Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones

Neutrophil extracellular traps induce aggregation of washed human platelets independently of... Background: The release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. Recently it became clear that NETs are also released in pathological conditions. NETs released in the blood can activate thrombosis and initiate a cascade of platelet responses. However, it is not well understood if these responses are mediated through direct or indirect interactions. We investigated whether cell-free NETs can induce aggregation of washed human platelets in vitro and the contribution of NET-derived extracellular DNA and histones to platelet activation response. Methods: Isolated human neutrophils were stimulated with PMA to produce robust and consistent NETs. Cell-free NETs were isolated and characterised by examining DNA-histone complexes and quantification of neutrophil elastase with ELISA. NETs were incubated with washed human platelets to assess several platelet activation responses. Using pharmacological inhibitors, we explored the role of different NET components, as well as main platelet receptors, and downstream signalling pathways involved in NET-induced platelet aggregation. Results: Cell-free NETs directly induced dose-dependent platelet aggregation, dense granule secretion and procoagulant phosphatidyl serine exposure on platelets. Surprisingly, we found that inhibition of NET-derived DNA and histones did not affect NET-induced platelet aggregation or activation. We further identified the molecular pathways involved in NET-activated platelets. The most potent single modulator of NET-induced platelet responses included NET-bound cathepsin G, platelet Syk kinase, and P2Y and αIIbβ3 receptors. Conclusions: In vitro-generated NETs can directly induce marked aggregation of washed human platelets. Pre-treatment of NETs with DNase or heparin did not reduce NET-induced activation or aggregation of human washed platelets. We further identified the molecular pathways activated in platelets in response to NETs. Taken together, we conclude that targeting certain platelet activation pathways, rather than the NET scaffold, has a more profound reduction on NET-induced platelet aggregation. Keywords: Neutrophil, Neutrophil extracellular traps, Platelet, Aggregation, DNA, Histones, Cathepsin G * Correspondence: pat.metharom@curtin.edu.au Platelet Research Laboratory, School of Pharmacy and Biomedical Sciences, Curtin Health and Innovation Research Institute, Faculty of Health Sciences, Curtin University, Bentley Campus, Office 160, Building 305, Kent Street, Bentley, Perth, WA 6102, Australia © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 2 of 15 Background intravascular fibrin formation and degrading tissue Neutrophils are well known for their crucial role in in- factor pathway inhibitor [21], while myeloperoxidase nate immunity, providing the first line of defence against (MPO) can prime platelets [22]. pathogens through multiple mechanisms [1]. The dis- Collectively, NETs are a potentially potent agonist of covery of a relatively new antimicrobial mechanism, platelet activation and promoter of coagulation, thereby whereby activated neutrophils expel their DNA and pro- amplifying and supporting thrombus formation. How- teins forming an extracellular matrix, termed neutrophil ever, despite many studies reporting NETs as promoters extracellular traps (NETs) [2], has gained much interest of thrombosis, these studies were conducted in whole recently. NETs possess antimicrobial function either by blood assays or in the presence of plasma, implicating a entrapping and immobilising pathogens or presentation role of plasma coagulation factors. Thus the capacity of of NET-bound antimicrobial proteins [2, 3]. However, intact cell-free NETs to directly activate washed platelets NETs can serve as more than just a host defence mech- is not clearly understood. In this study, we investigated anism, as studies have implicated the role of NETs in in- the effect of NETs on platelet function including aggre- flammatory and autoimmune diseases and pathological gation, secretion, and surface expression of receptors. conditions including thrombosis [4]. Notably, as the role We also begin to determine molecular mediators and of NETs in thrombosis is being investigated extensively signalling pathways by examining the effect of antago- in recent times, there is potential for NETs to not only nists of specific NET components and antiplatelet drugs, serve as therapeutical target for thrombotic diseases but on the impact of NETs on platelet activation. several other clinical conditions such as diabetes, sys- temic lupus erythematosus, pre-eclampsia and certain Methods types of cancers, all which are known to be associated Materials with increased risk of thrombosis [5–8]. Purified anti-human TLR2, TLR4 blocking antibodies Increasing number of studies are recognising NETs and their matching isotype controls were obtained from as a procoagulant surface, which is capable of pro- BioLegend, Inc., USA. Bay 61–3606 and Phorbol moting thrombosis both in vitro and in animal 12-myristate 13-acetate (PMA), ticagrelor and Cell De- models of deep vein thrombosis and arterial throm- tection ELISA PLUS kit were from Sigma-Aldrich, bosis [9–13]. The NET structure can serve as a scaf- Australia. ML-171, aspirin, RGDS, cathepsin G inhibitor fold for platelet adhesion and aggregation [9, 14]thus I, neutrophil elastase inhibitor (1-(3-methylbenzoyl)-1- providing a platform for the subsequent formation of H-indazole-3-carbonitrile), myeloperoxidase inhibitor 1 thrombi. Furthermore, NETs have been shown to dir- (4-Aminobenzoic acid hydrazide), losartan were ob- ectly promote the activation of intrinsic coagulation tained from Cayman Chemical, USA. Abciximab (Reo- pathway leading to thrombin generation [15]. Besides Pro) and low molecular weight heparin (Clexane intact NETs being capable of activating coagulation, enoxaparin sodium) were from Eli Lilly and Sanofi Aven- several components within the NET structure have tis Australia Pty Ltd., respectively. DNAse I solution was been reported to activate platelets and initiate or pro- purchased from STEMCELL Technologies Australia Pty mote coagulation. Cell-free DNA, which makes up Ltd. Collagen and thrombin were from Chrono-log Cor- the major backbone of NETs, has previously been poration, USA. Human PMN Elastase ELISA kit was ob- shown to activate thrombin generation via the intrin- tained from Abcam Biotechnology, Cambridge, UK. sic pathway of coagulation [16]. The second most abundant constituent and protein found on NETs, Preparation of washed human platelets extracellular histones, have been studied extensively Blood was drawn from healthy volunteers into a syringe and known to activate platelets and promote coagula- containing acid-citrate-dextrose (ACD; 1:7 (v/v) with in- tion through multiple mechanisms [9, 17–19]. For ex- formed consent in concordance with the Curtin Univer- ample, histones are capable of generating thrombin in sity Human Research Ethics Committee (approval the presence of plasma and activating platelet aggre- number HR54/2014). Blood donors were gation which has been suggested to be mediated medication-free 2 weeks prior to the day of donation. through toll-like receptor (TLR) 2 and TLR 4 [18]. Washed platelets were prepared, with some modifica- However, the involvement of TLRs in platelet aggre- tions, as previously described [23, 24]. Briefly, blood was gation is not clear as Clark et al. have shown that centrifuged at 150 x g for 20 min. Platelet-rich plasma LPS induced platelet activation but not aggregation (PRP) was collected and centrifuged at 800 x g for [20] Furthermore, neutrophil granular enzymes that 10 min in the presence of 1 μM prostaglandin E1 (PGE1; are bound to NETs such as neutrophil elastase (NE) Cayman Chemical). Platelets were then washed three and cathepsin G (Cat G), can separately promote co- times in CGS buffer (14.7 mM trisodium citrate, agulation and thrombus growth by facilitating 33.33 mM glucose and 123.2 mM NaCl, pH 7), in the Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 3 of 15 presence of PGE1 (1 μM). Platelets were adjusted to 1 × Platelet-dense granule secretion assay 10 /mL with calcium-free Tyrode-Hepes buffer (5 mM Platelet secretion was determined by measuring ATP re- HEPES, 5.5 mM glucose, 138 mM NaCl, 12 mM lease using luciferin/luciferase reagent (Chrono-Lume, NaHCO , 0.49 mM MgCl , 2.6 mM KCl, 0.36 mM Chrono-log Corporation, USA). Briefly, 90 μl of platelets 3 2 NaH PO , pH 7.4). Platelets were supplemented with (1 × 10 /mL) in Tyrodes-HEPES buffer (with calcium) 2 4 1.8 mM CaCl (final concentration) prior to were incubated with 10 μl of NETs with gentle shake at experimentation. 37 °C for 1 and 10 min before adding 5 μlof Chrono-Lume reagent. The luminescence was measured Preparation of neutrophils and cell-free neutrophil using Enspire Multimode Plate Reader (PerkinElmer, extracellular traps (NETs) USA). Where anti-platelet drugs were used, platelets Neutrophils were isolated from human blood using Poly- were pre-incubated with the drugs for 15 min at 37 °C morphPrep (Axis-Shield, Norway), with minor changes before incubating with NETs. to the manufacturer’s protocol. Briefly, blood anticoagu- lated with EDTA (2 mM) was layered over Polymorph- P-selectin exposure and αIIbβ3 activation Prep and centrifuged at 500 x g for 40 min. The Platelet activation was measured by detecting P-selectin neutrophil fraction was collected and washed twice at and active-form αIIbβ3 on the platelet surface using flow 4 °C in Hank’s buffered saline solution (without calcium cytometry. Where inhibitors were used, platelets were or magnesium) and resuspended in X-VIVO 15 media pre-incubated for 15 min at 37 °C before adding NETs. (Lonza, Switzerland). Neutrophil purity was > 95% as de- Whenever inhibitors of components of NETs (i.e. termined with a haematology analyser (Mindray, DNAse I, cathepsin G, myeloperoxidase, and elastase in- BC-VET2800). Cell-free NETs were isolated as previ- hibitors) were used, NETs were pre-incubated for ously described [25] with minor changes to the protocol. 30 min at 37 °C. The specificity of inhibitors used was This method of NET isolation does not involve using also examined for their effect on thrombin (0.1 U/mL) DNase or EDTA [26], which may confound platelet re- and collagen (5 μg/mL)–induced platelet activation. In- sponse to NETs. Briefly, neutrophils (2.5 × 10 /mL) were hibitor-, or vehicle-, treated washed human platelets stimulated with 500 nM PMA for 3 h at 37 °C and 5% (1 × 10 /mL) were treated with NETs (10% of final vol- CO . The supernatant, containing PMA, was discarded ume) and stained with phycoerythrin-conjugated mouse and the NET monolayer was detached with anti-human CD62P (P-selectin) and fluorescein phosphate-buffered saline (PBS). The cell debris was pel- isothiocyanate-conjugated mouse anti-human PAC1 leted by centrifugation at 480 x g for 10 min at 4 °C. The (active-form αIIbβ3), or suitable isotype control anti- supernatant was further centrifuged at 15,000 x g for bodies for 15 min in the dark. All antibodies were from 20 min at 4 °C to pellet DNA then resuspended in PBS BD Biosciences. Samples were analysed by flow cytome- at 100 μl per 1 × 10 of stimulated neutrophils to obtain try (BD LSRFortessa™ cell analyzer). cell-free NETs. Cell-free NETs were characterised by de- tecting DNA-histone complex and neutrophil elastase Phosphatidylserine (PS) exposure using Cell Detection ELISA PLUS kit (Sigma Aldrich) Platelets (3 × 10 /mL) were incubated with NETs for and Human PMN Elastase ELISA kit (Abcam), respect- 30 min at 37 °C with continuous stirring at 1200 rpm. ively. Cell-free NETs were incubated with platelets at Whenever inhibitors were used, NETs were 10% of final reaction volume (i.e. 1-volume NET solution pre-incubated for 30 min at 37 °C. Thrombin (0.1 U/ to 9-volume platelets). mL) was used as positive control. Platelets were then stained with Annexin V-FITC (BioLegend, USA) in bind- Platelet aggregation assay ing buffer according to manufacturer’s instructions for Washed platelets (3 × 10 /mL) in Tyrode-HEPES buffer 15 min in the dark. Samples were then washed in bind- supplemented with 1.8 mM calcium chloride were incu- ing buffer and analysed by flow cytometry (BD LSRFor- bated in the presence of cell-free NETs (10% of final re- tessa™ cell analyzer). action volume) and platelet aggregation was monitored at 37 °C with continuous stirring at 1200 rpm in a light Antibodies and western blot transmission aggregometer (Model 700 Aggregometer, Rabbit antibodies specific for p-Akt (Ser473), p-Erk1/2 Chrono-log Corporation, USA) for at least 20 min. (Thr202/Tyr204), p-Syk (Tyr352), p-Tyr1000 and Tyrode-HEPES buffer was used as a blank. Where inhib- α-actinin were obtained from Cell Signalling Technology itors were used, platelets were pre-incubated for 15 min (USA). Platelets (3 × 10 /mL) were incubated with NETs at 37 °C prior to incubation with NETs. Control samples (10% of final reaction volume) at 37 °C for 3 min with were incubated with the corresponding volume of continuous stirring at 1200 rpm. Platelets were then buffer. lysed in Laemmli sample buffer supplemented with Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 4 of 15 Protease/Phosphatase Inhibitor Cocktail (Cell Signalling (Fig. 1c). Platelet secretion was examined after 1 and Technology) and β-mercaptoethanol. Forty-five μlof 10 min incubation with NETs to identify the time re- protein sample was loaded per lane and separated by so- quired for secretion compared to aggregation. NETs in- dium dodecyl sulphate–polyacrylamide gel electrophor- duced significant platelet secretion by 1 min, while esis then transferred to a polyvinylidene difluoride aggregation did not occur until 5 min (Fig. 1b-c), indi- (PVDF) protein blotting membrane with 0.2 μm pore cating that NET-induced platelet secretion precedes ag- size (GE Healthcare Life Sciences). The PVDF mem- gregation. Furthermore, we also examined if platelet brane was blocked in 5% non-fat powdered milk in functional response occurred concomitantly with intra- Tris-buffered saline with 0.1% Tween 20 (or 3% bovine cellular signalling, particularly the phosphorylation of serum albumin (BSA, Bovogen Biologicals Pty Ltd., proteins at tyrosine residues. Indeed, NETs induced tyro- Australia) in TBS-T for detection of p-Tyr-1000) at sine phosphorylation of several substrates with strong room temperature for 1 h. After a brief rinse with migrating bands at ~ 134, 80 and 60 kDa compared to TBS-T, the membrane was incubated overnight at 4 °C vehicle control (Fig. 1d). with primary antibodies at 1:1000 dilution. The primary antibody was detected with secondary horseradish NETs induce surface expression of receptors and peroxidase-conjugated anti-rabbit antibody (Jackson Im- phosphatidyl serine exposure on platelets mune Research, USA) at 1:40000 dilution. The mem- We also examined α-granule secretion by measuring the brane was developed using Amersham ECL Prime surface expression of P-selectin. Platelets incubated with Western Blotting Detection Reagent (GE Healthcare Life NETs for 10 to 15 min showed a significant increase of Sciences, USA), and chemiluminescence was detected P-selectin surface expression as detected by flow cytom- using ChemiDoc imaging system (Bio-Rad, USA). etry (Fig. 2a-b). A conformational change in platelet αIIbβ3 receptor is required for platelet aggregation [27]. Statistical analysis Therefore we assessed platelet surface expression of Data were analysed using GraphPad PRISM 4.0 software. active-form αIIbβ3 using PAC1 monoclonal antibody. As Results are expressed as the mean ± standard error shown in Fig. 2a-b, NETs induced a conformational (SEM). One-way ANOVA with posthoc Bonferroni’s change of αIIbβ3 from resting to an activated state. As Multiple Comparison Test were used to examine the PS exposure on platelets can propagate coagulation [28], statistical significance between means. Differences were we assessed the ability of NETs to induce PS exposure considered significant at P < 0.05. on the platelet surface. Platelets were incubated with NETs for 30 min at 37 °C with continuous stirring at Results 1200 rpm before analysing PS expression. Annexin NETs induce aggregation, secretion and activation of V-FITC was used to stain PS and was analysed by flow washed human platelets cytometry. NETs induced a marked increase in PS ex- We first examined the ability of cell-free NETs to dir- pression on platelet’s surface (Fig. 2c-d), suggesting that ectly induce aggregation of washed human platelets in- NET-activated platelets can provide a procoagulant dependently of the coagulation pathway. Platelets were surface. washed three times in CGS buffer to remove any con- taminating plasma, then resuspended in Tyrode-HEPES buffer (with 1.8 mM calcium chloride). Light transmis- The role of NET-derived DNA, histones, cat G, and MPO in sion aggregometry was used to measure platelet aggrega- NET-induced platelet response tion. Autologous cell-free NETs at different dilutions As the major components of NETs are DNA and histones, induced marked platelet aggregation (Fig. 1a). Interest- we first investigated whether the effect of NETs on washed ingly, platelet aggregation response was apparent only platelets is mediated via NET-derived DNA and/or his- after 5 mins in the presence of the highest concentration tones. DNA-histone complexes were confirmed in of NETs used (10% of final reaction volume) (Fig. 1b). cell-free NETs using Cell Detection ELISA PLUS kit. For all experiments, it was imperative that NETs were Based on comparisons between whole neutrophil lysates used within the same day of isolation as freezing or stor- and NET dilution samples, the concentration of ing NETs at 4 °C for more than 24 h completely abol- DNA-histone complex in cell-free NETs used in majority ished their activity. of experiments (i.e. 10% final NETs) was equivalent to the As platelet secretion amplifies platelet aggregation, we amount present in approximately 3.8 ± 1.2 × 10 /mL of assessed the ability of NETs to induce platelet dense neutrophil lysate (Additional file 1:Figure S1). Addition- granule secretion of ATP/ADP using a luminescence ally, the 10% cell-free NET solution was determined to assay. NETs triggered significant ATP/ADP secretion contain 292 ± 172 pg/mL of elastase, another major pro- from platelet dense granules compared to vehicle control tein component of NETs (Additional file 1: Figure S2). Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 5 of 15 ac Fig. 1 NETs directly induce aggregation of washed human platelets. a Representative traces showing dose-dependent aggregation of washed human platelets (WP, 3 × 10 /mL) in response to cell free-NETs. 0.1, 1 and 10% NETs refers to NETs constituting 0.1, 1 or 10% of final reaction volume, respectively (e.g. 10% NET is 1-volume NET solution to 9-volume platelets). Platelet aggregation was measured by light transmission aggregometer (Chrono-log). NETs were used at 10% of final reaction volume for following experiments*. b Progress time curve displaying % of NET-induced platelet aggregation which steadily increased over 20 min. c ATP/ADP release from NET-stimulated platelets. WP (1 × 10 /mL) were incubated with NETs with a gentle shake at 37 °C for 1 and 10 min before adding Chrono-Lume reagent. Luminescence was measured using Enspire Multimode Plate Reader (PerkinElmer). NETs induced significant ATP/ADP secretion from platelets at 1 and 10 min. Results represent fold change in luminescence arbitrary absorbance unit relative to vehicle (PBS) control. Data expressed as mean ± SEM; **P < 0.01, ***P < 0.0001, N ≥ 5. d Platelet signalling in response to NETs was examined by Western blot analysis. WP (3 × 10 /mL) were incubated with collagen (5 μg/mL), vehicle (PBS) or NETs for 3 min. Twenty μl of total cell lysate was analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted for phospho-Tyrosine (p-Tyr-1000). Equal loading was verified by α-actinin. Western blots indicate tyrosine-phosphorylated proteins in NET-stimulated platelets. *10% NET solution is prepared from 1 × 10 /ml of PMA-activated neutrophils, however due to losses during preparation steps, 10% NET is estimated to contain histone/DNA complexes equivalent to 3.8 ± 1.2 × 10 /mL neutrophils (Additional file 1: Figure S1). The elastase content present in a 10% NET reaction volume is 292 ± 172 pg/mL Calf thymus histones (CTH) are well-established response to another agonist remained unaffected (Fig. platelet agonists [29] and were used as a positive con- 3b). trol. CTH (1, 5, 20 and 40 μg/mL) induced a In order to test the impact of DNA on NET-induced dose-dependent aggregation of washed human platelet platelet aggregation, NETs were pre-treated with DNase (Fig. 3a). Heparin can strongly bind to and abate the (20, 200 U/mL) for 30 min at 37 °C before adding to effect of histones on platelets [30–32]. Indeed, hep- platelets (where NETs was used at 10% of the final reac- arin 20 U/mL completely abated CTH but not tion volume). DNase can dismantle NET-DNA (Add- NET-induced platelet aggregation (Fig. 3 b, d). Colla- itional file 1: Figure 3). However, DNase did not reduce gen (5 μg/mL) was added to the CTH-heparin reac- the ability of NETs to induce aggregation of washed hu- tion in order to verify that the platelet functional man platelets (Fig. 3c & d), suggesting neither DNA nor Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 6 of 15 Fig. 2 NETs induce platelet activation and phosphatidyl serine (PS) exposure on washed human platelets. a Representative dot plot of CD62P (P-selectin), PAC1 (active-form αIIbβ3) fluorescence. WP (1 × 10 /mL) were treated with vehicle (PBS) or NETs then incubated with labelled monoclonal antibodies phycoerythrin (PE)-conjugated CD62P and fluorescein isothiocyanate (FITC)-PAC1 for 10–15 min in the dark. The reaction was stopped by fixing cells in 2% paraformaldehyde before analysing samples with flow cytometry (BD LSRFortessa™ cell analyzer). NETs induced expression of P-selectin and active-form αIIbβ3. b Fold change in the geometrical mean fluorescence of P-selectin-PE and PAC1-FITC in NET-activated platelets compared to vehicle-treated platelets. c WP (3 × 10 /mL) were incubated with vehicle (PBS) or NETs for 30 min at 37 °C with continuous stirring at 1200 rpm. PS was detected by incubating platelets with Annexin V-FITC in binding buffer for 15 min in the dark. Samples were then washed in binding buffer and analysed by flow cytometry (BD LSRFortessa). NETs induced PS exposure compared to vehicle control. d Fold change in geometrical mean of fluorescence of Annexin V-FITC in NET-activated platelets compared to vehicle-treated platelets. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Data are expressed as mean ± SEM,*P < 0.05, n≥ 4 Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 7 of 15 ab cd Fig. 3 Heparin and DNase did not reduce NET-induced platelet aggregation. a Representative traces showing dose-dependent aggregation of washed human platelets (WP, 3 × 10 /mL) in response to calf thymus histones (CTH; 1, 5, 20 and 40 μg/mL). b Representative aggregation traces showing the effect of heparin on NET and CTH-induced platelet aggregation. The reactions were allowed to proceed for 18 min, then collagen (5 μg/mL) was added to the CTH-Heparin reaction to test platelet functional response. c Representative aggregation traces showing the effect of DNase on NET-induced platelet aggregation. NETs were pre-treated with DNase (20, 200 U/mL) for 30 min at 37 °C before addition to WP (3 × 10 /mL). Traces are representative of 3 independent experiments. d Bar graphs depict % of platelet aggregation treated with NET alone or with Heparin and DNase. Percentage platelet aggregation was measured at 20 min. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Data are expressed as mean ± SEM,*P < .05; ns: non-significant, n ≥ 3 histones are major contributors to NET-induced washed that neither DNA nor histones contribute to platelet aggregation. Since histones can activate platelets, NET-induced washed platelet aggregation or activation. which has suggested to be mediated through toll-like re- Extracellular DNA and histones gained considerable ceptor (TLR) 2 and TLR4 [18], platelets were interest for their contribution to NET-induced throm- pre-incubated with TLR2- and TLR4-blocking antibodies bosis, while little attention has been paid to other com- or matching isotype controls (50 μg/mL) for 15 min be- ponents of NETs such as neutrophil proteases. Previous fore incubation with NETs. Similarly, anti-TLR2 and studies have reported that Cat G and MPO can modu- -TLR4 antibodies also did not affect NET-induced plate- late platelet response [36, 37]. To determine the effect of let activation (Additional file 1: Figure S4). It was re- NET-derived Cat G and MPO on platelet activation, we cently reported that calf thymus histones can activate used Cat G and MPO inhibitors at concentrations previ- platelets through GPVI receptor [29]. However, ously described in the literature [36, 37]. Although Cat pre-incubating platelets with a GPVI inhibitor, losartan G inhibition did not significantly affect the maximum (30 μM[33–35]) did not affect NET-induced platelet ag- aggregation response of platelet to NETs, we observed a gregation (Additional file 1: Figure S5). Our data suggest trend of increased aggregation lag time (data not Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 8 of 15 shown). Interestingly, Cat G inhibitor (Cat G I) but not 100 vs. 56 ± 10.1; ***P < 0.001, n = 5) (Fig. 6b), dense MPO inhibitor (MPO I) significantly reduced granule secretion (% Max secretion; at 1 min: 100 vs. NET-induced platelet expression of P-selectin (% Max 69.3 ± 9.8; P = 0.052; at 10 min: 100 vs. 62.1 ± 9.4; *P < response: 100 vs. 85 ± 3.2; *P < 0.05, n = 4), PAC1 (% 0.05, n = 5) (Fig. 6e-f), and expression of P-selectin (% Max response: 100 vs. 77.3 ± 3.7; *P < 0.05, n = 4) and PS Max response: 100 vs. 72.9 ± 2.9; ***P < 0.001, n = 3) (Fig. exposure (% Max response: 100 vs. 64.6 ± 12.1; *P < 0.05, 6d) and PAC1 (% Max response: 100 vs. 40.34 ± 11; ***P n = 4) (Fig. 4a-c). The concentration of Cat G I used < 0.001, n = 3) (Fig. 6c). Surprisingly, aspirin did not alter (0.5 μM) did not affect platelet physiological response to NET-induced platelet aggregation or expression of thrombin (0.1 U/mL) (Additional file 1: Figure S6), con- P-selectin and PAC1 (Fig. 6b-d, Additional file 1: Figure firming that the inhibitory response is specific to S8), however it significantly reduced NET-induced plate- NET-bound Cat G activation of platelets. Additionally, let dense granule secretion (% Max secretion; at 1 min: similarly to MPO, neutrophil elastase did not markedly 100 vs. 62.6 ± 7.8; **P < 0.01, n = 5; at 10 min: 100 vs. affect NET-induced platelet activation (Additional file 1: 63.5 ± 13.1; *P < 0.05, n = 5) (Fig. 6e-f). These findings Figure S7). suggest a broader role of ticagrelor, but not aspirin, in reducing NET-induced platelet response. SYK and NOX1 contribute to NET-induced platelet response NET-induced platelet response is dependent on integrin We began to delineate the platelet receptors and down- αIIbβ3 stream pathway involved in NET-induced platelet activa- Considering the role of NETs in mediating platelet adhe- tion. The non-receptor tyrosine kinase Syk mediates sion and spreading [30], we were interested in examining signalling from major platelet receptors, including the the effect of platelet adhesion receptor αIIbβ3in histone receptors GPVI and TLR2 [29]. Whereas NET-induced platelet response. Reopro, a monoclonal NADPH oxidase (NOX) regulates GPVI-induced react- antibody that binds to and inhibits the active form of ive oxygen species generation and subsequent thromb- αIIbβ3, dramatically reduced NET-induced platelet ag- oxane A2 (TxA2) production [38]. We demonstrate that gregation (% Max Agg: 100 vs. 31.2 ± 3.3; ***P < 0.001, n platelet Syk phosphorylation was augmented upon ex- = 4) (Fig. 7b) and dense granule secretion (% Max secre- posure to NETs, which was accompanied by upregula- tion; at 1 min: 100 vs. 50 ± 12.2; P = 0.075; at 10 min: tion of the downstream signalling molecules p-Akt and 100 vs. 59.1 ± 10.8; *P < 0.05, n = 5) (Fig. 7e-f). Moreover, p-Erk1/2 (Fig.5e). A Syk phosphorylation inhibitor (Bay Reopro completely inhibited NET-induced PAC1 expres- 61–3606, 5 μM) reduced NET-induced platelet aggrega- sion (Fig. 7c), most likely due to competitive binding to tion (% Max Agg: 100 vs. 74.21 ± 8.8; ***P < 0.001, n =7) αIIbβ3 which is also the target for the PAC1 antibody. (Fig. 5b), dense granule secretion (% Max secretion at However, Reopro did not reduce NET-induced platelet 1 min: 100 vs. 52.2 ± 9.2, ***P < 0.001; at 10 min: 100 vs. P-selectin expression (Fig. 7d), suggesting that NETs do 64.4 ± 6.3; **P < 0.01, n = 6) (Fig. 5e-f), and PAC1 expres- not trigger αIIbβ3 outside-in signalling in platelets. sion (% Max response: 100 vs. 81.7 ± 6.9; *P < 0.05, n =3) RGDS (100 μM), a peptide that binds to αIIbβ3 and (Fig. 5c), while P-selectin expression remained un- prevents conformational change triggered by inside-out changed (Fig. 5d). On the other hand, NOX1 inhibitor signalling, significantly reduced NET-induced platelet (ML171, 5 μM) did not alter NET-induced platelet ag- aggregation (% Max Agg: 100 vs. 43.7 ± 5.6; ***P < 0.001, gregation or activation (P-selectin and active-form n = 3) (Fig. 7b) and dense granule secretion (% Max se- αIIbβ3 expression) (Fig. 5a-d), however, it significantly cretion; at 1 min: 100 vs. 66 ± 8.7; *P < 0.05; at 10 min: reduced NET-induced platelet dense granule secretion 100 vs. 48.2 ± 13.8; **P < 0.01, n = 5) (Fig. 7e-f). Platelet (% Max secretion at 1 min: 100 vs. 62.4 ± 7.1; **P < 0.01; activation show that RGDS markedly reduced at 10 min: 100 vs. 58.6 ± 6.6; **P < 0.01, n = 6) (Fig. 5d). NET-induced platelet P-selectin expression (% Max re- Collectively, these results highlight the diversity of plate- sponse: 100 vs. 82.6 ± 3.7; **P < 0.01, n = 3) (Fig. 7d) and let pathways that are activated by NETs. PAC1 expression (% Max response: 100 vs. 38.4 ± 16.2; **P < 0.01, n = 3) (Fig. 7c). Overall, our results confirm P2Y but not cyclooxygenase pathway is required for the crucial role of αIIbβ3 in NET-induced platelet NET-induced platelet aggregation response. Drugs that target either P2Y (e.g., ticagrelor) or cyclo- oxygenase pathway (aspirin) are clinically available and Discussion crucial in the management of thrombosis [39]. There- Our study explored the effect of in vitro-generated NETs fore, we were interested in investigating their effect on on washed human platelets. As described in the NET-induced platelet response. Ticagrelor markedly re- methods, cell-free NETs were isolated from duced NET-induced platelet aggregation (% Max Agg: PMA-activated human neutrophils. PMA is a known Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 9 of 15 Fig. 4 Inhibition of Cat G, but not MPO, attenuates NET-induced platelet response. Flow cytometry analysis of P-selectin, PAC1, and PS exposure on platelets. NETs were pre-treated with vehicle or inhibitors for 30 min at 37 °C. a-b WP (1 × 10 /mL) were incubated with NETs pre-treated with vehicle (0.1% DMSO in PBS), Cat G I (0.5 μM) or MPO I (50 μM) for 10–15 min in the dark with labelled monoclonal antibodies that detect P-selectin (CD62P) and active αIIbβ3 (PAC1). The reaction was stopped by fixing cells in 2% PFA before analysing samples with flow cytometry (BD LSRFortessa). Bar graphs depict the % inhibition in P-selectin, and active αIIbβ3 expression in platelets treated with NETs pre-treated with different inhibitors compared to platelets treated with NETs that were pre- treated with vehicle. Results were normalized for each donor relative to NET-induced platelet response. c WP (3 × 10 /mL) were incubated with NETs pre-treated with vehicle (0.1% DMSO in PBS), Cat G I (0.5 μM) or MPO I (50 μM) for 30 min at 37 °C with continuous stirring at 1200 rpm. Platelets were then stained with Annexin V-FITC in binding buffer for 15 min in the dark. Samples were then washed in binding buffer and analysed by flow cytometry (BD LSRFortessa). Bar graph depicts the % inhibition in PS expression in platelets treated with NETs pre-treated with different inhibitors compared to platelets treated with NETs pre-treated with vehicle. Results were normalised for each donor relative to NET-induced platelet response. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Data are expressed as mean ± SEM,*P < .05; ns: non-significant, n =4 platelet agonist [40], however PMA was washed out with the culture media after 3 h incubation with neutrophils, thus it is highly unlikely that NET-induced platelet ag- gregation was confounded by PMA. Unlike the widely used method of cell-free NET preparation by Urban et al., [26], we did not use this method involving DNase/ EDTA, as EDTA can hinder platelet functional response [41]. We demonstrate that intact cell-free NETs exhibit the capacity to directly activate several platelet re- sponses, such as aggregation, dense and α-granule secre- tion (ADP release and P-selectin expression), PS exposure and activation of integrin αIIbβ3, which oc- curred independently of the presence of coagulation fac- tors or thrombin. NETs triggered a dose-dependent aggregation response in platelets with delayed lag time which correlates with the ability of NETs to first induce rapid platelet dense granule secretion. In addition to being a procoagulant platform, NETs in- duced PS exposure on platelet’s surface, a characteristic feature of procoagulant platelets. In the presence of small amounts of activated coagulation factors, PS can instigate thrombin generation, which can directly acti- vate platelets and conversion of fibrinogen to fibrin [42]. In washed platelets, strong or multiple agonists can trig- ger PS-exposing procoagulant platelets [43, 44]. The lat- ter is in line with our data and suggests that NETs are not a single agonist but a platform that presents a num- ber of agonists that can promote platelet activation via multiple pathways. Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 10 of 15 c d e f Fig. 5 (See legend on next page.) Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 11 of 15 (See figure on previous page.) Fig. 5 Syk, but not NOX1, inhibition attenuated NET-induced platelet aggregation. a Representative aggregation traces showing the effect of Syk phosphorylation (BAY61, 5 μM) and NOX1 (ML171, 5 μM) inhibitors on NET-induced platelet aggregation. WP (3 × 10 /mL) were pre-incubated with the inhibitors for 15 min at 37 °C before addition of NETs. Platelet aggregation was measured using transmission aggregometer (Chrono- log). b Bar graph comparing the effect of BAY61 and ML171 on NET-induced platelet aggregation. Platelet aggregation percentage was calculated after 20 min on a light transmission aggregometer. Results were normalized for each donor relative to NET-induced platelet aggregation. Data are expressed as mean ± SEM, ***P < .001; ns: non-significant, n =7. (c-d) Bar graphs comparing the effect of BAY61 (5 μM) and ML171 (5 μM) on platelet activation (measured by P-selectin and active αIIbβ3 expression, n = 3) and (e-f) ATP/ADP secretion (n = 6) elicited by NETs. Results were normalized for each donor relative to NET-induced platelet response. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant. e Syk phosphorylation is augmented by NETs. WP (3 × 10 /mL) were incubated with collagen (5 μg/mL), vehicle (PBS) or NETs for 3 min. Forty-five μl of total cell lysate was then analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted for p-Syk, p-Akt and p-Erk1/2. Equal loading was verified by α-actinin. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. The immunoblots are representative sample of 3 independent experiments NETs are made up of DNA and proteins (1:1.67 ± tenfold lower platelet aggregation response in washed 0.26 g, DNA to proteins) [26]. Histones account for 70% platelets compared to PRP [29]. of all NET-associated proteins [18, 26, 29, 45]. Previous Neutrophil granular proteins are a part of NETs and studies showed the ability of single-strand DNA to bind have separately been shown to activate platelets [37, 50]. platelets [46] while double strand DNA can induce Neutrophil serine proteases and histones are negatively platelet aggregation [47, 48]. Degradation of DNA with charged proteins [51] and would be tightly bound to the DNase has been shown to digest NETs and reduce plate- positively charged DNA backbone of NETs, thus most let aggregates under flow [30], or platelet adhesion to likely remain bound after isolation procedures [26]. The NETs under static condition [12]. On the other hand, pre-treatment of NETs with an MPO inhibitor did not sig- histones are well-established as platelet agonists that can nificantly reduce NET-induced upregulation of P-selectin, trigger a cascade of platelet responses with defined sur- active αIIbβ3, or PS exposure on platelets, suggesting face receptors and signalling pathways [18, 29, 45]. Hep- MPO does not play a major role in NET-induced platelet arin has been reported to bind to histones, thus activation which is consistent with previous studies preventing its binding to platelets [19, 32]. Surprisingly, reporting that MPO is not a robust activator of platelets, in this study DNase- and heparin-treated NETs were still but only induces partial activation or priming of platelets capable of aggregating washed platelets and induced ex- [37]. On the other hand, inhibiting Cat G resulted in a sig- pression of P-selectin and active αIIbβ3 to the same ex- nificant decrease in platelet surface expression of tent of untreated NETs. Although DNase and heparin P-selectin, active αIIbβ3 and PS. This suggests Cat G as a can destabilise the NET structure [9], the presence of molecular mediator of NET-induced platelet activation, freely suspended individual NET components – such as and potentially significant contributor to thrombus forma- cell free-DNA, histones, and neutrophil proteases – may tion, as previously described [36]. NE is the second most have greater capacity and exposure to directly bind and abundant NET-associated proteins after histones [26]and activate platelets, as opposed to being restricted on can potentiate Cat G-induced platelet aggregation [52], NETs. This presumption is in line with a study that however in our hands, NE inhibitor did not affect showed DNAse-treatment of NETs resulted in increased NET-induced platelet responses (data not shown). coagulation effect [15]. Moreover, nuclear histones have As the NET scaffold contains an array of associated different molecular mass and stoichiometry compared to proteins, some of which have been independently associ- NET-derived histones [26]. Therefore they may exhibit ated with platelet activation [26, 36, 37, 50, 52–54], a different biological activity. Indeed, a recent report has single inhibitor is highly unlikely to completely abrogate found that individual histones and DNA capable of indu- NET-induced platelet responses. However, we were in- cing coagulation, but not intact NETs that were released terested mainly in clarifying the major NET components, from human neutrophils [49]. platelet receptors and downstream signalling molecules Histones are known to induce platelet Syk kinase acti- that mediate NET-induced platelet secretion and aggre- vation through GPVI, and other tyrosine kinase-linked gation. Inhibition of the tyrosine kinase Syk activity, receptors [29]. We show that inhibition of Syk attenu- P2Y and αIIbβ3 reduced NET-induced platelet aggre- ated NET-induced platelet responses. However, inhibi- gation and secretion. While inhibition of NOX1 and tors of histone receptors on platelets (TLR2, TLR4 and TxA2 reduced NET-induced platelet dense granule se- GPVI) did not reduce NET-induced platelet aggregation. cretion, but not aggregation. Moreover, the sheer magnitude of platelet aggregation The role of NETs in initiating thrombosis in vivo has response to NETs in washed system precludes a signifi- been established in mice models of different diseases cant contribution of histones which are known to have a [55]. However, the molecular mechanisms that drive Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 12 of 15 c d e f Fig. 6 Inhibition of cyclooxygenase pathway in platelets attenuated NET-induced platelet secretion but not aggregation, while inhibition of P2Y affects both. a Representative aggregation traces showing the effect of ticagrelor (1 μM) and aspirin (100 μM) on NET-induced platelet aggregation. WP (3 × 10 /mL) were pre-incubated with the inhibitors for 15 min at 37 °C before addition of NETs. Platelet aggregation was measured by light transmission aggregometry (Chrono-log). b Bar graph comparing the effect of ticagrelor (1 μM) and aspirin (100 μM) on NET- induced platelet aggregation. Platelet aggregation percentage was calculated after 20 min on a light transmission aggregometer. Results were normalized for each donor relative to NET-induced platelet aggregation. Data are expressed as mean ± SEM, ***P < .001; ns: non-significant, n =3. (c-d) Bar graphs comparing the effect of ticagrelor (1 μM) and aspirin (100 μM) on platelet activation (measured by P-selectin and active αIIbβ3 expression) (n = 3) and (e-f) ATP/ADP secretion (n = 5) elicited by NETs. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Results were normalized for each donor relative to NET-induced platelet response. Data are expressed as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001, ns: non-significant NET-induced thrombosis are not well understood. As initiate thrombosis [56, 57]. We propose that platelets the recent study by Noubouossie et al., has demon- adhere mainly to NET-derived DNA, then multiple strated that intact NETs do not directly initiate coagula- NET-bound proteins induce platelet aggregation and PS tion [49], we propose that platelets but not coagulation exposure which then can propagate coagulation and factors are more likely to be the main target of NETs in thrombin generation. In addition to their pivotal role in thrombosis. Our study did not account for the disrupt- thrombosis, platelets can also orchestrate inflammation ing effect of NETs on endothelium which can also [58]. Therefore, although dismantling NETs may reduce Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 13 of 15 cd ef Fig. 7 Inhibition of αIIbβ3 attenuates NET-induced platelet response. a Representative aggregation traces showing the effect of Reopro (25 μg/ mL) and RGDS (100 μM) on NET-induced platelet aggregation. WP (3 × 10 /mL) were pre-incubated with the inhibitors for 15 min at 37 °C before addition of NETs. Both Reopro and RGDS reduced platelet aggregation. Platelet aggregation was measured by light transmission aggregometry (Chrono-log). b Bar graph comparing the effect of Reopro (25 μg/mL) and RGDS (100 μM) on NET-induced platelet aggregation. Platelet aggregation percentage was calculated after 20 min on a light transmission aggregometer. Results were normalised for each donor relative to NET-induced platelet aggregation. Data are expressed as mean ± SEM,***P < .001. c-f Bar graphs comparing the effect of Reopro (25 μg/mL) and RGDS (100 μM) on platelet activation (measured by P-selectin and active αIIbβ3 expression, n = 3) and ATP/ADP secretion (n = 5) elicited by NETs. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Results were normalised for each donor relative to NET-induced platelet response. Data are expressed as mean ± SEM, *P < 0.05; **P < 0.01, ns: non-significant NET-induced thrombosis [59], inhibition of platelet ac- resulted in decreased neutrophil-platelet complexes in tivity may not only reduce thrombosis but also the kidney vasculature, along with improved vascular platelet-mediated inflammation. Indeed, Jansen et al., function in tumour-bearing mice [60]. Thus in these have recently shown that platelet inhibition with clopi- contexts NETs can also be considered as scaffold for dogrel was superior to DNase in reducing granulocyte platelets that drives inflammatory reactions. activation, NET formation and acute kidney injury in a renal reperfusion injury mice model [31]. Apart from Conclusion NETs inducing thrombosis, Cedervall et al. also showed This study showed for the first time that in vitro gener- that the use of DNase to disrupt tumour-induced NETs ated NETs can directly induce marked platelet Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 14 of 15 aggregation. We further identified the molecular path- Received: 1 March 2018 Accepted: 15 May 2018 ways activated in platelet responses to NETs. It is im- portant to note that aspirin, a widely used antiplatelet, was not as effective at reducing NET-induced platelet References 1. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and aggregation as ticagrelor or Reopro. Finally, pretreat- inflammation. Nat Rev Immunol. 2013;13:159–75. ment of NETs with DNase or heparin did not reduce 2. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, NET-induced activation or aggregation of human Weinrauch Y, Zychlinsky A. Neutrophil extracellular traps kill Bacteria. Science. 2004;303:1532–5. washed platelets. Taken together, we conclude that tar- 3. McDonald B, Urrutia R, Yipp Bryan G, Jenne Craig N, Kubes P. 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Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones

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Life Sciences; Cell Biology; Protein-Ligand Interactions; Receptors; Cytokines and Growth Factors
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1478-811X
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10.1186/s12964-018-0235-0
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Abstract

Background: The release of neutrophil extracellular traps (NETs), a mesh of DNA, histones and neutrophil proteases from neutrophils, was first demonstrated as a host defence against pathogens. Recently it became clear that NETs are also released in pathological conditions. NETs released in the blood can activate thrombosis and initiate a cascade of platelet responses. However, it is not well understood if these responses are mediated through direct or indirect interactions. We investigated whether cell-free NETs can induce aggregation of washed human platelets in vitro and the contribution of NET-derived extracellular DNA and histones to platelet activation response. Methods: Isolated human neutrophils were stimulated with PMA to produce robust and consistent NETs. Cell-free NETs were isolated and characterised by examining DNA-histone complexes and quantification of neutrophil elastase with ELISA. NETs were incubated with washed human platelets to assess several platelet activation responses. Using pharmacological inhibitors, we explored the role of different NET components, as well as main platelet receptors, and downstream signalling pathways involved in NET-induced platelet aggregation. Results: Cell-free NETs directly induced dose-dependent platelet aggregation, dense granule secretion and procoagulant phosphatidyl serine exposure on platelets. Surprisingly, we found that inhibition of NET-derived DNA and histones did not affect NET-induced platelet aggregation or activation. We further identified the molecular pathways involved in NET-activated platelets. The most potent single modulator of NET-induced platelet responses included NET-bound cathepsin G, platelet Syk kinase, and P2Y and αIIbβ3 receptors. Conclusions: In vitro-generated NETs can directly induce marked aggregation of washed human platelets. Pre-treatment of NETs with DNase or heparin did not reduce NET-induced activation or aggregation of human washed platelets. We further identified the molecular pathways activated in platelets in response to NETs. Taken together, we conclude that targeting certain platelet activation pathways, rather than the NET scaffold, has a more profound reduction on NET-induced platelet aggregation. Keywords: Neutrophil, Neutrophil extracellular traps, Platelet, Aggregation, DNA, Histones, Cathepsin G * Correspondence: pat.metharom@curtin.edu.au Platelet Research Laboratory, School of Pharmacy and Biomedical Sciences, Curtin Health and Innovation Research Institute, Faculty of Health Sciences, Curtin University, Bentley Campus, Office 160, Building 305, Kent Street, Bentley, Perth, WA 6102, Australia © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 2 of 15 Background intravascular fibrin formation and degrading tissue Neutrophils are well known for their crucial role in in- factor pathway inhibitor [21], while myeloperoxidase nate immunity, providing the first line of defence against (MPO) can prime platelets [22]. pathogens through multiple mechanisms [1]. The dis- Collectively, NETs are a potentially potent agonist of covery of a relatively new antimicrobial mechanism, platelet activation and promoter of coagulation, thereby whereby activated neutrophils expel their DNA and pro- amplifying and supporting thrombus formation. How- teins forming an extracellular matrix, termed neutrophil ever, despite many studies reporting NETs as promoters extracellular traps (NETs) [2], has gained much interest of thrombosis, these studies were conducted in whole recently. NETs possess antimicrobial function either by blood assays or in the presence of plasma, implicating a entrapping and immobilising pathogens or presentation role of plasma coagulation factors. Thus the capacity of of NET-bound antimicrobial proteins [2, 3]. However, intact cell-free NETs to directly activate washed platelets NETs can serve as more than just a host defence mech- is not clearly understood. In this study, we investigated anism, as studies have implicated the role of NETs in in- the effect of NETs on platelet function including aggre- flammatory and autoimmune diseases and pathological gation, secretion, and surface expression of receptors. conditions including thrombosis [4]. Notably, as the role We also begin to determine molecular mediators and of NETs in thrombosis is being investigated extensively signalling pathways by examining the effect of antago- in recent times, there is potential for NETs to not only nists of specific NET components and antiplatelet drugs, serve as therapeutical target for thrombotic diseases but on the impact of NETs on platelet activation. several other clinical conditions such as diabetes, sys- temic lupus erythematosus, pre-eclampsia and certain Methods types of cancers, all which are known to be associated Materials with increased risk of thrombosis [5–8]. Purified anti-human TLR2, TLR4 blocking antibodies Increasing number of studies are recognising NETs and their matching isotype controls were obtained from as a procoagulant surface, which is capable of pro- BioLegend, Inc., USA. Bay 61–3606 and Phorbol moting thrombosis both in vitro and in animal 12-myristate 13-acetate (PMA), ticagrelor and Cell De- models of deep vein thrombosis and arterial throm- tection ELISA PLUS kit were from Sigma-Aldrich, bosis [9–13]. The NET structure can serve as a scaf- Australia. ML-171, aspirin, RGDS, cathepsin G inhibitor fold for platelet adhesion and aggregation [9, 14]thus I, neutrophil elastase inhibitor (1-(3-methylbenzoyl)-1- providing a platform for the subsequent formation of H-indazole-3-carbonitrile), myeloperoxidase inhibitor 1 thrombi. Furthermore, NETs have been shown to dir- (4-Aminobenzoic acid hydrazide), losartan were ob- ectly promote the activation of intrinsic coagulation tained from Cayman Chemical, USA. Abciximab (Reo- pathway leading to thrombin generation [15]. Besides Pro) and low molecular weight heparin (Clexane intact NETs being capable of activating coagulation, enoxaparin sodium) were from Eli Lilly and Sanofi Aven- several components within the NET structure have tis Australia Pty Ltd., respectively. DNAse I solution was been reported to activate platelets and initiate or pro- purchased from STEMCELL Technologies Australia Pty mote coagulation. Cell-free DNA, which makes up Ltd. Collagen and thrombin were from Chrono-log Cor- the major backbone of NETs, has previously been poration, USA. Human PMN Elastase ELISA kit was ob- shown to activate thrombin generation via the intrin- tained from Abcam Biotechnology, Cambridge, UK. sic pathway of coagulation [16]. The second most abundant constituent and protein found on NETs, Preparation of washed human platelets extracellular histones, have been studied extensively Blood was drawn from healthy volunteers into a syringe and known to activate platelets and promote coagula- containing acid-citrate-dextrose (ACD; 1:7 (v/v) with in- tion through multiple mechanisms [9, 17–19]. For ex- formed consent in concordance with the Curtin Univer- ample, histones are capable of generating thrombin in sity Human Research Ethics Committee (approval the presence of plasma and activating platelet aggre- number HR54/2014). Blood donors were gation which has been suggested to be mediated medication-free 2 weeks prior to the day of donation. through toll-like receptor (TLR) 2 and TLR 4 [18]. Washed platelets were prepared, with some modifica- However, the involvement of TLRs in platelet aggre- tions, as previously described [23, 24]. Briefly, blood was gation is not clear as Clark et al. have shown that centrifuged at 150 x g for 20 min. Platelet-rich plasma LPS induced platelet activation but not aggregation (PRP) was collected and centrifuged at 800 x g for [20] Furthermore, neutrophil granular enzymes that 10 min in the presence of 1 μM prostaglandin E1 (PGE1; are bound to NETs such as neutrophil elastase (NE) Cayman Chemical). Platelets were then washed three and cathepsin G (Cat G), can separately promote co- times in CGS buffer (14.7 mM trisodium citrate, agulation and thrombus growth by facilitating 33.33 mM glucose and 123.2 mM NaCl, pH 7), in the Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 3 of 15 presence of PGE1 (1 μM). Platelets were adjusted to 1 × Platelet-dense granule secretion assay 10 /mL with calcium-free Tyrode-Hepes buffer (5 mM Platelet secretion was determined by measuring ATP re- HEPES, 5.5 mM glucose, 138 mM NaCl, 12 mM lease using luciferin/luciferase reagent (Chrono-Lume, NaHCO , 0.49 mM MgCl , 2.6 mM KCl, 0.36 mM Chrono-log Corporation, USA). Briefly, 90 μl of platelets 3 2 NaH PO , pH 7.4). Platelets were supplemented with (1 × 10 /mL) in Tyrodes-HEPES buffer (with calcium) 2 4 1.8 mM CaCl (final concentration) prior to were incubated with 10 μl of NETs with gentle shake at experimentation. 37 °C for 1 and 10 min before adding 5 μlof Chrono-Lume reagent. The luminescence was measured Preparation of neutrophils and cell-free neutrophil using Enspire Multimode Plate Reader (PerkinElmer, extracellular traps (NETs) USA). Where anti-platelet drugs were used, platelets Neutrophils were isolated from human blood using Poly- were pre-incubated with the drugs for 15 min at 37 °C morphPrep (Axis-Shield, Norway), with minor changes before incubating with NETs. to the manufacturer’s protocol. Briefly, blood anticoagu- lated with EDTA (2 mM) was layered over Polymorph- P-selectin exposure and αIIbβ3 activation Prep and centrifuged at 500 x g for 40 min. The Platelet activation was measured by detecting P-selectin neutrophil fraction was collected and washed twice at and active-form αIIbβ3 on the platelet surface using flow 4 °C in Hank’s buffered saline solution (without calcium cytometry. Where inhibitors were used, platelets were or magnesium) and resuspended in X-VIVO 15 media pre-incubated for 15 min at 37 °C before adding NETs. (Lonza, Switzerland). Neutrophil purity was > 95% as de- Whenever inhibitors of components of NETs (i.e. termined with a haematology analyser (Mindray, DNAse I, cathepsin G, myeloperoxidase, and elastase in- BC-VET2800). Cell-free NETs were isolated as previ- hibitors) were used, NETs were pre-incubated for ously described [25] with minor changes to the protocol. 30 min at 37 °C. The specificity of inhibitors used was This method of NET isolation does not involve using also examined for their effect on thrombin (0.1 U/mL) DNase or EDTA [26], which may confound platelet re- and collagen (5 μg/mL)–induced platelet activation. In- sponse to NETs. Briefly, neutrophils (2.5 × 10 /mL) were hibitor-, or vehicle-, treated washed human platelets stimulated with 500 nM PMA for 3 h at 37 °C and 5% (1 × 10 /mL) were treated with NETs (10% of final vol- CO . The supernatant, containing PMA, was discarded ume) and stained with phycoerythrin-conjugated mouse and the NET monolayer was detached with anti-human CD62P (P-selectin) and fluorescein phosphate-buffered saline (PBS). The cell debris was pel- isothiocyanate-conjugated mouse anti-human PAC1 leted by centrifugation at 480 x g for 10 min at 4 °C. The (active-form αIIbβ3), or suitable isotype control anti- supernatant was further centrifuged at 15,000 x g for bodies for 15 min in the dark. All antibodies were from 20 min at 4 °C to pellet DNA then resuspended in PBS BD Biosciences. Samples were analysed by flow cytome- at 100 μl per 1 × 10 of stimulated neutrophils to obtain try (BD LSRFortessa™ cell analyzer). cell-free NETs. Cell-free NETs were characterised by de- tecting DNA-histone complex and neutrophil elastase Phosphatidylserine (PS) exposure using Cell Detection ELISA PLUS kit (Sigma Aldrich) Platelets (3 × 10 /mL) were incubated with NETs for and Human PMN Elastase ELISA kit (Abcam), respect- 30 min at 37 °C with continuous stirring at 1200 rpm. ively. Cell-free NETs were incubated with platelets at Whenever inhibitors were used, NETs were 10% of final reaction volume (i.e. 1-volume NET solution pre-incubated for 30 min at 37 °C. Thrombin (0.1 U/ to 9-volume platelets). mL) was used as positive control. Platelets were then stained with Annexin V-FITC (BioLegend, USA) in bind- Platelet aggregation assay ing buffer according to manufacturer’s instructions for Washed platelets (3 × 10 /mL) in Tyrode-HEPES buffer 15 min in the dark. Samples were then washed in bind- supplemented with 1.8 mM calcium chloride were incu- ing buffer and analysed by flow cytometry (BD LSRFor- bated in the presence of cell-free NETs (10% of final re- tessa™ cell analyzer). action volume) and platelet aggregation was monitored at 37 °C with continuous stirring at 1200 rpm in a light Antibodies and western blot transmission aggregometer (Model 700 Aggregometer, Rabbit antibodies specific for p-Akt (Ser473), p-Erk1/2 Chrono-log Corporation, USA) for at least 20 min. (Thr202/Tyr204), p-Syk (Tyr352), p-Tyr1000 and Tyrode-HEPES buffer was used as a blank. Where inhib- α-actinin were obtained from Cell Signalling Technology itors were used, platelets were pre-incubated for 15 min (USA). Platelets (3 × 10 /mL) were incubated with NETs at 37 °C prior to incubation with NETs. Control samples (10% of final reaction volume) at 37 °C for 3 min with were incubated with the corresponding volume of continuous stirring at 1200 rpm. Platelets were then buffer. lysed in Laemmli sample buffer supplemented with Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 4 of 15 Protease/Phosphatase Inhibitor Cocktail (Cell Signalling (Fig. 1c). Platelet secretion was examined after 1 and Technology) and β-mercaptoethanol. Forty-five μlof 10 min incubation with NETs to identify the time re- protein sample was loaded per lane and separated by so- quired for secretion compared to aggregation. NETs in- dium dodecyl sulphate–polyacrylamide gel electrophor- duced significant platelet secretion by 1 min, while esis then transferred to a polyvinylidene difluoride aggregation did not occur until 5 min (Fig. 1b-c), indi- (PVDF) protein blotting membrane with 0.2 μm pore cating that NET-induced platelet secretion precedes ag- size (GE Healthcare Life Sciences). The PVDF mem- gregation. Furthermore, we also examined if platelet brane was blocked in 5% non-fat powdered milk in functional response occurred concomitantly with intra- Tris-buffered saline with 0.1% Tween 20 (or 3% bovine cellular signalling, particularly the phosphorylation of serum albumin (BSA, Bovogen Biologicals Pty Ltd., proteins at tyrosine residues. Indeed, NETs induced tyro- Australia) in TBS-T for detection of p-Tyr-1000) at sine phosphorylation of several substrates with strong room temperature for 1 h. After a brief rinse with migrating bands at ~ 134, 80 and 60 kDa compared to TBS-T, the membrane was incubated overnight at 4 °C vehicle control (Fig. 1d). with primary antibodies at 1:1000 dilution. The primary antibody was detected with secondary horseradish NETs induce surface expression of receptors and peroxidase-conjugated anti-rabbit antibody (Jackson Im- phosphatidyl serine exposure on platelets mune Research, USA) at 1:40000 dilution. The mem- We also examined α-granule secretion by measuring the brane was developed using Amersham ECL Prime surface expression of P-selectin. Platelets incubated with Western Blotting Detection Reagent (GE Healthcare Life NETs for 10 to 15 min showed a significant increase of Sciences, USA), and chemiluminescence was detected P-selectin surface expression as detected by flow cytom- using ChemiDoc imaging system (Bio-Rad, USA). etry (Fig. 2a-b). A conformational change in platelet αIIbβ3 receptor is required for platelet aggregation [27]. Statistical analysis Therefore we assessed platelet surface expression of Data were analysed using GraphPad PRISM 4.0 software. active-form αIIbβ3 using PAC1 monoclonal antibody. As Results are expressed as the mean ± standard error shown in Fig. 2a-b, NETs induced a conformational (SEM). One-way ANOVA with posthoc Bonferroni’s change of αIIbβ3 from resting to an activated state. As Multiple Comparison Test were used to examine the PS exposure on platelets can propagate coagulation [28], statistical significance between means. Differences were we assessed the ability of NETs to induce PS exposure considered significant at P < 0.05. on the platelet surface. Platelets were incubated with NETs for 30 min at 37 °C with continuous stirring at Results 1200 rpm before analysing PS expression. Annexin NETs induce aggregation, secretion and activation of V-FITC was used to stain PS and was analysed by flow washed human platelets cytometry. NETs induced a marked increase in PS ex- We first examined the ability of cell-free NETs to dir- pression on platelet’s surface (Fig. 2c-d), suggesting that ectly induce aggregation of washed human platelets in- NET-activated platelets can provide a procoagulant dependently of the coagulation pathway. Platelets were surface. washed three times in CGS buffer to remove any con- taminating plasma, then resuspended in Tyrode-HEPES buffer (with 1.8 mM calcium chloride). Light transmis- The role of NET-derived DNA, histones, cat G, and MPO in sion aggregometry was used to measure platelet aggrega- NET-induced platelet response tion. Autologous cell-free NETs at different dilutions As the major components of NETs are DNA and histones, induced marked platelet aggregation (Fig. 1a). Interest- we first investigated whether the effect of NETs on washed ingly, platelet aggregation response was apparent only platelets is mediated via NET-derived DNA and/or his- after 5 mins in the presence of the highest concentration tones. DNA-histone complexes were confirmed in of NETs used (10% of final reaction volume) (Fig. 1b). cell-free NETs using Cell Detection ELISA PLUS kit. For all experiments, it was imperative that NETs were Based on comparisons between whole neutrophil lysates used within the same day of isolation as freezing or stor- and NET dilution samples, the concentration of ing NETs at 4 °C for more than 24 h completely abol- DNA-histone complex in cell-free NETs used in majority ished their activity. of experiments (i.e. 10% final NETs) was equivalent to the As platelet secretion amplifies platelet aggregation, we amount present in approximately 3.8 ± 1.2 × 10 /mL of assessed the ability of NETs to induce platelet dense neutrophil lysate (Additional file 1:Figure S1). Addition- granule secretion of ATP/ADP using a luminescence ally, the 10% cell-free NET solution was determined to assay. NETs triggered significant ATP/ADP secretion contain 292 ± 172 pg/mL of elastase, another major pro- from platelet dense granules compared to vehicle control tein component of NETs (Additional file 1: Figure S2). Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 5 of 15 ac Fig. 1 NETs directly induce aggregation of washed human platelets. a Representative traces showing dose-dependent aggregation of washed human platelets (WP, 3 × 10 /mL) in response to cell free-NETs. 0.1, 1 and 10% NETs refers to NETs constituting 0.1, 1 or 10% of final reaction volume, respectively (e.g. 10% NET is 1-volume NET solution to 9-volume platelets). Platelet aggregation was measured by light transmission aggregometer (Chrono-log). NETs were used at 10% of final reaction volume for following experiments*. b Progress time curve displaying % of NET-induced platelet aggregation which steadily increased over 20 min. c ATP/ADP release from NET-stimulated platelets. WP (1 × 10 /mL) were incubated with NETs with a gentle shake at 37 °C for 1 and 10 min before adding Chrono-Lume reagent. Luminescence was measured using Enspire Multimode Plate Reader (PerkinElmer). NETs induced significant ATP/ADP secretion from platelets at 1 and 10 min. Results represent fold change in luminescence arbitrary absorbance unit relative to vehicle (PBS) control. Data expressed as mean ± SEM; **P < 0.01, ***P < 0.0001, N ≥ 5. d Platelet signalling in response to NETs was examined by Western blot analysis. WP (3 × 10 /mL) were incubated with collagen (5 μg/mL), vehicle (PBS) or NETs for 3 min. Twenty μl of total cell lysate was analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted for phospho-Tyrosine (p-Tyr-1000). Equal loading was verified by α-actinin. Western blots indicate tyrosine-phosphorylated proteins in NET-stimulated platelets. *10% NET solution is prepared from 1 × 10 /ml of PMA-activated neutrophils, however due to losses during preparation steps, 10% NET is estimated to contain histone/DNA complexes equivalent to 3.8 ± 1.2 × 10 /mL neutrophils (Additional file 1: Figure S1). The elastase content present in a 10% NET reaction volume is 292 ± 172 pg/mL Calf thymus histones (CTH) are well-established response to another agonist remained unaffected (Fig. platelet agonists [29] and were used as a positive con- 3b). trol. CTH (1, 5, 20 and 40 μg/mL) induced a In order to test the impact of DNA on NET-induced dose-dependent aggregation of washed human platelet platelet aggregation, NETs were pre-treated with DNase (Fig. 3a). Heparin can strongly bind to and abate the (20, 200 U/mL) for 30 min at 37 °C before adding to effect of histones on platelets [30–32]. Indeed, hep- platelets (where NETs was used at 10% of the final reac- arin 20 U/mL completely abated CTH but not tion volume). DNase can dismantle NET-DNA (Add- NET-induced platelet aggregation (Fig. 3 b, d). Colla- itional file 1: Figure 3). However, DNase did not reduce gen (5 μg/mL) was added to the CTH-heparin reac- the ability of NETs to induce aggregation of washed hu- tion in order to verify that the platelet functional man platelets (Fig. 3c & d), suggesting neither DNA nor Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 6 of 15 Fig. 2 NETs induce platelet activation and phosphatidyl serine (PS) exposure on washed human platelets. a Representative dot plot of CD62P (P-selectin), PAC1 (active-form αIIbβ3) fluorescence. WP (1 × 10 /mL) were treated with vehicle (PBS) or NETs then incubated with labelled monoclonal antibodies phycoerythrin (PE)-conjugated CD62P and fluorescein isothiocyanate (FITC)-PAC1 for 10–15 min in the dark. The reaction was stopped by fixing cells in 2% paraformaldehyde before analysing samples with flow cytometry (BD LSRFortessa™ cell analyzer). NETs induced expression of P-selectin and active-form αIIbβ3. b Fold change in the geometrical mean fluorescence of P-selectin-PE and PAC1-FITC in NET-activated platelets compared to vehicle-treated platelets. c WP (3 × 10 /mL) were incubated with vehicle (PBS) or NETs for 30 min at 37 °C with continuous stirring at 1200 rpm. PS was detected by incubating platelets with Annexin V-FITC in binding buffer for 15 min in the dark. Samples were then washed in binding buffer and analysed by flow cytometry (BD LSRFortessa). NETs induced PS exposure compared to vehicle control. d Fold change in geometrical mean of fluorescence of Annexin V-FITC in NET-activated platelets compared to vehicle-treated platelets. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Data are expressed as mean ± SEM,*P < 0.05, n≥ 4 Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 7 of 15 ab cd Fig. 3 Heparin and DNase did not reduce NET-induced platelet aggregation. a Representative traces showing dose-dependent aggregation of washed human platelets (WP, 3 × 10 /mL) in response to calf thymus histones (CTH; 1, 5, 20 and 40 μg/mL). b Representative aggregation traces showing the effect of heparin on NET and CTH-induced platelet aggregation. The reactions were allowed to proceed for 18 min, then collagen (5 μg/mL) was added to the CTH-Heparin reaction to test platelet functional response. c Representative aggregation traces showing the effect of DNase on NET-induced platelet aggregation. NETs were pre-treated with DNase (20, 200 U/mL) for 30 min at 37 °C before addition to WP (3 × 10 /mL). Traces are representative of 3 independent experiments. d Bar graphs depict % of platelet aggregation treated with NET alone or with Heparin and DNase. Percentage platelet aggregation was measured at 20 min. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Data are expressed as mean ± SEM,*P < .05; ns: non-significant, n ≥ 3 histones are major contributors to NET-induced washed that neither DNA nor histones contribute to platelet aggregation. Since histones can activate platelets, NET-induced washed platelet aggregation or activation. which has suggested to be mediated through toll-like re- Extracellular DNA and histones gained considerable ceptor (TLR) 2 and TLR4 [18], platelets were interest for their contribution to NET-induced throm- pre-incubated with TLR2- and TLR4-blocking antibodies bosis, while little attention has been paid to other com- or matching isotype controls (50 μg/mL) for 15 min be- ponents of NETs such as neutrophil proteases. Previous fore incubation with NETs. Similarly, anti-TLR2 and studies have reported that Cat G and MPO can modu- -TLR4 antibodies also did not affect NET-induced plate- late platelet response [36, 37]. To determine the effect of let activation (Additional file 1: Figure S4). It was re- NET-derived Cat G and MPO on platelet activation, we cently reported that calf thymus histones can activate used Cat G and MPO inhibitors at concentrations previ- platelets through GPVI receptor [29]. However, ously described in the literature [36, 37]. Although Cat pre-incubating platelets with a GPVI inhibitor, losartan G inhibition did not significantly affect the maximum (30 μM[33–35]) did not affect NET-induced platelet ag- aggregation response of platelet to NETs, we observed a gregation (Additional file 1: Figure S5). Our data suggest trend of increased aggregation lag time (data not Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 8 of 15 shown). Interestingly, Cat G inhibitor (Cat G I) but not 100 vs. 56 ± 10.1; ***P < 0.001, n = 5) (Fig. 6b), dense MPO inhibitor (MPO I) significantly reduced granule secretion (% Max secretion; at 1 min: 100 vs. NET-induced platelet expression of P-selectin (% Max 69.3 ± 9.8; P = 0.052; at 10 min: 100 vs. 62.1 ± 9.4; *P < response: 100 vs. 85 ± 3.2; *P < 0.05, n = 4), PAC1 (% 0.05, n = 5) (Fig. 6e-f), and expression of P-selectin (% Max response: 100 vs. 77.3 ± 3.7; *P < 0.05, n = 4) and PS Max response: 100 vs. 72.9 ± 2.9; ***P < 0.001, n = 3) (Fig. exposure (% Max response: 100 vs. 64.6 ± 12.1; *P < 0.05, 6d) and PAC1 (% Max response: 100 vs. 40.34 ± 11; ***P n = 4) (Fig. 4a-c). The concentration of Cat G I used < 0.001, n = 3) (Fig. 6c). Surprisingly, aspirin did not alter (0.5 μM) did not affect platelet physiological response to NET-induced platelet aggregation or expression of thrombin (0.1 U/mL) (Additional file 1: Figure S6), con- P-selectin and PAC1 (Fig. 6b-d, Additional file 1: Figure firming that the inhibitory response is specific to S8), however it significantly reduced NET-induced plate- NET-bound Cat G activation of platelets. Additionally, let dense granule secretion (% Max secretion; at 1 min: similarly to MPO, neutrophil elastase did not markedly 100 vs. 62.6 ± 7.8; **P < 0.01, n = 5; at 10 min: 100 vs. affect NET-induced platelet activation (Additional file 1: 63.5 ± 13.1; *P < 0.05, n = 5) (Fig. 6e-f). These findings Figure S7). suggest a broader role of ticagrelor, but not aspirin, in reducing NET-induced platelet response. SYK and NOX1 contribute to NET-induced platelet response NET-induced platelet response is dependent on integrin We began to delineate the platelet receptors and down- αIIbβ3 stream pathway involved in NET-induced platelet activa- Considering the role of NETs in mediating platelet adhe- tion. The non-receptor tyrosine kinase Syk mediates sion and spreading [30], we were interested in examining signalling from major platelet receptors, including the the effect of platelet adhesion receptor αIIbβ3in histone receptors GPVI and TLR2 [29]. Whereas NET-induced platelet response. Reopro, a monoclonal NADPH oxidase (NOX) regulates GPVI-induced react- antibody that binds to and inhibits the active form of ive oxygen species generation and subsequent thromb- αIIbβ3, dramatically reduced NET-induced platelet ag- oxane A2 (TxA2) production [38]. We demonstrate that gregation (% Max Agg: 100 vs. 31.2 ± 3.3; ***P < 0.001, n platelet Syk phosphorylation was augmented upon ex- = 4) (Fig. 7b) and dense granule secretion (% Max secre- posure to NETs, which was accompanied by upregula- tion; at 1 min: 100 vs. 50 ± 12.2; P = 0.075; at 10 min: tion of the downstream signalling molecules p-Akt and 100 vs. 59.1 ± 10.8; *P < 0.05, n = 5) (Fig. 7e-f). Moreover, p-Erk1/2 (Fig.5e). A Syk phosphorylation inhibitor (Bay Reopro completely inhibited NET-induced PAC1 expres- 61–3606, 5 μM) reduced NET-induced platelet aggrega- sion (Fig. 7c), most likely due to competitive binding to tion (% Max Agg: 100 vs. 74.21 ± 8.8; ***P < 0.001, n =7) αIIbβ3 which is also the target for the PAC1 antibody. (Fig. 5b), dense granule secretion (% Max secretion at However, Reopro did not reduce NET-induced platelet 1 min: 100 vs. 52.2 ± 9.2, ***P < 0.001; at 10 min: 100 vs. P-selectin expression (Fig. 7d), suggesting that NETs do 64.4 ± 6.3; **P < 0.01, n = 6) (Fig. 5e-f), and PAC1 expres- not trigger αIIbβ3 outside-in signalling in platelets. sion (% Max response: 100 vs. 81.7 ± 6.9; *P < 0.05, n =3) RGDS (100 μM), a peptide that binds to αIIbβ3 and (Fig. 5c), while P-selectin expression remained un- prevents conformational change triggered by inside-out changed (Fig. 5d). On the other hand, NOX1 inhibitor signalling, significantly reduced NET-induced platelet (ML171, 5 μM) did not alter NET-induced platelet ag- aggregation (% Max Agg: 100 vs. 43.7 ± 5.6; ***P < 0.001, gregation or activation (P-selectin and active-form n = 3) (Fig. 7b) and dense granule secretion (% Max se- αIIbβ3 expression) (Fig. 5a-d), however, it significantly cretion; at 1 min: 100 vs. 66 ± 8.7; *P < 0.05; at 10 min: reduced NET-induced platelet dense granule secretion 100 vs. 48.2 ± 13.8; **P < 0.01, n = 5) (Fig. 7e-f). Platelet (% Max secretion at 1 min: 100 vs. 62.4 ± 7.1; **P < 0.01; activation show that RGDS markedly reduced at 10 min: 100 vs. 58.6 ± 6.6; **P < 0.01, n = 6) (Fig. 5d). NET-induced platelet P-selectin expression (% Max re- Collectively, these results highlight the diversity of plate- sponse: 100 vs. 82.6 ± 3.7; **P < 0.01, n = 3) (Fig. 7d) and let pathways that are activated by NETs. PAC1 expression (% Max response: 100 vs. 38.4 ± 16.2; **P < 0.01, n = 3) (Fig. 7c). Overall, our results confirm P2Y but not cyclooxygenase pathway is required for the crucial role of αIIbβ3 in NET-induced platelet NET-induced platelet aggregation response. Drugs that target either P2Y (e.g., ticagrelor) or cyclo- oxygenase pathway (aspirin) are clinically available and Discussion crucial in the management of thrombosis [39]. There- Our study explored the effect of in vitro-generated NETs fore, we were interested in investigating their effect on on washed human platelets. As described in the NET-induced platelet response. Ticagrelor markedly re- methods, cell-free NETs were isolated from duced NET-induced platelet aggregation (% Max Agg: PMA-activated human neutrophils. PMA is a known Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 9 of 15 Fig. 4 Inhibition of Cat G, but not MPO, attenuates NET-induced platelet response. Flow cytometry analysis of P-selectin, PAC1, and PS exposure on platelets. NETs were pre-treated with vehicle or inhibitors for 30 min at 37 °C. a-b WP (1 × 10 /mL) were incubated with NETs pre-treated with vehicle (0.1% DMSO in PBS), Cat G I (0.5 μM) or MPO I (50 μM) for 10–15 min in the dark with labelled monoclonal antibodies that detect P-selectin (CD62P) and active αIIbβ3 (PAC1). The reaction was stopped by fixing cells in 2% PFA before analysing samples with flow cytometry (BD LSRFortessa). Bar graphs depict the % inhibition in P-selectin, and active αIIbβ3 expression in platelets treated with NETs pre-treated with different inhibitors compared to platelets treated with NETs that were pre- treated with vehicle. Results were normalized for each donor relative to NET-induced platelet response. c WP (3 × 10 /mL) were incubated with NETs pre-treated with vehicle (0.1% DMSO in PBS), Cat G I (0.5 μM) or MPO I (50 μM) for 30 min at 37 °C with continuous stirring at 1200 rpm. Platelets were then stained with Annexin V-FITC in binding buffer for 15 min in the dark. Samples were then washed in binding buffer and analysed by flow cytometry (BD LSRFortessa). Bar graph depicts the % inhibition in PS expression in platelets treated with NETs pre-treated with different inhibitors compared to platelets treated with NETs pre-treated with vehicle. Results were normalised for each donor relative to NET-induced platelet response. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Data are expressed as mean ± SEM,*P < .05; ns: non-significant, n =4 platelet agonist [40], however PMA was washed out with the culture media after 3 h incubation with neutrophils, thus it is highly unlikely that NET-induced platelet ag- gregation was confounded by PMA. Unlike the widely used method of cell-free NET preparation by Urban et al., [26], we did not use this method involving DNase/ EDTA, as EDTA can hinder platelet functional response [41]. We demonstrate that intact cell-free NETs exhibit the capacity to directly activate several platelet re- sponses, such as aggregation, dense and α-granule secre- tion (ADP release and P-selectin expression), PS exposure and activation of integrin αIIbβ3, which oc- curred independently of the presence of coagulation fac- tors or thrombin. NETs triggered a dose-dependent aggregation response in platelets with delayed lag time which correlates with the ability of NETs to first induce rapid platelet dense granule secretion. In addition to being a procoagulant platform, NETs in- duced PS exposure on platelet’s surface, a characteristic feature of procoagulant platelets. In the presence of small amounts of activated coagulation factors, PS can instigate thrombin generation, which can directly acti- vate platelets and conversion of fibrinogen to fibrin [42]. In washed platelets, strong or multiple agonists can trig- ger PS-exposing procoagulant platelets [43, 44]. The lat- ter is in line with our data and suggests that NETs are not a single agonist but a platform that presents a num- ber of agonists that can promote platelet activation via multiple pathways. Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 10 of 15 c d e f Fig. 5 (See legend on next page.) Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 11 of 15 (See figure on previous page.) Fig. 5 Syk, but not NOX1, inhibition attenuated NET-induced platelet aggregation. a Representative aggregation traces showing the effect of Syk phosphorylation (BAY61, 5 μM) and NOX1 (ML171, 5 μM) inhibitors on NET-induced platelet aggregation. WP (3 × 10 /mL) were pre-incubated with the inhibitors for 15 min at 37 °C before addition of NETs. Platelet aggregation was measured using transmission aggregometer (Chrono- log). b Bar graph comparing the effect of BAY61 and ML171 on NET-induced platelet aggregation. Platelet aggregation percentage was calculated after 20 min on a light transmission aggregometer. Results were normalized for each donor relative to NET-induced platelet aggregation. Data are expressed as mean ± SEM, ***P < .001; ns: non-significant, n =7. (c-d) Bar graphs comparing the effect of BAY61 (5 μM) and ML171 (5 μM) on platelet activation (measured by P-selectin and active αIIbβ3 expression, n = 3) and (e-f) ATP/ADP secretion (n = 6) elicited by NETs. Results were normalized for each donor relative to NET-induced platelet response. Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: non-significant. e Syk phosphorylation is augmented by NETs. WP (3 × 10 /mL) were incubated with collagen (5 μg/mL), vehicle (PBS) or NETs for 3 min. Forty-five μl of total cell lysate was then analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted for p-Syk, p-Akt and p-Erk1/2. Equal loading was verified by α-actinin. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. The immunoblots are representative sample of 3 independent experiments NETs are made up of DNA and proteins (1:1.67 ± tenfold lower platelet aggregation response in washed 0.26 g, DNA to proteins) [26]. Histones account for 70% platelets compared to PRP [29]. of all NET-associated proteins [18, 26, 29, 45]. Previous Neutrophil granular proteins are a part of NETs and studies showed the ability of single-strand DNA to bind have separately been shown to activate platelets [37, 50]. platelets [46] while double strand DNA can induce Neutrophil serine proteases and histones are negatively platelet aggregation [47, 48]. Degradation of DNA with charged proteins [51] and would be tightly bound to the DNase has been shown to digest NETs and reduce plate- positively charged DNA backbone of NETs, thus most let aggregates under flow [30], or platelet adhesion to likely remain bound after isolation procedures [26]. The NETs under static condition [12]. On the other hand, pre-treatment of NETs with an MPO inhibitor did not sig- histones are well-established as platelet agonists that can nificantly reduce NET-induced upregulation of P-selectin, trigger a cascade of platelet responses with defined sur- active αIIbβ3, or PS exposure on platelets, suggesting face receptors and signalling pathways [18, 29, 45]. Hep- MPO does not play a major role in NET-induced platelet arin has been reported to bind to histones, thus activation which is consistent with previous studies preventing its binding to platelets [19, 32]. Surprisingly, reporting that MPO is not a robust activator of platelets, in this study DNase- and heparin-treated NETs were still but only induces partial activation or priming of platelets capable of aggregating washed platelets and induced ex- [37]. On the other hand, inhibiting Cat G resulted in a sig- pression of P-selectin and active αIIbβ3 to the same ex- nificant decrease in platelet surface expression of tent of untreated NETs. Although DNase and heparin P-selectin, active αIIbβ3 and PS. This suggests Cat G as a can destabilise the NET structure [9], the presence of molecular mediator of NET-induced platelet activation, freely suspended individual NET components – such as and potentially significant contributor to thrombus forma- cell free-DNA, histones, and neutrophil proteases – may tion, as previously described [36]. NE is the second most have greater capacity and exposure to directly bind and abundant NET-associated proteins after histones [26]and activate platelets, as opposed to being restricted on can potentiate Cat G-induced platelet aggregation [52], NETs. This presumption is in line with a study that however in our hands, NE inhibitor did not affect showed DNAse-treatment of NETs resulted in increased NET-induced platelet responses (data not shown). coagulation effect [15]. Moreover, nuclear histones have As the NET scaffold contains an array of associated different molecular mass and stoichiometry compared to proteins, some of which have been independently associ- NET-derived histones [26]. Therefore they may exhibit ated with platelet activation [26, 36, 37, 50, 52–54], a different biological activity. Indeed, a recent report has single inhibitor is highly unlikely to completely abrogate found that individual histones and DNA capable of indu- NET-induced platelet responses. However, we were in- cing coagulation, but not intact NETs that were released terested mainly in clarifying the major NET components, from human neutrophils [49]. platelet receptors and downstream signalling molecules Histones are known to induce platelet Syk kinase acti- that mediate NET-induced platelet secretion and aggre- vation through GPVI, and other tyrosine kinase-linked gation. Inhibition of the tyrosine kinase Syk activity, receptors [29]. We show that inhibition of Syk attenu- P2Y and αIIbβ3 reduced NET-induced platelet aggre- ated NET-induced platelet responses. However, inhibi- gation and secretion. While inhibition of NOX1 and tors of histone receptors on platelets (TLR2, TLR4 and TxA2 reduced NET-induced platelet dense granule se- GPVI) did not reduce NET-induced platelet aggregation. cretion, but not aggregation. Moreover, the sheer magnitude of platelet aggregation The role of NETs in initiating thrombosis in vivo has response to NETs in washed system precludes a signifi- been established in mice models of different diseases cant contribution of histones which are known to have a [55]. However, the molecular mechanisms that drive Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 12 of 15 c d e f Fig. 6 Inhibition of cyclooxygenase pathway in platelets attenuated NET-induced platelet secretion but not aggregation, while inhibition of P2Y affects both. a Representative aggregation traces showing the effect of ticagrelor (1 μM) and aspirin (100 μM) on NET-induced platelet aggregation. WP (3 × 10 /mL) were pre-incubated with the inhibitors for 15 min at 37 °C before addition of NETs. Platelet aggregation was measured by light transmission aggregometry (Chrono-log). b Bar graph comparing the effect of ticagrelor (1 μM) and aspirin (100 μM) on NET- induced platelet aggregation. Platelet aggregation percentage was calculated after 20 min on a light transmission aggregometer. Results were normalized for each donor relative to NET-induced platelet aggregation. Data are expressed as mean ± SEM, ***P < .001; ns: non-significant, n =3. (c-d) Bar graphs comparing the effect of ticagrelor (1 μM) and aspirin (100 μM) on platelet activation (measured by P-selectin and active αIIbβ3 expression) (n = 3) and (e-f) ATP/ADP secretion (n = 5) elicited by NETs. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Results were normalized for each donor relative to NET-induced platelet response. Data are expressed as mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001, ns: non-significant NET-induced thrombosis are not well understood. As initiate thrombosis [56, 57]. We propose that platelets the recent study by Noubouossie et al., has demon- adhere mainly to NET-derived DNA, then multiple strated that intact NETs do not directly initiate coagula- NET-bound proteins induce platelet aggregation and PS tion [49], we propose that platelets but not coagulation exposure which then can propagate coagulation and factors are more likely to be the main target of NETs in thrombin generation. In addition to their pivotal role in thrombosis. Our study did not account for the disrupt- thrombosis, platelets can also orchestrate inflammation ing effect of NETs on endothelium which can also [58]. Therefore, although dismantling NETs may reduce Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 13 of 15 cd ef Fig. 7 Inhibition of αIIbβ3 attenuates NET-induced platelet response. a Representative aggregation traces showing the effect of Reopro (25 μg/ mL) and RGDS (100 μM) on NET-induced platelet aggregation. WP (3 × 10 /mL) were pre-incubated with the inhibitors for 15 min at 37 °C before addition of NETs. Both Reopro and RGDS reduced platelet aggregation. Platelet aggregation was measured by light transmission aggregometry (Chrono-log). b Bar graph comparing the effect of Reopro (25 μg/mL) and RGDS (100 μM) on NET-induced platelet aggregation. Platelet aggregation percentage was calculated after 20 min on a light transmission aggregometer. Results were normalised for each donor relative to NET-induced platelet aggregation. Data are expressed as mean ± SEM,***P < .001. c-f Bar graphs comparing the effect of Reopro (25 μg/mL) and RGDS (100 μM) on platelet activation (measured by P-selectin and active αIIbβ3 expression, n = 3) and ATP/ADP secretion (n = 5) elicited by NETs. In all assays, NETs constituted 10% of final reaction volume and contains 292 ± 172 pg/mL of NET-elastase. Results were normalised for each donor relative to NET-induced platelet response. Data are expressed as mean ± SEM, *P < 0.05; **P < 0.01, ns: non-significant NET-induced thrombosis [59], inhibition of platelet ac- resulted in decreased neutrophil-platelet complexes in tivity may not only reduce thrombosis but also the kidney vasculature, along with improved vascular platelet-mediated inflammation. Indeed, Jansen et al., function in tumour-bearing mice [60]. Thus in these have recently shown that platelet inhibition with clopi- contexts NETs can also be considered as scaffold for dogrel was superior to DNase in reducing granulocyte platelets that drives inflammatory reactions. activation, NET formation and acute kidney injury in a renal reperfusion injury mice model [31]. Apart from Conclusion NETs inducing thrombosis, Cedervall et al. also showed This study showed for the first time that in vitro gener- that the use of DNase to disrupt tumour-induced NETs ated NETs can directly induce marked platelet Elaskalani et al. Cell Communication and Signaling (2018) 16:24 Page 14 of 15 aggregation. We further identified the molecular path- Received: 1 March 2018 Accepted: 15 May 2018 ways activated in platelet responses to NETs. It is im- portant to note that aspirin, a widely used antiplatelet, was not as effective at reducing NET-induced platelet References 1. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and aggregation as ticagrelor or Reopro. Finally, pretreat- inflammation. Nat Rev Immunol. 2013;13:159–75. ment of NETs with DNase or heparin did not reduce 2. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, NET-induced activation or aggregation of human Weinrauch Y, Zychlinsky A. Neutrophil extracellular traps kill Bacteria. Science. 2004;303:1532–5. washed platelets. Taken together, we conclude that tar- 3. McDonald B, Urrutia R, Yipp Bryan G, Jenne Craig N, Kubes P. 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