The formyl peptide fMLF primes platelet activation and augments thrombus formation

Abstract Essentials The role of formyl peptide receptor 1 (FPR1) and its ligand, fMLF, in the regulation of platelet function, hemostasis, and thrombosis is largely unknown. Fpr1‐deficient mice and selective inhibitors for FPR1 were used to investigate the function of fMLF and FPR1 in platelets. N‐formyl‐methionyl‐leucyl‐phenylalanine primes platelet activation and augments thrombus formation, mainly through FPR1 in platelets. Formyl peptide receptor 1 plays a pivotal role in the regulation of platelet function. Background Formyl peptide receptors (FPRs) play pivotal roles in the regulation of innate immunity and host defense. The FPRs include three family members: FPR1, FPR2/ALX, and FPR3. The activation of FPR1 by its high‐affinity ligand, N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLF) (a bacterial chemoattractant peptide), triggers intracellular signaling in immune cells such as neutrophils and exacerbates inflammatory responses to accelerate the clearance of microbial infection. Notably, fMLF has been demonstrated to induce intracellular calcium mobilization and chemotaxis in platelets that are known to play significant roles in the regulation of innate immunity and inflammatory responses. Despite a plethora of research focused on the roles of FPR1 and its ligands such as fMLF on the modulation of immune responses, their impact on the regulation of hemostasis and thrombosis remains unexplored. Objective To determine the effects of fMLF on the modulation of platelet reactivity, hemostasis, and thrombus formation. Methods Selective inhibitors for FPR1 and Fpr1‐deficient mice were used to determine the effects of fMLF and FPR1 on platelets using various platelet functional assays. Results N‐formyl‐methionyl‐leucyl‐phenylalanine primes platelet activation through inducing distinctive functions and enhances thrombus formation under arterial flow conditions. Moreover, FPR1 regulates normal platelet function as its deficiency in mouse or blockade in human platelets using a pharmacological inhibitor resulted in diminished agonist‐induced platelet activation. Conclusion Since FPR1 plays critical roles in numerous disease conditions, its influence on the modulation of platelet activation and thrombus formation may provide insights into the mechanisms that control platelet‐mediated complications under diverse pathological settings.


| INTRODUC TI ON
Platelets are small circulating blood cells that play indispensable roles in the regulation of hemostasis to prevent excessive bleeding upon vascular injury. However, their unwarranted activation under pathological conditions leads to the formation of blood clots (thrombi) within the circulation. 1 This results in reduced/retarded blood supply to vital organs including the heart and brain, which leads to heart attacks or strokes, respectively. 2 Moreover, platelet activation during microbial infection results in their aggregation, thrombus formation in the microvasculature, and, in later stages, sequestration of platelets in organs such as the lungs, instigating thrombocytopenia and bleeding complications. 3 In addition to their prominent roles in hemostasis and thrombosis, platelets play a crucial role in the regulation of innate immunity, inflammatory responses, and clearance of microbial infection. [4][5][6] Platelets contain a broad spectrum of receptors that induce inflammatory responses during microbial infection and other pathological conditions. In addition, platelets secrete various inflammatory and immunomodulatory molecules from their granules upon activation.
They also possess antimicrobial proteins including thrombocidins, cathelicidins, and human β-defensins that trigger direct microbicidal activities. 4 Furthermore, platelets can directly bind and internalize invading microbes. 7 The presence of major inflammatory molecules such as formyl peptide receptors (FPRs) and toll-like receptors facilitates platelets to recognize a diverse array of endogenous damageassociated molecular patterns and exogenous pathogen-associated molecular patterns. Collectively, these properties render platelets effector and sentinel cells in primary host defense against invading pathogenic microbes. 7,8 The FPRs belong to a family of G protein-coupled receptors and are predominantly expressed in immune cells, where they play a prominent role in the regulation of inflammatory responses and host defense. In humans, three FPR family members have been identified: FPR1, FPR2/ALX, and FPR3. 9 Although they were originally identified by their capability to recognize N-formyl peptides produced from bacteria or mitochondria of damaged cells, FPRs can bind a wide variety of structurally and functionally diverse ligands. These include bacterial and mitochondrial formyl peptides, nonformylated peptides/proteins, and small lipid molecules. 10 While FPR1 binds to bacterial-derived N-formyl peptides with high affinity, FPR2/ALX largely binds to mitochondrial formyl peptides. 9,11 The ligation of N-formyl peptides to FPRs in immune cells triggers a range of signaling cascades resulting in numerous biological activities. For example, the stimulation of FPR1 by fMLF in neutrophils induces degranulation, chemotaxis, production of superoxide anions, calcium mobilization, cytokine release, and expression of various surface markers. 12,13 During microbial infection, invading bacteria release Nformyl peptides that facilitate the recruitment of immune cells to the site of infection and accelerate the clearance of microbial infection and repair of tissue damage. 14 Czapiga et al 15 reported the presence of FPR1 in platelets and its ability to induce chemotactic and migratory responses upon ligation with N-formyl peptides, which emphasize a crucial role for FPR1 in platelet-mediated immune responses. Notably, bacterial or synthetic fMLF has been shown to act as a potent chemotactic agent through FPR1 in platelets. Despite numerous reports on the immune functions of FPR1, its impact upon ligation with fMLF on the modulation of hemostasis and thrombosis remains uncharacterized. Recently, we have reported the presence of FPR2/ALX in human platelets and its significance in the regulation of LL37-induced platelet activation and thrombus formation. 16 Here, we report the ability of fMLF to prime platelets and augment thrombus formation, and the significance of FPR1 in the regulation of platelet function in the presence and absence of fMLF.

| Preparation of human platelet-rich plasma and isolated platelets
The University of Reading Research Ethics Committee approved all the experimental procedures using human blood from healthy volunteers. The blood samples were collected from healthy, aspirinfree volunteers after obtaining written informed consent. The blood was collected into VACUETTE blood collection tubes containing 3.2% (w/v) sodium citrate. The blood samples were then centrifuged at 102 g for 20 minutes at room temperature to obtain platelet-rich plasma (PRP). The PRP was rested at 30°C for 30 minutes prior to use. For the preparation of isolated platelets, the blood was mixed with ACD [2.5% (w/v) sodium citrate, 2% (w/v) D-glucose, and 1.5% (w/v) citric acid] at 1 (ACD): 9 (blood) ratio and centrifuged at 102 g for 20 minutes. The PRP was collected, mixed with appropriate volume of ACD and prostaglandin I 2 (PGI 2 ), and centrifuged at 1413 g for 10 minutes at room temperature. The resultant platelet pellet was resuspended in modified Tyrodes-HEPES buffer (134 mmol/L NaCl, 2.9 mmol/L KCl, 0.34 mmol/L Na 2 HPO 4 .12H 2 O, 12 mmol/L NaHCO 3 , 20 mmol/L HEPES, and 1 mmol/L MgCl 2 , pH 7.3) with appropriate volume of ACD and PGI 2 and centrifuged once again at 1413 g for 10 minutes at room temperature. The resultant platelet Essentials • The role of formyl peptide receptor 1 (FPR1) and its ligand, fMLF, in the regulation of platelet function, hemostasis, and thrombosis is largely unknown.
• Fpr1-deficient mice and selective inhibitors for FPR1 were used to investigate the function of fMLF and FPR1 in platelets.
• Formyl peptide receptor 1 plays a pivotal role in the regulation of platelet function.
pellet was resuspended to a final density of 4 × 10 8 cells/mL in modified Tyrode's-HEPES buffer and allowed to rest for 30 minutes at 30°C prior to use.

| Mouse blood collection and platelet preparation
The mouse strains of Fpr1 −/−17 and Fpr2/3 −/−18 on a C57BL/6 background obtained from William Harvey Research Institute, London, UK, and wild-type C57BL/6 mice from Envigo, UK, were used in this study. The mice were sacrificed with CO 2 and the blood was directly collected by cardiac puncture into a syringe containing 3.2% (w/v) sodium citrate at 1 (citrate):9 (blood) ratio. The blood was then centrifuged at 203 g for 8 minutes at room temperature and the PRP was collected. The remaining blood was resuspended in 500 μL of modified Tyrode's-HEPES buffer and centrifuged once again at 203 g for 5 minutes. The resultant PRP with PGI 2 then centrifuged at 1028 g for 5 minutes. The resultant platelet pellet was resuspended in modified Tyrode's-HEPES buffer at a density of 2 × 10 8 cells/mL and rested for 30 minutes at 30°C prior to use.

| In vitro thrombus formation assay
The in vitro thrombus formation assay under arterial flow conditions was performed as described previously. 19,20 Briefly, human DiOC 6labeled (Sigma Aldrich) human whole blood was preincubated with a vehicle, MLF or fMLF (5 μmol/L) or cyclosporin H (CsH) (10 μmol/L), for 10 minutes before perfusion over collagen (Nycomed) (400 μg/ mL)-coated Vena8™ Biochips (Cellix Ltd) at a shear rate of 20 dynes/cm 2 . Z-stack fluorescence images of thrombi were obtained every 30 s for up to 10 minutes using a Nikon eclipse (TE2000-U) microscope (Nikon Instruments). The fluorescence intensity was calculated by analyzing the data using ImageJ software (National Institutes of Health). were then added to wells and incubated at room temperature for 1 h.

| Platelet adhesion assay
Nonadhered platelets were discarded, and the wells were washed three times with modified Tyrode's-HEPES buffer. One hundred and fifty microliters of citrate lysis buffer (3.53 mmol/L p-nitrophenyl phosphate, 71.4 mmol/L trisodium citrate, 28.55 mmol/L citric acid, 0.1% [v/v] Triton X-100; pH 5.4) were added and the plate was incubated for 1 h at room temperature. The reaction was stopped by adding 2 M NaOH and the absorbance was measured at 405 nm using a Fluostar Optima spectrofluorimeter.

| Tail bleeding assay
The tail bleeding assay was performed as described previously. 21 The British Home Office has approved the experimental procedures.
In brief, C57BL/6 (10-12 weeks old; Envigo, UK) or Fpr1 −/− mice were anesthetized using ketamine (80 mg/kg) and xylazine (5 mg/kg) administered via the intraperitoneal route and placed on a heated mat (37°C). The tail tip (3 mm) was dissected and immersed in sterile saline. The time to cessation of bleeding was measured and the assay was terminated at 20 minutes.

| Platelet aggregation assay
The platelet aggregation assays were performed by optical aggregometry using a two-channel platelet aggregometer (Chrono-Log). Human isolated platelets or PRP (270 μL) were added into a siliconized cuvette and prewarmed at 37°C for 90 s. Upon addition of an agonist, the platelets were allowed to aggregate under continuous stirring at 1200 rpm for 5 minutes at 37ºC and the level of aggregation was monitored. The platelets were pretreated with different concentrations of fMLF (1, 5, 10, and 20 μmol/L) for 5 minutes before the addition of CRP-XL (0.25 μg/mL), collagen (0.5 μg/mL), or thrombin (0.01 U/mL) and the level of aggregation was monitored.
Data were analyzed by calculating the percentage of maximum platelet aggregation obtained at 5 minutes.

| Adenosine triphosphate secretion assay
To assess the level of dense granule secretion in platelets, adenosine triphosphate (ATP) secretion was measured using a luciferin-luciferase luminescence substrate (Chrono-Log) by lumiaggregometry (Chrono-log). The level of ATP released from platelets upon stimulation with a platelet agonist, CRP-XL (0.25 μg/mL), in the presence and absence of different concentrations of Boc-MLF was measured by observing the level of luminescence released.

| Statistical analysis
The data obtained in this study are represented as mean ± SEM.
The statistical significance was analyzed using two-tailed unpaired Student t test for two-sample comparisons for the data obtained from the flow cytometric assay for FPR1 expression and platelet receptor characterization and cyclic adenosine monophosphate (cAMP) assay. For multiple comparisons such as for data obtained from in vitro thrombus formation, fMLF binding, ATP release, platelet aggregation, and activation, the statistical significance was established using 1-way or 2-way ANOVA followed by Bonferroni's correction.
Data obtained from the tail bleeding assay were analyzed using a nonparametric Mann-Whitney test. All statistical analyses were performed using Graphpad Prism 7 software (GraphPad Software Inc.).

| Platelets express FPR1
Recently, we have reported the presence of FPR2/ALX in human and its orthologue, Fpr2/3 in human platelets. 16 Similarly, the expression of FPR1 in human platelets at protein level, and in megakaryocytes at transcript level, has been previously reported. 15 Furthermore, the presence of FPR1 transcripts in human and mouse platelets was demonstrated. 22 In line with these findings, here we confirmed the presence of FPR1 on the surface of human isolated platelets by flow cytometry and in isolated platelet lysates using immunoblot analysis. Notably, the activation of platelets using 1 μg/mL cross-linked collagen-related peptide (CRP-XL) increased the level of FPR1 on the surface as determined by flow cytometry ( Figure 1AI), while the level of proteins identified by immunoblots remained unchanged ( Figure 1AII). These data confirm the presence of FPR1 in platelets, possibly in granules or the open canalicular system or both, and their increase on the surface upon activation similar to FPR2/ALX. 16

| N-formyl-methionyl-leucyl-phenylalanine selectively binds to FPR1 on the platelet surface
A fluorescently-labeled fMLF was used to investigate its binding to FPR1 on the surface of platelets by flow cytometry. To ascertain the selective binding of fMLF to FPR1, platelets obtained from Notably, the absence of Fpr2/3 protein in platelets obtained from Fpr2/3 −/− mice was confirmed previously. 16 The binding of FITC-fMLF (5 μmol/L) was significantly reduced in Fpr1 −/− mouse isolated platelets compared to the control and Fpr2/3 −/− mouse platelets ( Figure 1BII). These data confirm the selective binding of fMLF to Fpr1 in mouse platelets.

| N-formyl-methionyl-leucyl-phenylalanine stimulates platelet activation
To determine whether fMLF is able to stimulate platelet activation upon binding to FPR1, a range of platelet functional assays were performed. Platelet activation triggers inside-out signaling to integrin αIIbβ3 on the platelet surface and converts it to a high-affinity state to allow fibrinogen binding and subsequent platelet aggregation. 23 To examine whether fMLF influences the inside-out signaling to integrin αIIbβ3 in platelets, the level of fibrinogen binding on the platelet surface was measured as a marker for inside-out signaling to integrin αIIbβ3. Indeed, fMLF has increased the level of fibrinogen binding in human isolated platelets in a concentration-dependent manner ( Figure 1CI). A minimum concentration of 0.5 μmol/L fMLF has shown significant increase in fibrinogen binding compared to the resting platelets. Similarly, the level of Pselectin exposure on the platelet surface was measured as a marker for α-granule secretion by flow cytometry. The results indicate that fMLF has induced α-granule secretion in human isolated platelets in a concentration-dependent manner ( Figure 1CII). Similar to the isolated platelets, fMLF has increased the level of fibrinogen binding and P-selectin exposure in human platelets when PRP was used in a concentration-dependent manner ( Figure 1D). In addition, fMLF has increased the level of platelet adhesion to immobilized collagen under static conditions ( Figure 2A). Together, these data confirm that the fMLF significantly primes platelet activation and adhesion.

| N-formyl-methionyl-leucyl-phenylalanine augments thrombus formation
Microbial infection and various inflammatory diseases including sepsis are associated with the risk of disseminated intravascular coagulation or thrombosis in the microvasculature. 24 To investigate whether fMLF has a direct impact on thrombosis, its effect on thrombus formation under arterial flow conditions was analyzed.
Human DiOC 6 -labeled whole blood was preincubated with a control (nonformylated peptide, MLF) or fMLF (5 μM) for 10 minutes prior to perfusion over collagen-coated Vena8™ biochips. Thrombus formation was monitored for 10 minutes by acquiring fluorescent images at every 30 s . Indeed, fMLF has increased the mean fluorescence intensity of thrombi by approximately 60% compared to the controls ( Figure 2B). These data demonstrate the direct impact of fMLF on augmenting the thrombus formation under arterial flow conditions in human whole blood. The effect of fMLF on other blood cells, mainly leukocytes, and their subsequent influence on thrombus formation cannot be ruled out under these circumstances.

| Agonist-induced platelet aggregation is amplified by fMLF
Following the determination of the effects of fMLF on thrombus formation and platelet activation, aggregation assays were performed to establish its effects on isolated platelets. Human isolated platelets were incubated with various concentrations of fMLF (1, 5, 10, and 20 μmol/L) prior to stimulation with subthreshold concentrations of different platelet agonists such as CRP-XL (0.25 μg/mL), collagen (0.5 μg/mL), and thrombin (0.01 U/mL), and the level of aggregation was monitored for 5 minutes by optical aggregometry. Notably, fMLF has failed to induce a noticeable platelet aggregation on its own ( Figure 2C) although its preincubation with platelets markedly enhanced agonist-induced platelet aggregation. Maximum aggregation (100%) was obtained in human isolated platelets that were treated with 20 μmol/L fMLF and 0.25 μg/mL CRP-XL for 5 minutes ( Figure 2C).
Similar results were obtained with collagen-induced ( Figure 2D) and thrombin-induced ( Figure 2E) platelet aggregation. These data confirm the ability of fMLF to prime platelets and amplify their aggregation upon stimulation with different agonists although it was unable to aggregate platelets on its own under the current settings.
F I G U R E 1 Expression of FPR1 in platelets and the impact of fMLF on platelet activation. A, the expression of FPR1 on the surface of resting or 1 μg/mL CRP-XL-activated human isolated platelets was analyzed using FPR1-selective and fluorescent-labeled secondary antibodies by flow cytometry (AI). Data represent mean ± SEM (n = 8). Similarly, the presence of FPR1 in human isolated platelet lysates was confirmed by immunoblot analysis using selective antibodies (AII). B, the absence of Fpr1 was confirmed in isolated platelet lysates obtained from Fpr1 −/− in comparison to the control and Fpr2/3 −/− mice by immunoblot analysis using selective antibodies (BI). Isolated platelets obtained from control, Fpr1 −/−, and Fpr2/3 −/− mice were preincubated with 10 μmol/L FITC-conjugated fMLF and their level of binding to the platelet surface was measured by flow cytometry (BII). Data represent mean ± SEM (n = 5). C, different concentrations of fMLF were used to determine their impact on platelet activation in human isolated platelets by quantifying the level of fibrinogen binding (CI) and P-selectin exposure (CII) using flow cytometry. Data represent mean ± SEM (n = 10). D, different concentrations of fMLF were used to determine their effects on platelet activation in human platelet-rich plasma (PRP) by quantifying the level of fibrinogen binding (DI) and P-selectin exposure (DII) using flow cytometry. Data represent mean ± SEM (n = 10). The blots shown are representative of three separate experiments. Protein 14-3-3ζ was detected as a loading control in the immunoblots. The statistical significance was calculated by 1-way ANOVA followed by Bonferroni's correction in most of the experiments except the data shown in panel A, which were analyzed by 2-tailed unpaired Student t test (*P < 0.01, **P < 0.001, and ***P < 0.0001). FITC, fluorescently labeled; fMLF, N-formyl-methionyl-leucyl-phenylalanine; FPR1, N-formyl peptide receptor-1

| N-formyl-methionyl-leucyl-phenylalanine selectively acts through FPR1 in platelets
A large number of studies indicate that fMLF binds primarily to FPR1 and exerts its effects in immune cells. [25][26][27] The functional dependence of fMLF on FPR1 in platelets was determined using a pharmacological inhibitor for FPR1, Boc-MLF in human platelets, and platelets obtained from Fpr1 −/− mice through measuring the levels of fibrinogen binding and P-selectin exposure by flow cytometry.
Similar to human platelets, fMLF increased the level of fibrinogen binding [ Figure 3AI (isolated platelets) and AII (whole blood)] and P-selectin exposure [ Figure 3BI (isolated platelets) and BII (whole  Figure 3DI) and P-selectin exposure ( Figure 3DII). Together, these data emphasize the involvement of FPR1 in the regulation of fMLF-mediated effects in platelets.

| Inhibition of FPR1 reduces the agonist-induced platelet activation
In order to study the importance of FPR1 in the regulation of nor-

| Deletion of FPR1 affects mouse platelet activation
To examine further the impact of FPR1 in platelets, the whole blood

| The inhibition or deletion of FPR1 increases the level of cyclic AMP
Cyclic AMP (cAMP) is a potent inhibitor of platelet function and its level is generally reduced upon platelet activation. Stimulants of cAMP generation are known to inhibit platelet activation. 28 The FPRs are G i protein-coupled receptors, 29 which are known to inhibit adenylate cyclase and thus, lead to a reduction in cAMP levels. Therefore, the deletion of genes for G i -coupled receptors in mice generally increases the basal levels of cAMP in target cells. 30,31 To investigate whether the effects of FPR1 in platelets are driven through cAMP-dependent signaling, the level of cAMP Notably, E coli-derived fMLF 34 has been implicated in bacterial cystitis, 35 pneumococcal pneumonia, 36 inflammatory bowel disease, 37 pouchitis, colitis 38 and juvenile peridontitis. 39 It has been shown that inhalation or injection of fMLF can cause bronchial inflammation 40 and induce rapid neutropenia, thereby increasing susceptibility to infection. 41 The plasma levels of fMLF were increased in these conditions, and also in high-fat-diet-treated mice due to altered microbiome where it impaired the glucose tolerance and insulin secretion. 42 Many of these infectious and inflammatory conditions are associ- The impact of fMLF on platelet adhesion and aggregation. A, different concentrations of fMLF were used to analyze their influence on human platelet adhesion on immobilized collagen under static conditions. Data represent mean ± SEM (n = 10). B, the effect of fMLF on the modulation of thrombus formation was analyzed by using human DiOC6-labeled whole blood that was preincubated with nonformylated MLF or fMLF (5 μmol/L) for 10 min prior to perfusion over collagen-coated (400 μg/mL) Vena8™ Biochips. Images shown are representative of three separate experiments (10× magnification; scale bar -10 μm). Likewise, the effects of fMLF on C, cross-linked collagenrelated peptide (CRP-XL)-induced, , D, collagen-induced, or E, thrombin-induced platelet activation were measured using isolated human platelets by optical aggregometry. Data represent mean ± SEM (n = 5). The statistical significance was calculated by 1-way ANOVA followed by Bonferroni's correction in most of the experiments except the data shown in panels B, which were analyzed by 2-tailed unpaired Student t test (*P <0.01, **P <0.001, and ***P <0.0001). fMLF, N-formyl-methionyl-leucyl-phenylalanine; MLF, methionyl-leucyl-phenylalanine been reported to be at least in micromolar ranges. 46 In this study, we demonstrate that fMLF is able to prime platelets and augment thrombus formation in micromolar concentrations. Therefore, the increased levels of fMLF under the aforementioned pathological conditions may lead to platelet activation and contribute to thrombotic complications.  48 In addition, the fibrinogen binding to integrin α IIb β 3 in platelets and Mac-1 in neutrophils was suggested to play an F I G U R E 5 Blockade of FPR1 using a pharmacological inhibitor reduces platelet spreading, activation and thrombus formation. A, platelet adhesion and spreading on fibrinogen-coated glass surface was analyzed in the absence or presence of Boc-MLF (1, 5, 10, and 20 μmol/L) by confocal microscopy (60× magnification; scale bar -10 μm). The number of adhered (AI) and spread platelets (AII) and the relative surface area of spread platelets (AIII) were determined by analyzing the images using ImageJ. Ten random fields of view were recorded for each sample. Data represent mean ± SEM (n = 3). B, the levels of fibrinogen binding (BI) and P-selectin exposure (BII) were analyzed in human platelet-rich plasma (PRP) by flow cytometry upon stimulation with CRP-XL (0.5 μg/mL), in the presence and absence of different concentrations of CsH (1, 5, 10, 20, and 30 μmol/L). Data represent mean ± SEM (n = 3). C, the impact of CsH on the modulation of thrombus formation was analyzed using human DiOC6-labeled whole blood that was preincubated with a vehicle control or 10 μmol/L CsH for 10 min prior to perfusion over collagen-coated (400 μg/mL) Vena8™ Biochips. Images shown are representative of three separate experiments (10× magnification; scale bar -10 μm). Data represent mean ± SEM (n = 3). P values shown are as calculated by 1-way ANOVA followed by Bonferroni's correction except for data shown in C, which were analyzed by 2-tailed unpaired Student t test, respectively. (*P < 0.01, **P < 0.001, and ***P < 0.0001) CRP-XL, cross-linked collagen-related peptide; CsH, cyclosporin H; MLF, methionyl-leucyl-phenylalanine , was analyzed using a cAMP assay kit. *Represents the significant difference between the various concentrations of agonists within the Fpr1 +/+ group. # Represents the significant difference between Fpr1 +/+ and Fpr1 −/− groups. Data represent mean ± SEM (n = 4). The statistical significance was calculated by 2-way ANOVA followed by Bonferroni's correction in most of the experiments except for the data shown in panels E and F, which were calculated by non parametric Mann-Whitney test, and 2-tailed unpaired Student t test, respectively (*P <0.01, **P <0.001, and ***P <0.0001). ADP, adenosine diphosphate; cAMP, cyclic adenosine monophosphate; CRP-XL, crosslinked collagen-related peptide; FPR1, N-formyl peptide receptor-1   I  I  II  II   I  II  I  II   I  II   B C D E F (such as CRP-XL and ADP) platelet activation was established. The blockade of FPR1 resulted in reduced ATP release upon activation with CRP-XL, which not only affects secondary platelet activation but may also influence the modulation of inflammatory responses. 60 Similarly, the inhibition of FPR1 impaired the ability of platelet spreading on fibrinogen, which is essential for thrombosis and subsequent wound repair. 61 The specificity of inhibitors to FPR1 may be limited by their ability to bind FPR2/ALX nonspecifically at higher concentrations. Therefore, the prominent role of FPR1 on the regulation of platelet activation was also corroborated using platelets obtained from Fpr1-deficient mice, wherein the effects of fMLF and platelet agonists were largely diminished.
The selective binding of fMLF to Fpr1 was confirmed using platelets obtained from Fpr1-deficient mice. In addition, the hemostasis in Fpr1-deficient mice was affected further emphasizing its significance in the regulation of platelet function. Fpr1-deficient mice displayed severe inflammation, higher mortality during pneumococcal meningitis, 62 increased susceptibility to Listeria monocytogenes infection, and impaired antibacterial host defense. 17 In addition, Fpr1-deficient mice demonstrate major roles in sterile inflammation triggered by mitochondrial N-formyl peptides as demonstrated by the attenuation of inflammation in response to sterile acute lung injury in these mice. 64 In line with these studies, here we demonstrate the dysfunction of platelets and affected hemostasis in Fpr1-deficient mice, which may also substantiate the reduced inflammatory responses due to significant contribution of platelets in inflammation and innate immunity.
As a Gi protein-coupled receptor, FPR1 triggers downstream signaling via various molecules such as phospholipase C, PI3K/AKT, and MAPK, and modulates calcium mobilization in neutrophils. 13 The ability of fMLF to induce calcium mobilization in platelets has been previously reported. 15 Here, we report the impact of FPR1 on cAMP-mediated signaling in platelets using Boc-MLF and platelets obtained from Fpr1-deficient mice. Indeed, the inhibition of FPR1 in human platelets or its deletion in mouse platelets resulted in elevated levels of cAMP, which is a potent inhibitor for platelet activation. This confirmed the involvement of cAMP-dependent signaling in the regulation of FPR1-mediated effects in platelets.

Given the significance of FPRs and their ligands under various
clinical settings, the therapeutic potential of these are being widely investigated. Notably, honokiol, a plant-derived bioactive agent, has recently been demonstrated to reduce the proinflammatory responses induced by fMLF in neutrophils through inhibiting FPR1. 65 Recently, we have reported the presence of FPR2/ALX in platelets, and its significance in the activation of platelets and thrombus formation upon ligation with an antimicrobial cathelicidin, LL37. 16 In conclusion, similar to LL37, this study demonstrates a prominent role for fMLF for priming platelet activation and augmenting thrombus formation under arterial flow conditions. Using an FPR1 selective inhibitor and Fpr1-deficient mice, the functional dependence of fMLF on this receptor was established. Therefore, the priming effects of fMLF on platelets may significantly contribute to the development of thrombotic and proinflammatory diatheses in pathological settings where the level of fMLF is elevated. Hence, FPR1 may act as a potential therapeutic target and its ligands may provide a powerful platform to develop novel therapeutic agents in order to treat and prevent thrombotic and inflammatory complications in diverse pathophysiological settings.

ACK N OWLED G M ENTS
We would like to thank the British Heart Foundation (Grant number: Higher Education for their funding support for this research.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest with the contents of this article.