessed using the paired sample Student t-test (normality was assessed by Shapiro-Wilk test): p <.05 (*), p <.01 (**), p <.001 (***) Resting and thrombin-stimulated (1 U/ml) platelets were compared for degranulation (anti-CD42b/APC) and integrin α IIb β 3 activation (anti-PAC-1/FITC) in the presence of 5 µM Efb 68-87 or scrambled Efb 68-87 . A representative forward scattering (FSC)/side scattering (SSC) dot plot of isolated platelets in the presence of Efb 68-87 at resting (blue) and thrombin-stimulated (red) is also shown. The statistical significance between Efb 68-87 and the control peptide was assessed using one-way ANOVA with Bonferroni post-test; p <.01 (**), p <.001 (***), error bars represent mean ± SEM from four independent experiments. (C) Platelet signalling was studied by immunoblotting in the presence of Efb 68-87 or Efb 68-87 ctrl peptides (10 μM). Platelets were activated with 0.1 U/ml thrombin (in the presence of 1 mM EGTA to avoid platelet aggregation and so facilitate protein extraction). Following lysis and SDS-PAGE, lysates were immunoblotted for protein kinase C (PKC) phosphorylated substrates of classical protein kinase C (PKC) isoforms, phosphorylated Src and actin. Data were quantified by densitometry using ImageJ 1.47v, presented as intensity ratio over actin staining (i.e. loading control) and are mean ± SEM from five independent experiments. No statistical significance was detected using a paired sample Student t-test (normality was assessed by Shapiro-Wilk test) assessing whether Efb 68-87 can be used to capture platelets from whole blood. Platelets bound to surfaces coated with Efb 68-87 at venous (200 s −1 ) ( Figure 3B), but not arterial shear stress (1000 s −1 ) ( Figure S2). Interestingly, platelet adhesion and thrombus formation on fibrous collagen were not affected by Efb 68-87 ( Figure 3C), suggesting that Efb 68-87 does not interfere with platelet function.

| Efb 68-87 does not interfere with platelet activation and hemostatic function
We investigated functional implications of Efb 68-87 for platelet function. Platelet aggregation in response to the agonists collagen and thrombin was not affected by platelet pre-treatment with Efb 68-87 compared to Efb 68-87 (ctrl) ( Figure 4A). The aggregation in response to secondary agonists ADP and U46619 (a stable analogue of thromboxane A 2 ) was also unaffected by Efb 68-87 (Figure 3). In addition, platelet activation and degranulation were tested by flow cytometry. Data showed that the presence of Efb 68-87 did not significantly change integrin α IIb β 3 activation (PAC1 antibody) or platelet degranulation (P-selectin externalization) compared to Efb 68-87 (ctrl) ( Figure 4B). Finally, potential effect of Efb 68-87 incubation on intracellular platelets signaling was tested by immunoblotting ( Figure 4C).
Basal (resting platelets) and activated (thrombin-stimulated platelets) levels of PKC activity and Src kinase activation in the presence of Efb 68-87 and Efb 68-87 (ctrl) were assessed using an anti-phospho PKC substrate antibody (detecting various proteins phosphorylated by classical platelet PKC isoforms α and β) and anti-phospho Src antibody (detecting Src in its active/phosphorylated form). Treatment with Efb 68-87 did not affect activity of these two major platelet signaling pathways. Collectively, these results confirm that Efb 68-87 binding does not interfere with platelet function.

| Efb 68-87 inhibits formation of plateletleukocyte aggregates and reduces formation of neutrophil extracellular traps
The formation of heterotypic cellular complexes between platelets and leukocytes (i.e. platelet-leukocyte aggregates or PLAs) is an important driver of thromboinflammation. Since PLA formation depends on binding of platelet P-selectin to leukocyte PSGL-1, we tested whether Efb 68-87 can block the formation of PLAs in human whole blood. PLAs were detected by flow cytometry as an event highly stained by both anti-CD42b and anti-CD45 antibodies ( Figure 5A). PLA levels were significantly increased by stimulation of whole blood with the GPVI receptor agonist CRP, but not the PAR1 agonist TRAP6 or the Toll-like receptor (TLR) agonist LPS used separately ( Figure S4).
The study of Efb 68-87 's effect on PLA formation was performed with combined stimulation by CRP, TRAP6 and LPS. Substantial inhibition of PLA formation was observed at 1 μM or higher concentrations of Efb 68-87 ( Figure 5B). Since NET formation is another process dependent on P-selectin binding to PSGL-1, 24 we next investigated whether Efb 68-87 interferes with the stimulation of NET formation by platelets.
Neutrophils isolated from human peripheral blood were allowed to adhere and were then treated with platelets. Resting platelets were compared to platelets treated with TRAP6 (5 µM). Stimulated platelets were able to induce a significant formation of NETs (Figure 6), which was inhibited by Efb 68-87 , but not with the control peptide for  6,36 The interaction of P-selectin with PSGL-1 is the key molecular event for PLA formation. 4 Other key surface receptors involved in PLA formation are activated in response to P-selectin-PSGL-1 interaction.
The activation of the leukocyte integrin α M β2 (macrophage-1 antigen or MAC-1) by P-selectin-PSGL-1 interaction, for example, is a key step in the formation of heterotypic complexes between platelets and leukocytes. 37 In addition to linking inflammation with platelet activation and thrombus formation, and supporting the concept of thromboinflammation, 38  timely and unresolved challenge for vascular drug discovery. In this study, we therefore followed a different approach. Based on our previous work showing that the bacterial protein Efb binds directly to P-selectin, inhibits its interaction with PSGL-1 and interferes with PLA formation, 25 we set out to identify an Efb-derived peptide that retains the ability to bind P-selectin and inhibit PLA formation.
The peptide we identified is Efb 68-87 , a twenty amino acid sequence from the N-terminal domain of Efb and part of one of two previously identified Efb binding sites for fibrinogen. 30 Efb 68-87 binding to platelets is not increased by the addition of exogenous fibrinogen and is therefore fibrinogen-independent ( Figure 1D). On the other hand, Efb 30-67 interacts with platelets only in the presence of exogenous fibrinogen. Efb  corresponds to the other previously identified fibrinogen binding site. 30 Interestingly, Efb 68-87 shows similar binding affinity to resting and stimulated platelets, but the Efb 68-87 binding capacity of platelets (i.e. amount of peptide bound per platelet) is significantly increased by stimulation ( Figure 1B,C). This binding profile is compatible with a binding site that is present at a low level on the surface of resting platelets but that undergoes an activation-dependent increase in level. This is characteristic of P-selectin levels which increase on the surface of platelets as a consequence of stimulation-dependent degranulation (i.e. migration to the cell periphery and fusion with the plasma membrane of Pselectin-rich alpha granules). 46 These data were supported by dot blot experiments, which confirmed direct binding of Efb 68-87 to Pselectin ( Figure 2C). Efb 68-99 was also able to bind to P-selectin in the dot blot experiments, although the level of staining was visibly lower. Further experiments would be required to assess whether the residues between Tyr88 and Ala99 may interfere with the binding of P-selectin. It is also noteworthy that contrarily to Efb 30-105 (also known as Efb-N), 25 Efb 68-87 does not interact with multimerin-1 and thrombospondin-1. Since the interaction of Efb-N with multimerin-1 and thrombospondin-1 was detected with the same approach used in this study (i.e. peptide conjugation and platelet protein pull-down), the most likely explanation is that the binding sites for these proteins We demonstrated in addition that Efb 68-87 does not bind to other blood cells ( Figure 3A) and that Efb 68-87 does not interfere with the activation and function of platelets in response to physiological stimuli ( Figure 4). Efb 68-87 can therefore be used as a tool to selectively label platelets without affecting their responsiveness, which could find application in research and diagnostic laboratory practice.
The ability to sequester platelets from whole blood ( Figure 3B) could be investigated further to develop a novel platelet isolation method.
Since Efb 68-87 displayed increased binding to activated platelets ( Figure 2B), it may be possible to develop a method to selectively capture activated platelets from whole blood. The selective reduction of the count of circulating activated platelets could find clinical application because an increase in circulating activated platelets is observed in several cardiovascular diseases and is proposed as a disease mechanism leading to thrombosis. 49,50 In summary, we present here the identification and validation of Efb 68-87 , a novel P-selectin antagonist derived from the N-terminal domain of the S. aureus protein Efb. In addition to confirming the selective binding to P-selectin, we have established the ability of Efb 68-87 to interfere with PLA and platelet-induced NET formation in vitro without affecting platelet signaling and platelet functional responses. As the formation of PLAs and NETs is critical for the progression of vascular inflammation and its association with thrombotic complications, Efb 68-87 has the potential to become a clinical tool for the treatment of conditions ranging from major blood vessel atherosclerosis to microcirculatory dysfunction. Future clinical studies on this peptide may lead to the development of a novel treatment to help in the battle against thromboinflammatory conditions.

ACK N OWLED G EM ENTS
The authors thank the DFG-funded Cytometry and Cell Sorting Core Unit at the UKE hospital (Hamburg). This work was funded by the British Heart Foundation for the financial support of SW, SB and GP (FS/17/13/3269), the Werner Otto Foundation for the financial support of NW and GP (BN3/97) and the European Research Council for the support of GP (EU project 101025074). Open access funding enabled and organized by ProjektDEAL.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.