DOI: 10.1161/CIRCULATIONAHA.114.013743

NOD2 Receptor is Expressed in Platelets and Enhances Platelet Activation and Thrombosis Running title: Zhang et al.; Platelets Express Functional NOD2 Si Zhang, MD, PhD1; Shenghui Zhang, MD, PhD1; Liang Hu, MSc1; Lili Zhai, BSc1; Ruyi Xue, MD, PhD2; Jianqin Ye, MSc1; Leilei Chen, BSc1; Guanjun Cheng, MD, PhD3; Jozef Mruk, MD4; Satya P. Kunapuli, PhD5; Zhongren Ding, MD, PhD1 1

Key Laboratory of Molecular Medicine, Ministry of Education, and Depar Department rtm men entt of

Biochemistry and Molecular Biology, Fudan University Shanghai Medical Co College, ollleg ege, e, S Shanghai, hang ha ngha ng haii, ha China; 2Dept of Internal Medicine, and Institute of Liver Disease, Fudan University Zhongshan Hosptial, Hosp Ho spti sp tial ti a , Sh Sha Shanghai, anghai, China; 3Thoracic Onc Oncology nccol olog o y Research Labo Laboratory, ora rato t ry, University of to Pennsylvania, Peennsylv lvan aniaa, Ph an P Philadelphia, ilad il ad del elph phia ph ia,, PA PA;; 4De Dept p ooff In pt Inte Internal tern nal M Medicine, edic ed icin ic i e,, U in University niv iver ersi sity si ty ooff Ka Kans Kansas n as S ns School choo ch oool of o Medicine, Medicine, Witchita, Witc Wi tchhitaa, KS; KS;; 5Dept De ooff P Physiology, hysio olo loggy, an and nd So Soll Sh S Sherry heerry yT Thrombosis hrom mbos bosis sis Re R Research seearrch hC Center, entterr, r, Temple University School Medicine, Philadelphia, T emple mpl U nive ni vers ve rsit rs ityy Sc Scho hool ho oll ooff M eddici cine ci ne,, Ph ne Phil illad del elph ph hiaa, PA

Address Ad Addr dres esss for for Correspondence: Corr Co rres espo pond nden ence ce:: Zhongren Ding, MD, PhD Key Laboratory of Molecular Medicine, Ministry of Education Department of Biochemistry and Molecular Biology Fudan University Shanghai Medical College Shanghai 200032, China Tel: + 86 21 5423 7896 Fax: + 86 21 6403 3738 E-mail: [email protected] Journal Subject Codes: Thrombosis:[92] Platelets, Thrombosis:[172] Arterial thrombosis, Platelet adhesion:[177] Secretion, Platelet adhesion:[178] Aggregation, Platelet adhesion:[181] Signal transduction 1 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

Abstract

Background—Pattern recognition receptor NOD2 (nucleotide binding oligomerization domain 2) is well investigated in immunity, its expression and function in platelets has never been explored. Method and Results—Using RT-PCR and Western blot we show that both human and mouse platelets express NOD2, and its agonist MDP induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentration of thrombin or collagen, as well as clot retraction. These potentiating effects of MDP were not seen in platelets from NOD2-deficient mice. Plasma from septic ppatients ati tiien ntss aalso lso ls o potentiates platelet aggregation induced by thrombin orr collagen NOD2-dependently. Using intravital administration accelerated ntrrav avit itall microscopy, micr icrosc sccopy, op we found thatt MDP admin in nisttration accelera raate t d in vivo vivo thrombosis in FeCl FeC Cl3-injured mesenteric Cl mes e en ente teeriic arteriole arte ar teri te riol ri olee thrombosis th hro ombossiss mouse moouse model. mode odel. Platelet Plaateelet depletion Pl dep pleetiion aand nd ttransfusion rannsfu ra nsfu fusi sion ionn experiments ex xpe peri rime ri m ntts co me con confirmed nfirrme medd that tha NOD2 NOD OD22 from from m platelets pla late tele lets le ts contributes coonttrib trib but utes es to to the t e in th i vivo vivvo tthrombosis hrom hr ombo om bosiss inn mice. bo micee. NOD2 activation activat attio ionn al also s aaccelerates so c el cc eler erat er ates es pl platelet-dependent lat a el elet et-d et -d dep epen en nde dent nt hhemostasis. emos em osta os t si ta sis. s. W Wee fu furt further rthe rt herr fo he foun found undd th un that hat a platelets express RIP2 (receptor-interacting protein 2), and provided evidences suggesting that MAPK and NO/sGC/cGMP/PGK pathways downstream of RIP2 mediate the role of NOD2 in platelets. Finally, MDP stimulates proinflammatory cytokine IL-1ȕ maturation and accumulation in human and mouse platelets NOD2-dependently. Conclusions—NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during the conditions of inflammation. To our knowledge, this is the first study on NOD-like receptors in platelets which links thrombotic events to inflammation. Key words: NOD2, RIP2, platelet, thrombosis, mitogen-activated protein kinase

2 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

Humans are constantly challenged by numerous bacteria and viruses. As the primary barrier defending the humans against the pathogen invasion, innate immune system recognizes the conserved molecular structure in microbes, and initiates inflammatory and antimicrobial response relying on pattern recognition receptors. Among the major families of pattern recognition receptors, Toll-like receptors and NOD-like receptors are the key players in innate immunity1, 2. In contrast to Toll-like receptors, which are found on plasma membrane, NOD-like receptors are cytoplasmic receptors. All NOD-like receptors are structurally similar, containing a central nucleotide-binding oligomerization domain (NOD), C-terminal leucine-rich repeat domain, and a variable N-terminal protein-protein interaction domain which interacts with downstream effectors1. NOD1 and NOD2 are the two important Nod-like receptors recepto to orss ffitting ittti ting ng tthe he he typical ypical structure with NOD1 containing one caspase recruitment domain (CARD), while NOD2 containing co ont ntai aini ai ning ni ngg two two CARD CARD AR domains1, 2. NOD11 iiss bbroadly roa oadl d y di dist distributed, sttri ribu bute tedd, te d, w whereas hereaas NOD NOD2 OD2 iss main OD m mainly ain i ly eexpressed xppre ress sssed d iin n mono m monocytes, onoocy yte tess, macrophages, intestinal cells and Paneth ma acr c op opha hage gess, ge s, ddendritic enddrit dritiic ccells, ellss, in el nte test stiinal st inall eepithelial pith pi thel th ellia i l ce ell l s an nd Pa ane n th th ccells e lss2. Although el Allth t ou o gh NOD1 NOD OD11 and an nd NOD2 have a high high degree deg egre r e of similarity, re sim mil i ar arit i y, they the heyy recognize reco re co ogn gniz i e di iz ddifferent ffer ff eren er entt ba en act c er eria iall ce ia cell ll w alll pe al peptidoglyca an bacterial wall peptidoglycan components. NOD1 recognizes d-glutamyl-meso-diaminopimelic acid (iE-DAP) primarily from Gram-negative bacteria whereas NOD2 detects muramyl dipeptide (MDP), the minimal bioactive motif in peptidoglycan from all bacteria2-4. In addition to their critically important roles in host defending pathogen invasion, they are also crucial to the pathogenesis of a myriad of inflammatory diseases. Homozygous mutations of NOD2 are highly correlated with the incidence of Crohn's disease5, 6. Though primarily involved in hemostasis and thrombosis, increasing evidences demonstrate that platelets also play key roles in immune and inflammatory responses and the

3 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

related diseases7. Many pattern recognition receptors, mainly Toll-like receptors, which were originally discovered in classic immune cells, are also expressed in platelets8-10, and play important roles in platelet activation, thrombosis and hemostasis11-13. As the two most important members in NOD-like receptor family, NOD1 and NOD2 were intensively investigated in classic immune cells and in inflammatory diseases2, 5, 14-16, their expression and functions in platelets have never been reported. In this study, we report that NOD2 is also expressed in platelets and functions in platelet activation and thrombosis.

Materials and Methods nt.. Detailed Materials and Methods are described in the online-only Data Supplement Supplement. Platelets and peripheral blood mononuclear cells (PBMC) preparation Alll ex experiments xpe peri rime ri ment me n s us usin using ing human subjects were perfor performed for orme m d in accordance wi me with ith the Declaration of Helssin i ki andd app ppro pp rooveed byy the h In he Inst stit st itut it u io ut iona naal Re Revi v ew B vi oard rd F udan ud a Un an Univ iver iv erssit er sity. Pl Plat atel at elet el etss an et andd PB PBMC MC Helsinki approved Institutional Review Board Fudan University. Platelets weeree pprepared repare re reed as ddetailed etai et ailledd in th tthee on onli liine ne--on -only Da Data ta Supplement. Suppl uppllemen nt.. were online-only Preparation Prep Pr epar ep arat ar atio at ion io n of p platelet late la tele te lett po le poor or p plasma lasm la smaa fr sm from om p patients atie at ient ie ntss wi nt with th b bacterial acte ac teri te rial ri al iinfection nfec nf ecti ec tion ti on Blood from patients with bacteremia and clinical sepsis but without DIC and clinical septic shock was obtained from Fudan University Zhongshan Hospital. Ethical permission for all donations was obtained from the Zhongshan Hospital ethics board. Of 14 patients, 7 were infected with Staphylococcus aureus, 4 with Escherichia coli, and 3 with Enterococci. All patients were clinically cured after appropriate treatment. The control blood was obtained from the same donors at least two weeks after cure of infection. Platelet poor plasma was prepared and stored at -80°C before use. Animal studies NOD2-deficient mice were purchased from Jackson Laboratory (Bar Harbor, ME). All animal 4 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

procedures were performed according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). Mouse platelets were prepared as described previously17. Platelet functional studies Platelet aggregation, secretion, spreading and clot retraction were measured as previously described18, 19 and detailed in the online-only Data Supplement. Bleeding assay Bleeding assay was measured as described previously20 with more information in the online-only Data Supplement. cGMP assay cGMP cG GMP iin n pl plat platelets ate telet etts w was as assayed using commercial commercially allly l aavailable vailable cGM cGMP MP 1255I ra radioimmunoassay kit as reported epoorted previ previously iou ousl s y211. NO Od detection etec et ecti tiion Platelet NO le level eve vell wa wass me m measured assur ured e uusing ed s ng ffluorescent si luor lu ores or esce es cent ce nt NO sen sensor enso en soor DA DAFDAF-FM F FM D DA A on on a ccollagen o la ol lage g n matrix aass ge previously reported22. Briefly, platelets were first preincubated with NO fluorescent indicator DAF-FM DA (1 μM) in Tyrode’s buffer at 37°C for 30 min. After stimulation with MDP for 15 min, platelets were immobilized on a collagen matrix slide and fluorescence was observed by confocal microscopy with 495 nm excitation and 515 nm emission filters. RT-PCR Total RNA was isolated and 1 ȝg of total RNA was reversely transcribed to cDNA using RNA isolation kit and RT-PCR kit (TaKaRa, Japan), respectively. PCR reactions were performed using specific primers (Supplementary Table 1).

5 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

Intravital microscopy of FeCl3-injured thrombus formation in mouse mesenteric arteriole Intravital microscopy of FeCl3 thrombus formation in mouse mesenteric arteriole was performed as described previously18, 23. More information is provided in the online-only Data Supplement. Lethal pulmonary thromboembolism mouse model induced by epinephrine and collagen C57BL/6 mice were administrated rabbit anti-mouse platelet serum via the tail vein to produce an approximate 90% decrease of the count of circulating platelets. On the second day, the WT and NOD2-/- mice were anesthetized with 10% chloral hydrate and blood was collected from the abdominal aorta. Blood was centrifuged at 200 g for 3 min to obtain platelet rich plasma (PRP), followed by centrifugation at 700 g for 3 min to get platelet pellets, and then platelets were esuspended in 1 mL Tyrode’s solution. Then, each 0.5 mL platelet suspension was ass incubated inc n ub ubat ated at ed with with resuspended MDP (5 ȝg/mL) or normal saline at 37ºC for 30 min, centrifuged, n and finally platelet pellets were esuusp speende ende dedd in in T yrod yr o e's solution. The MDP- or saline-treated salin al ne-treated platelets plattel e ets were werre transfused into the we resuspended Tyrode's pl lattelet-deplete tedd m ice ce (0. 0.12 0. 1255 mL 12 mL pper er mou m ouse)). A fteer 330 0m in,, th in he m icce ce we eree iinjected njeecte nj ectedd with with a m ix ixtu x ure platelet-depleted mice (0.125 mouse). After min, the mice were mixture off recombinant rec ecom o bi om bina nant na nt collagen col olla lage genn (4 ge ( mg/kg) mg/ g/kg kg)) and kg and ep epinephrine pin ineephr hrin hr in ne (0.3 ( .33 mg/kg) (0 mg/ g/kgg) dissolved diss sssolv lved ed in in 0.1 0 1 mL saline 0. salline ine via via th thee ail vein. Thee ddeath e th ea h rrate atte wa as re reco ord rded ed within wit i hi hinn 15 min. min n. tail was recorded IL-1ȕ assay Platelet IL-1ȕ was assayed by flow cytometry, Western blot and ELISA. More information is available in the online-only Data Supplement. Statistical analysis All data are expressed as mean ± SD. Differences between the groups were analyzed by one-way ANOVA, followed by a Tukey test for multiple comparison unless otherwise stated. The MannWhitney test was used for comparison between two groups, and the Fisher exact test was used for comparison of death rate. Statistical analysis was performed using Prism 5 (GraphPad Inc, San

6 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

Diego). P < 0.05 was considered to be statistically significant.

Results Human and mouse platelets express NOD2 Human platelets express NOD2 receptor both at the RNA and protein levels as detected by RTPCR (Figure 1A1) and Western blot (Figure 1B), similarly to PBMC (Figure 1). In contrast to PBMC, which expresses both NOD1 and NOD2, only NOD2 was detected in human platelets. The detected NOD2 on platelets is not from the contaminating PBMC, because the PBMCspecific marker CD14, which is robustly expressed in PBMC, is not detectable from platelet sample ample (Figure 1A2). Similarly, we also detected robust expression of NOD2, but buut not n t NOD1, no NOD1 NO D1,, in D1 mouse platelets by RT-PCR (Figure 1C) and Western blot (Figure 1D). NOD2 platelet dense NO OD2 receptor reccep e torr agonist agoonist MDP potentiates plat ag tele elet aggregation n aand nd dd en granule release ense NOD2 white and NOD2 elicits NOD NO D2 function functio on has ha been be n extensively ext xten ensi en sivvel elyy sstudied tuudiedd iin nw hite bblood loodd ccells, lood ellls, an el nd N OD2 aactivation ctiv ct ivaatio iv ation el elic icit citss proinflammatory white blood immunity. proi pr oinf oi nfla nf l mm la mat ator oryy rresponses espponnse ses in w hit itee bl loo od ccells, ell lls, ll s pplaying s, laayi ying ng a ccritical riiti tica caal rrole o e in ol n iinnate nn natte im mmun munity ty. ty NOD2 receptor MDP NOD2 functions functtio ons iin n platelets plat pl atel at e etts have h ve not ha not o been bee eenn studied. stud st udie ieed. d N OD D2 re ece cept p orr aagonist pt goni go nist M DP aalone lone lo n did not induce platelet aggregation, in washed human platelets, even at 100 μg/mL (data not shown). But in the range of 1 - 10 μg/mL, MDP concentration-dependently potentiated human platelet aggregation and ATP release induced by low concentrations of thrombin or collagen (Figure 2A). NOD2 activation induced by MDP was confirmed by the dimerizaton of NOD2 upon treatment of platelets with 10 μg/mL MDP (Figure 2B). Similarly, 10 μg/mL MDP also significantly potentiated mouse platelet aggregation and ATP release induced by low concentration of thrombin or collagen. The potentiating effect of MDP on platelet activation is NOD2 dependent, because it was not observed using platelets from NOD2-/- mice (Figure 2C &

7 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

D). In an ex vivo study, MDP intraperitoneally given to mice also potentiated platelet aggregation and ATP release induced by thrombin and collagen in a NOD2-dependent manner (data not shown). NOD2 receptor agonist MDP potentiates platelet clot retraction Platelet dependent clot retraction is a late phase outside-in signaling event associated with second wave of interaction between talin and integrin E3 intracellular domain during platelet activation24. When clot retraction was examined, we found that NOD2 agonist MDP accelerated clot retraction in human platelet suspension (Figure 3). Similarly, MDP also accelerated clot retraction in mouse platelet suspension (Figure 3). Consistent with the effects of NOD2 activation on platelet aggregation and ATP release, MDP-induced clot retraction n acc ccellerat cc eratio tio ionn is acceleration NOD2-dependent, as it did not occur in NOD2-/- mice (Figure 3). Taken together, these data cl leaarl rlyy indicate indi dica di cate that ca tha hatt NOD2 activation inducedd by by MDP MDP potentiates potentiatees platelet pllattel elet e activation. clearly NO OD2 D agonist st M DP d DP oes no oes nott af ffe fecct ct p latellett spr readin rea ingg in NOD2 MDP does affect platelet spreading Plat Pl Platelet atel at elet el et spreading spr prea eadi ding ng is is an an early ear a lyy phase phas ph asee outside-in outs ou t id ts idee-in ein signaling sig gnaali ling ng event eve vent ntt downstream dow owns nstr ns treeam tr eam of platelet pla late tellet te let Į ĮIIbE3 IIIbE3 ntegrin activ iv vat atio ionn24. In ccontrast io onntr traast to eenhancing nhan nh anccin an ng cl clot lot rretraction, etra et r ct ra ctio ion, io n, N NOD2 O 2 ag OD agon agonist onis on istt MD is MDP P ha hadd no effect integrin activation on human platelet spreading on fibrinogen at 10 μg/mL (Figure S1), the concentration which activates NOD2 receptor as evidenced by receptor dimerization (Figure 2B). Similarly, mouse platelet spreading was also unaffected by MDP stimulation (Figure S2). Moreover, NOD2 deficiency also did not affect mouse platelet spreading (Figure S2), contrasting to the impaired clot retraction in NOD2-/- mice (Figure 3). NOD2 deficiency impairs thrombus formation and hemostasis in vivo To explore the role of NOD2 in thrombus formation in vivo, we examined the FeCl3-injured thrombus formation in mesenteric arteriole in WT and NOD2-/- mice using intravital

8 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

microscopy. As shown in Figure 4A & B, WT mice receiving MDP exhibit increased thrombus formation in mesenteric arterioles whereas NOD2-/- mice do not, indicating the enhancing role of NOD2 activation in thrombosis. Consistently, decreased blood loss after tail snip was observed in WT mice receiving MDP but not NOD2-deficient mice receiving the same dose of MDP (Figure 4C), suggesting that NOD2 contributes to hemostasis as well as thrombosis under MDP stimulation. These results are consistent with the potentiating effects of MDP on platelet aggregation and secretion. NOD2 is also expressed in endothelial cells and macrophages15, which may also contribute to thrombosis and hemostasis upon MDP stimulation in septic settings. To corroborate that hat the NOD2 expressed in platelets prompts thrombosis and hemostasis in vivoo after aft fter ft er M MDP DP dosing, we preincubated platelets with normal saline or MDP in vitro and transfused into platelet-depleted pllattel elet et-dep dep eple letedd mice. le mi We then challenged the mice mice with epinephr mic epinephrine h inee an hr andd collagen, and examined ex xamined am m the mortality morttali liity ty using usi sing ng the the lethal letthal lung lungg thromboembolism th hromb mboeemb mb mboolissm mo m mouse u e mode us m model ode dell25. Th Thee de deat death th ra rate atee in mice mi ice c rreceiving e ei ec eivi vinng vi ng M MDP-treated DP-treeat DPated ed pplatelets late la tele te lets le ts iiss 69 69.2 69.2% .2% .2 % (n n = 13 13), 3), ) ssignificantly igni ig niifi f cant cant ntly ly hhigher ig ghe herr th than han n 228.6% 8.66% iin n tthe hee mice receiving ng ssaline-treated allin inee-tr trea tr e te tedd pl plat platelets a el at e et ets (n = 114; 4 P < 0. 4; 00.05, 05 5, on onee ta tail tailed iled il e F ed Fisher's ishe is her' he r s exact ex xac actt test). test te st). st ) In contrast, MDP-treated platelets from NOD2-deficient mice did not significantly increase death rate in platelet-depleted WT mice challenged with epinephrine and collagen compared with the mice receiving saline-treated NOD2-/- platelets (30.8% vs 25%, n = 13 and 12, respectively; P > 0.05, one tailed Fisher's exact test). We also examined the FeCl3-injured thrombus in mesenteric arteriole in platelet-depleted mice. Mice receiving MDP-treated platelets exhibited increased thrombus formation than the mice receiving saline-treated platelets. In contrast, MDP-treated platelets from NOD2-deficient mice did not significantly increase thrombus formation in platelet-depleted WT mice compared with the mice receiving saline-treated NOD2-/- platelets

9 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

(Figure S3). These data clearly indicates that platelet-expressed NOD2 plays a significant role in thrombosis and hemostasis, correlating well with NOD2 potentiating effect on platelet activation (Figure 2). NOD2 deficiency attenuated platelet hyperreactivity induced by bacterial infection During bacterial infection, MDP is shed from bacteria and released into the blood, resulting in a strong increase in MDP concentration and activation of NOD2 signal cascade in vivo. To check whether NOD2 activation affects platelet activity during bacterial infection, we treated PRP from WT or NOD2-/- mice with plasma from patients with bacteremia, and then detected platelet aggregation. NOD2 deficiency attenuated the potentiating effects of septic plasma on platelet aggregation induced by thrombin and collagen (Figure 5). Our results demonstra demonstrated raate tedd a cr crit critical itic it icaal ic al role ole of NOD2 in platelet hyperreactivity caused by bacterial infection. Platelets Pllat atel elet el etss express et exp ex presss RIP2, RIP2, RIP2/MAPK pathwayy is activated downstream dow wnstr trrea eam m of NOD2 Our Ourr results r sults thus re us far far indicate ind n iccat atee that that NOD2 NOD2 OD2 activation actiiva vationn enhances enha haances ncess platelet pla lattelet activation, activvat ac atio ionn, io n, aand nd tthus hus prompts hu prom prom mpts thrombosis hro omb mbos osis iss and and hemostasis. hem mosstaasi sis. s Next, Nex xt, we we sought soug soug ughht ht to to elucidate eluc lucid cidate datee the the mechanism meccha hani n sm ni sm of of NOD2-mediated NOD2 NO D22-m -meedia ediaate tedd platelet activation. activ vat atio io on. M Mitogen itog it o en og n aactivated ctiv ct ivat iv a ed pprotein at rote ro tein te in kkinase i as in asee (M (MAP (MAPK) APK) AP K) iiss w well elll do el docu documented cume cu m nt me n ed iin n pl pplatelet atelet activation and thrombosis. RIP2/MAPK pathway activation has been reported to mediate NOD2induced inflammation and anti-infection response in white blood cells1, 2. We therefore examined whether RIP2/MAPK pathway is involved in NOD2-mediated platelet activation. Though a variety of cells express RIP2, it is not clear whether platelets express RIP2. Using RT-PCR and Western blot, we found that both human and mouse platelets express robust RIP2, similarly to PBMC (Figure 6A). To our knowledge, this is the first report that platelets express RIP2. Furthermore, upon MDP (10 μg/mL) stimulation, platelet RIP2 was phosphorylated at 5 min lasting for more than 10 min (Figure 6B). Erk, p38, and JNK were also

10 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

phosphorylated with the peak phosphorylation apparently later than RIP2 (Figure 6B). These results agree with previous study with white blood cells and indicate that MAPK pathway is activated downstream of RIP2 in platelets upon NOD2 activation triggered by MDP. MAPK inhibition abolishes the potentiating effects of NOD2 on platelet activation After showing the activation of RIP2/MAPK pathway in platelets downstream of MDP-induced NOD2 activation, we further examined whether MAPK signaling mediates the potentiating effects of NOD2 on platelet activation. As shown in Figure 6C, inhibition of Erk, p38, and JNK with PD98059, SB203580, and SP600125, respectively, abolished the potentiating effect of MDP on platelet aggregation induced by thrombin or collagen. Similarly, the accelerated platelet-dependent clot retraction elicited by MDP was also prevented by inhibit inhibitors torss fo forr Er Erk, k, pp38, 38, and JNK (Figure 6D). These data confirmed that MAPK phosphorylation downstream of NOD2/RIP2 MDP NO OD2 D2/R /RIP /R IP22 mediates IP medi me diaates di at the potentiating effects off M DP on platelett ac aactivation. tiiva vattion. ti MDP MDP elevatess cGMP cGM MP and and NO in in platelets platelets in pla n a NOD2 NOD2 OD2 dependent depe de pend pe nden en nt manner ma annerr 6,27 cGMP observed platelets stimulated with thrombin, and cG GMP eelevation leva le vati va tion on hhas as bbeen as eeen ob bserv rved ed iin n pl plat atel at eletts st el stim imul im ulat a ed w at itth coll ccollagen, oll llag a en,, th ag thr romb romb m in n, an nd vWF vWF226,27 .

cGMP-PKG G-M MAP APK K pa ppathway t wa th wayy ha as be een n rreported epor ep orte or tedd to me te m diiat atee pl pplatelet atel at e ett aactivation el ctiv ct ivat iv atio at ionn do io down wnst wn stre st r am of cGMP-PKG-MAPK has been mediate downstream GPIb-IX28-30. After showing MAPK phosphorylation downstream of NOD2 activation upon MDP stimulation, we measured cGMP levels in platelets stimulated with MDP. As shown in Figure 7A, NOD2 agonist MDP concentration-dependently increased cGMP in human platelets up to 3.4 times in the range of 1 - 10 μg/mL, the concentrations which activated NOD2 and potentiated platelet activation concentration-dependently (Figure 2). MDP also increased cGMP in mouse platelets. At 10 μg/mL, MDP increased intracellular cGMP 3.7 times in mouse platelets, similarly to human platelets (Figure 7B). The cGMP-enhancing effect of MDP is mediated by NOD2, as it was abolished by NOD2 deficiency (Figure 7B). These results show

11 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

that cGMP is downstream of NOD2, and may contribute to platelet activation as in the case of GPIb-IX-mediated platelet activation, which was confirmed by that PKG inhibitor KT5823 abolished the potentiating effect of MDP on platelet aggregation (Figure 7C). However, we did not observe the inhibition of KT5823 on MAPK phosphorylation elicited by MDP (data not shown), suggesting a MAPK-independent mechanism for the stimulatory role for cGMP/PKG in platelet activation29. Platelets express functional iNOS and NO/sGC which medicate cGMP elevation during platelet activation31. As expected, MDP-treated human platelets showed elevated intracellular NO levels, as detected by fluorescent NO sensor DAF-FM DA (Figure 7D). NOD2 deficiency abolished MDP-induced elevation of intracellular NO levels, showing NOD2 dependency (Figure 7E). Thus, we propose that cGMP elevation in plate elets letss eli lici li cite ci ted te d by by platelets elicited NOD2 activation is mediated by RIP2/iNOS/NO/sGC signaling32 (Figure 7F). NO NOD2 OD2 rreceptor eccep e torr ag ago agonist onist MDP stimulates plate platelet elet let processing ooff IL-1 IL-1ȕ -1 1ȕ Platelets P lattel e ets repres represent esen en nt ann important impor mporta tannt ta nt linkage lin nkag kage between betw ween thrombosis thromb thr mbos mb osiis and and d inflammation. inf nfla lamm mmat mm attio ion. n. To To assess a sesss the as the h role ole ooff pl pplatelet attel elet et N NOD2 OD2 in OD2 n iinflammation, nfla nf laamm mmat atiion at ion, n, w wee m measured easu ea suureed pproinflammatory roi oinnfla oi nf amm mmat ator at o y cy or cytokine yto toki k ne ki ne IIL-1ȕ L-1ȕ Lȕ pr production rodu rodu ucttio ion inn human pla platelets ate tele leetss sstimulated t mu ti mulaate tedd wi with th M MDP. DP.. In DP n tthe he rrange ange an ge ooff 10 - 1100 00 ng/mL, ng/ g/mL mL,, MDP mL MDP concentrationconc co ncen nc e trationdependently stimulates IL-1ȕ accumulation at protein level in human platelets (Figure 8A). The effect of MDP is time-dependently from 1 to 4 hours (Figure 8A). This effect appeared to be mediated by caspase-1 activation, because caspase-1 inhibitor FMK002 abolished MDPstimulated IL-1ȕ maturation (Figure 8B) and accumulation (Figure 8C) in human platelets. MDP also stimulated IL-1ȕ accumulation in mouse platelets, which is NOD2-dependent (Figure 8D). MDP NOD2-dependently stimulates platelets IL-1ȕ maturation provided a direct evidence that NOD2 links the prothrombotic and proinflammatory roles of platelets.

12 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

Discussion Beyond the well-recognized pivotal role in thrombosis and hemostasis, platelets also play a critical role in inflammatory and infectious diseases33, and increasing evidences indicate that bacterial infection predisposes atherosclerosis and thrombotic events34-36. Among the mechanisms underlying the infection-associated prothrombotic state, Toll-like receptors have been reported to medicate platelet activation in response to bacterial challenge12. As a pattern recognition receptor, although NOD2 is well investigated in immunity, its expression and function in platelets have never been reported. The present study demonstrates that (1) both human and mouse platelets express robust NOD2; (2) NOD2 receptor activation potentiates platelet activation and enhances in vivo thrombosis; (3) Plasma from septic patients patien en ntss potentiates pot oten ot enti nti tiat ates at es platelet aggregation NOD2-dependently; 4) MAPK and iNOS/NO/sGC/cGMP/PKG pathways do own wnst stre st ream am of of RIP2 RIP RI P2 mediate the role of NOD2 in P2 in platelet platelet activati tiion o ; (5 (5)) NOD2 receptor downstream activation; ac ctiiva v tion trigg gger errs pl lat atel ellet pprocessing roccess ro cesssing ng ooff pro oinnflamm mmattorry cyto mm ccytokine ytokkine IIL-1ȕ. L-1ȕ Lȕ. To our ourr knowledge, kno n wl w ed edgge, activation triggers platelet proinflammatory his iiss th tthee fi firs rstt re epoort oon n NO NODD li Dlikke rece rreceptor eceept ptor or inn pl pplatelets, ateeletts, s oour ur ffindings ur ind n in ings gs pprovided rovi rovi v de dedd no nnovel vell in ve nsiigh g t in nto this first report NOD-like insight into platelet activation activ vat atio io on mechanism, m ch me chan anis ism, is m N OD22 fu OD func ncti nc tion ti on,, and on and further f rtthe fu herr highlighted h gh hi ghli l gh li hteed pl plat atel at elet e N et OD22 as a OD NOD2 function, platelet NOD2 critical receptor bridging thrombosis/hemostasis and immunity. MAPK is activated during platelet stimulation and play critical roles in platelet activation. Previously MAPK activation has been attributed to NOD2 signaling downstream of RIP2 in immune cells1, 2. Therefore, we checked MAPK pathway activation downstream of NOD2 in platelets using MDP as an agonist. We found that Erk, p38, and JNK were phosphorylated in MDP-stimulated platelets. Importantly, we found that platelets expressed robust RIP2, and RIP2 was phosphorylated earlier than Erk, p38, and JNK upon MDP stimulation, indicating that MAPK is activated downstream of RIP2 activation. In addition, we

13 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

found that MAPK inhibition abolished the potentiating effects of MDP on platelet aggregation, further confirming the critical roles of MAPK in NOD2-mediated platelet activation. These results also suggest that p38, Erk or JNK may be sequentially activated or coactivation of them is needed for platelet aggregation induced by low concentration of agonists. This hypothesis is supported by the previous studies showing that p38 sequentially activates Erk, JNK1 deficiency impairs Erk2 phosphorylation37, 38, and p38 or JNK inhibition abolished platelet aggregation under low concentration of agonist stimulation39, 40. Biphasic role of cGMP has been reported in agonist-induced platelet activation, low concentration of cGMP stimulates platelet activation during the early process41 via the cGMP/PKG/MAPK pathway29. After showing that NOD2 medicates platelet act activation tiv ivat atio at ionn vi viaa MAPK pathway downstream of RIP2, we next studied the role of cGMP in NOD2-mediated platelet pl lattel elet et aactivation. ctiv ct ivaatio iv on. n. W Wee found that NOD2 activat activation tio ionn dramatically eenhanced n an nh nce cedd platelet cGMP. In line with w ith h our findi findings, ing ngss, the he act activation ctiivat ct ivat atio ionn of T io Toll oll llike ikee re receptor, ecep pto or, aanother noth noth herr ppattern atttern rrecognition eccogn gnit ittio ionn re rreceptor c ptoor ce or w which hiich potentiates platelet platelet levels activates pote po tent te ntia nt i te ia tess pl plat ateelett aactivation, ctiv vat atio ion, n, aalso lsoo incr ls iincreases ncrreaase sess pl plat a eleet at et ccGMP GM MP le eve vels ls aand n ac nd ctiv ctiv vat a es e pplatelets lattele la leetss vvia iaa cGMP-dependent cGMP-depen nde dent n protein nt pro r te t in i kkinase inas in a e (PKG) as ( KG (P KG)) pathway path pa thwa th wayy11. Consistently, wa C ns Co nsis isste tent ntly nt ly,, we also ly a so found al fou o nd that tha hatt NOD2 N D2 NO agonist MDP potentiates platelet aggregation PKG-dependently. Previously, Shimada et al ever reported that macrophages from NOD2-deficient mice exhibited impaired NO production iNOS-dependently in response to Chlamydophila pneumoniae16. NO production from RIP2-deficient mice was also decreased in response to Chlamydophila pneumonia, suggesting the RIP2-dependent NO production16. Platelets express functional iNOS and NO/sGC medicates cGMP elevation during platelet activation31; thus, we propose that cGMP elevation in platelets elicited by NOD2 activation is mediated by RIP2/iNOS/NO/sGC signaling32, which is supported by our findings that MDP increases platelet

14 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

NO level NOD2-dependently. Generally, RIP2 activation is regarded as the result of its phosphorylation via direct interaction with dimerization form of NOD2 after NOD2 activation42. Therefore, we propose that NOD2 promotes platelet activation via MAPK and iNOS/NO/sGC/cGMP pathways downstream of RIP2. However, how RIP2 mediates iNOS activation in platelets is still not clear and deserves further investigation. As a key proinflammatory cytokine, IL-1ȕ is linked to the pathogenesis of a variety of thromboinflammatory diseases including sepsis, Crohn’s disease, ulcerative colitis, type 2 diabetes and atherosclerosis. To exploit the proinflammatory role of platelet NOD2 and its connecting role in thrombosis and inflammation/immunity during bacterial infection, we ou use pplatelets laate tele lets le ts provided data showing that MDP stimulates IL-1ȕ accumulation in human and m mouse NOD2-dependently; we also found that plasma from patients with bacteremia enhances platelet acctiiva vati tion ti onn NOD2-dependendly. NOD O 22-ddependendly. de fin inddings, P. gingiv iv valiss cchallenge ha hallenge has been activation In line with our findings, gingivalis epo ort r ed to prom om motte at theero oge gene nesi siss via via IL L-1 ssignaling ig gnalling433, S. S. ppneumonia neum monia iinfection nffec ecti tioon ti on w worsens o se or s ns reported promote atherogenesis IL-1 at atherosclerosis the hero ro osc s le lero rosi ro siss and and exacerbates ex xaccer erba bate t s ischemic te isch is chem ch em micc brain brainn injury injjury in jury r plateletplaate tellett- aand nd IIL-1-dependently L 1L1-ddepe depe pend nden nd en ntlly35, whil w while hilee ILIL 1ȕ deletion de ecr crea eaase sess the th severity seve se veriity ooff at ve the hero rosc ro scle sc lero le rosi ro siis44. decreases atherosclerosis It’s well-known that platelets participate in the pathogenesis of atherosclerosis45. The role of NOD2 in atherosclerosis is limited and inconsistent. Though two groups found that NOD2 prompt atherosclerosis15, 46, Yuan et al recently reported that MDP/NOD2 axis activation reduces atherosclerosis47. The role of platelet NOD2 in the growth and destabilization of atherosclerosis is not clear. The prothrombotic and proinflammatory roles of platelet NOD2 demonstrated by our present study suggest that platelet NOD2 may facilitate the initiation and development of atherosclerosis. The different MDP concentrations needed for NOD2-mediated prothrombotic and

15 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

proinflammatory response attracted our attention. At low MDP concentrations (10 - 100 ng/mL), platelet NOD2 triggers IL-1ȕ maturation; while at high MDP concentrations (1 - 10 μg/mL), platelet NOD2 potentiates platelets activation, but does not stimulate IL-1ȕ maturation (data not shown). Although the underlying mechanism accounting for this different concentration dependence remains to be elucidated, the present study implies that platelet NOD2 is likely to switch its roles at different stages of infection. At the initial stage of infection, in response to low concentrations of MDP from pathogens, platelet NOD2 appears to mainly exert proinflammatory effects. With the pathogen accumulation as the infection progresses, high concentration of MDP from bacteria triggers the prothrombotic response of platelet NOD2. In conclusion, NOD2, which is mainly expressed in white blood cells and plays ndd pla l ys ccritical la riti ri tica ti c l ca roles oles in innate immune against infection, is also expressed in platelets and potentiates platelet activation, acctiiva vati tion ti onn, thrombosis, thrromb th mboosis, mb os and prompts proinflammatory proinflamm mato atory response. Our Our u study stu tuddy dy also proposed a novel signaling pathway, MDP-NOD2-RIP2-MAPK/cGMP, activation, which explain ignnal a ing path hwaay, M DP P-N -NOD NOD OD22-RI RIP2 RI P2-M -M MAP PK/cG GMP,, fo forr pplatelet latel atelet et act ctiv ivaation iv n, wh whic ichh ma ic mayy ex expl p a in pl the prothrombotic NOD2 innate he pr prot o hr ot hrom ombboti om botiic state stat st atte in i infectious inf nffec ecttio ious us diseases. dis i ea ease ses. Considering se Con nsideeriing the the critical crittic ical a rroles al oles ooff NO oles OD2 iin n in inna naatee immune platelets mmune against agaain nst infection inf nfec e ti ec t on o aand nd the he crucial cru ruciial a rrole olee of ol o pl plat atel at e etts in tthrombosis, el hrom hr ombo om bosi bo sis, si s, hhemostasis, emos em osta os tasi ta sis, si s aand s, nd immunee response, further studies could shed new insight into the pathogenesis and treatment of inflammatory and thrombotic diseases.

Funding Sources: This work was partially supported by National Natural Science of Foundation of China (Grant No. 81173053, 81373411, 81100344), Drug Innovative Program from Shanghai Municipal Science and Technology Commission (Grant No. 11431920103), Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning to ZD and SZ, and grants HL93231 and HL118593 from National Institutes of Health to SPK. Conflict of Interest Disclosures: None.

16 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

References: 1. Chen G, Shaw MH, Kim YG, Nunez G. NOD-like receptors: role in innate immunity and inflammatory disease. Annu Rev Pathol. 2009;4:365-398. 2. Philpott DJ, Sorbara MT, Roberston SJ, Croitoru K, Girardin SE. NOD proteins: regulators of inflammation in health and disease. Nat Rev Immunol. 2014;14:9-23. 3. Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, Thomas G, Philpott DJ, Sansonetti PJ. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003;278:8869-8872. 4. Mo J, Boyle JP, Howard CB, Monie TP, Davis BK, Duncan JA. Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP. J Biol Chem. 2012;287:23057-23067. 5. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, Britton H, Moran T, Hanauer SB,, Nu Nunez Karaliuskas R, Duerr RH, Achkar a JP, Brant SR, Bayless TM, Kirschner BS, Hanaue uerr SB S Nune nezz Crohn's disease. G, Cho JH. A frameshift mutation in NOD2 associated with susceptibility to Croh hn's di dise seas se ase. as e. Nature. 2001;411:603-606. Kobayashi Chamaillard Henegariu Nunezz G, Flavell RA. Nod26. Kobay yashi KS, S, C hamaillard M, Ogura Y, Heneg egariu a O, Inohara N, Nune dependent immunity intestinal de epe pend nden nd entt re rregulation gulaati tioon on of innate and adaptive immu muni mu nitty in the intest tin inal tract. tra racct. ct Science. 2005;307:731-734. 20 0055;3 307:7 7311-7 7344. Lii C, Li J, L Lii Y Y,, Lan Lang Yougbare Zhu G,, Ch Chen Nii H. Platelets 7.. L ng S, Y oug ugba bare ree II,, Z hu G hen P,, N hen H Crosstalk Cros ossstalkk bbetween etw ween Pl lat atel eleetss andd tthe he Immune System: with Discoveries. mmu mune n S ne yste ys tem: m: Old Old Systems Sys yste tems ms w ithh New it New Di Dis scov over ov erriees. Ad Advv Hematol. Hemat Hem matoll. 20 22012;2012:384685. 12;2 12 ; 01 ;2 012: 2:38 2: 3846 4685 46 85.. 8. Shiraki R,, In Inoue Kawasaki Takei A,, Ka Kadotani M,, Oh Ohnishi Ejiri Kobayashi Inou o e N, ou N K aw was asak a i S, ak S T ak kei e A Kado dota do taani n M Ohni nish ni shii Y, E sh jiri ji ri JJ,, Ko oba baya yash ya shi S, Hirata sh K, Kawashima S, Yo Yokoyama M. E Expression Toll-like Thromb K K awas aw ashi hima ma S Yoko koya yama ma M xpre xp ress ssio ionn of T olll-li ol like ke rreceptors eceept ec ptor orss on hhuman uman um an pplatelets. late la tele lets ts Th Thro romb mb Res. 2004;113:379-385. 9. Aslam R, Speck ER, Kim M, Crow AR, Bang KW, Nestel FP, Ni H, Lazarus AH, Freedman J, Semple JW. Platelet Toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-alpha production in vivo. Blood. 2006;107:637-641. 10. Andonegui G, Kerfoot SM, McNagny K, Ebbert KV, Patel KD, Kubes P. Platelets express functional Toll-like receptor-4. Blood. 2005;106:2417-2423. 11. Zhang G, Han J, Welch EJ, Ye RD, Voyno-Yasenetskaya TA, Malik AB, Du X, Li Z. Lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via TLR4/MyD88 and the cGMP-dependent protein kinase pathway. J Immunol. 2009;182:7997-8004. 12. Blair P, Rex S, Vitseva O, Beaulieu L, Tanriverdi K, Chakrabarti S, Hayashi C, Genco CA, Iafrati M, Freedman JE. Stimulation of Toll-like receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase. Circ Res.

17 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

2009;104:346-354. 13. Panigrahi S, Ma Y, Hong L, Gao D, West XZ, Salomon RG, Byzova TV, Podrez EA. Engagement of platelet toll-like receptor 9 by novel endogenous ligands promotes platelet hyperreactivity and thrombosis. Circ Res. 2013;112:103-112. 14. Maeda S, Hsu LC, Liu H, Bankston LA, Iimura M, Kagnoff MF, Eckmann L, Karin M. Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing. Science. 2005;307:734-738. 15. Liu HQ, Zhang XY, Edfeldt K, Nijhuis MO, Idborg H, Back M, Roy J, Hedin U, Jakobsson PJ, Laman JD, de Kleijn DP, Pasterkamp G, Hansson GK, Yan ZQ. NOD2-mediated innate immune signaling regulates the eicosanoids in atherosclerosis. Arterioscler Thromb Vasc Biol. 2013;33:2193-2201. 16. Shimada K, Chen S, Dempsey PW, Sorrentino R, Alsabeh R, Slepenkin AV, Peterson E, Doherty TM, Underhill D, Crother TR, Arditi M. The NOD/RIP2 pathway is essential for host 2009;5:e1000379. defenses against Chlamydophila pneumoniae lung infection. PLoS Pathog. 2009;5:e e10 1000 0037 379. 9. isomerase ERp57 17. Wu Y, Ahmad SS, Zhou J, Wang L, Cully MP, Essex DW. The disulfide isom merras asee ER ERp5 p577 p5 mediates platelet aggregation, hemostasis, and thrombosis. Blood. 2012;119:1737-1746. 18. Zhang Zhang L,, G Gu Cheng 18 8. Zh Zha ang SH ang SH, Zh hang an Y, Shen J, Zhang S, Chen L u J, Mruk JS,, Ch C en ng G, G Zhu L, Kunapuli SP, Ding Z.. Tumo Tumor vascular agent 5,6-dimethylxanthenone-4-acetic acid platelet Di ing gZ mor va mo asc s ul ular ar ddisrupting isru is rupt ptin pt ingg ag in gen entt 5, ,66-di dime meth th hylxaant nthe heno he n ne ne-4 -4-a -4 -ace ceti ticc ac ti cid iinhibits nhib nh ibit itss pl plat atel elet el e et activation inhibition thromboxane signaling and phosphodiesterase. ac ctiiva v tion andd tthrombosis hroombo hr bosi siis vi via ia in inhi hibi b ti bi t onn ooff th hro omb boxxan ne A2 A2 si ign gnaaliing an nd ph hossph phod odie od iest ie stter eraase. ase. J Thromb Thro Th roomb Haemost. Haeemo m st. 22013;11:1855-1866. 0113;11::18555-1 - 86 66. 6 X,, Mi Flevaris Lu Liu H,, Ru Z,, Wa X,, Ki Kieffer Chen Du Xii X. 19. Su X M JJ,, Ya Yann J, Fle eva v ri riss P, P L u Y, L iu H Ruan Z Wang ng X Kief effe fer N, C henn S, he S D u X, X synthetic the integrin cytoplasmic C-terminal RGT, a synth het etic ic ppeptide epti ep tiide ccorresponding orre or resppon re ondi ding di ngg tto o th he in nteegr grin in bbeta etaa 3 cy et cyto topl to plas a mi as micc CC term te rmin rm inal in al ssequence, e uence, eq selectively inhibits signaling platelets interaction ele lect ctiv ivel elyy in inhi hibi bits ts ooutside-in utsi ut side de-in in si sign gnal alin ingg in hhuman uman um an pl plat atel elet etss by ddisrupting isru is rupt ptin ingg th thee in inte tera ract ctio ionn of iintegrin nteg nt egri rinn alpha IIb beta 3 with Src kinase. Blood. 2008;112:592-602. 20. Fay WP, Parker AC, Ansari MN, Zheng X, Ginsburg D. Vitronectin inhibits the thrombotic response to arterial injury in mice. Blood. 1999;93:1825-1830. 21. Zhang S, Hu L, Du H, Guo Y, Zhang Y, Niu H, Jin J, Zhang J, Liu J, Zhang X, Kunapuli SP, Ding Z. BF0801, a novel adenine derivative, inhibits platelet activation via phosphodiesterase inhibition and P2Y12 antagonism. Thromb Haemost. 2010;104:845-857. 22. Fang C, Stavrou E, Schmaier AA, Grobe N, Morris M, Chen A, Nieman MT, Adams GN, LaRusch G, Zhou Y, Bilodeau ML, Mahdi F, Warnock M, Schmaier AH. Angiotensin 1-7 and Mas decrease thrombosis in Bdkrb2-/- mice by increasing NO and prostacyclin to reduce platelet spreading and glycoprotein VI activation. Blood. 2013;121:3023-3032. 23. Zhang Y, Ye J, Hu L, Zhang S, Zhang SH, Li Y, Kunapuli SP, Ding Z. Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12. J Thromb

18 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

Haemost. 2012;10:2149-2157. 24. Shen B, Zhao X, O'Brien KA, Stojanovic-Terpo A, Delaney MK, Kim K, Cho J, Lam SC, Du X. A directional switch of integrin signalling and a new anti-thrombotic strategy. Nature. 2013;503:131-135. 25. Nieswandt B, Schulte V, Bergmeier W, Mokhtari-Nejad R, Rackebrandt K, Cazenave JP, Ohlmann P, Gachet C, Zirngibl H. Long-term antithrombotic protection by in vivo depletion of platelet glycoprotein VI in mice. J Exp Med. 2001;193:459-469. 26. Riba R, Sharifi M, Farndale RW, Naseem KM. Regulation of platelet guanylyl cyclase by collagen: evidence that Glycoprotein VI mediates platelet nitric oxide synthesis in response to collagen. Thromb Haemost. 2005;94:395-403. 27. Gambaryan S, Kobsar A, Hartmann S, Birschmann I, Kuhlencordt PJ, Muller-Esterl W, Lohmann SM, Walter U. NO-synthase-/NO-independent regulation of human and murine platelet soluble guanylyl cyclase activity. J Thromb Haemost. 2008;6:1376-1384. 28. Li Z, Xi X, Du X. A mitogen-activated protein kinase-dependent signaling pat pathway athw hway hw ay iin n th thee activation of platelet integrin alpha IIbbeta3. J Biol Chem. 2001;276:42226-42232. 2001;276:42226-422322. 29. Li Z,, Delaney y MK, O'Brien KA, Du X. Signalingg during platelet adhesion and activation. Arterioscler Arrteeri rios oscl os cler cl er Thromb Thrrom ombb Vasc Biol. 2010;30:2341-2349. 2010;30:2341-234 349. 34 30. Yin Lii Z Z,, Be Berndt Lowell CA, X.. S Src tyrosine mediates 30. Y in H, Liu u JJ,, L Bern rndt rn dt MC MC,, Lowe L owe well C A, Duu X rc ffamily rc amil am ilyy ty yro osiine ne kkinase in nase ase Ly Lynn m edi diat di atees VWF/GPIb-IX-induced via signaling VWF/ VW F GPIb-I -IXX in nduceed pplatelet lat ateeleet aactivation ctiv iv vat atiion vi ia tthe he ccGMP GMP GM P si sign gnaalin ng ppathway. athwa way. wa y. Blood. Bloo ood. 2008;112:1139-1146. 20008 0 ;1 ;112 12:1 :1 113 1399-111446. 46 31. Marjanov Marjanovic Stojanovic Brovkovych Skidgel X.. Si Signaling-mediated vic JJA, A, S to ojaano ovi vicc A, B rovk ro vk kovvyc ychh VM VM,, Sk S id dge gell RA RA,, Du X Sign gnal gn alin ingin g me gmedi d ated functional stimulating platelet func fu ncti tion onal al activation act ctiv ivat atio ionn of inducible ind nduc ucib ible le nitric-oxide nit itri ricc-ox oxid idee synthase syn ynth thas asee an andd it itss ro role le iin n st stim imul ulat atin ingg pl plat atel elet et activation. J Biol Chem. 2008;283:28827-28834. 32. Stojanovic A, Marjanovic JA, Brovkovych VM, Peng X, Hay N, Skidgel RA, Du X. A phosphoinositide 3-kinase-AKT-nitric oxide-cGMP signaling pathway in stimulating platelet secretion and aggregation. J Biol Chem. 2006;281:16333-16339. 33. Morrell CN, Aggrey AA, Chapman LM, Modjeski KL. Emerging roles for platelets as immune and inflammatory cells. Blood. 2014;123:2759-2767. 34. Smeeth L, Thomas SL, Hall AJ, Hubbard R, Farrington P, Vallance P. Risk of myocardial infarction and stroke after acute infection or vaccination. N Engl J Med. 2004;351:2611-2618. 35. Denes A, Pradillo JM, Drake C, Sharp A, Warn P, Murray KN, Rohit B, Dockrell DH, Chamberlain J, Casbolt H, Francis S, Martinecz B, Nieswandt B, Rothwell NJ, Allan SM. Streptococcus pneumoniae worsens cerebral ischemia via interleukin 1 and platelet glycoprotein Ibalpha. Ann Neurol. 2014;75:670-683.

19 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

36. Dalager-Pedersen M, Sogaard M, Schonheyder HC, Nielsen H, Thomsen RW. Risk for myocardial infarction and stroke after community-acquired bacteremia: a 20-year population-based cohort study. Circulation. 2014;129:1387-1396. 37. Li Z, Zhang G, Feil R, Han J, Du X. Sequential activation of p38 and ERK pathways by cGMP-dependent protein kinase leading to activation of the platelet integrin alphaIIb beta3. Blood. 2006;107:965-972. 38. Adam F, Kauskot A, Nurden P, Sulpice E, Hoylaerts MF, Davis RJ, Rosa JP, Bryckaert M. Platelet JNK1 is involved in secretion and thrombus formation. Blood. 2010;115:4083-4092. 39. Saklatvala J, Rawlinson L, Waller RJ, Sarsfield S, Lee JC, Morton LF, Barnes MJ, Farndale RW. Role for p38 mitogen-activated protein kinase in platelet aggregation caused by collagen or a thromboxane analogue. J Biol Chem. 1996;271:6586-6589. 40. Kauskot A, Adam F, Mazharian A, Ajzenberg N, Berrou E, Bonnefoy A, Rosa JP, Hoylaerts MF, Bryckaert M. Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation. J Biol Chem. 2007;282:31990-31999. stimulatory 41. Li Z, Xi X, Gu M, Feil R, Ye RD, Eigenthaler M, Hofmann F, Du X. A stimula lato torry to ry rrole olee fo ol forr cGMP-dependent protein kinase in platelet activation. Cell. 2003;112:77-86. 42. Franchi Warner Function 422. Fr Fran anch an chii L, ch L W arne ar n r N, Viani K, Nunez G. Func ncttioon of Nod-likee rreceptors nc ecep ep pto tors r in microbial recognition eco coggnition an andd ho host st ddefense. efen ef en nse s . Immunol Im mmu muno noll Rev. Revv. 2009;227:106-128. Re 2009 20 09;2 227:106 066-1 128 2 . 43. receptor mediates 43 3. Chi Chi H, Messas Mes e saas E, Levine Levin ne RA, RA, Graves Grrav aves ess DT, DT T, Amar Amaar S. IInterleukin-1 nteerl nt erleuukin n-1 rec cep pto torr sign ssignaling ig allin ingg me media atess atherosclerosis associated with bacterial exposure murine apolipoprotein atthe hero ro osc s le lero rosi ro siss as si ssooci ciaateed w itth ba bact cteeria ct eriaal ex expo posuure po r aand/or nd or a high nd/o hhigh-fat ighh-f - at ddiet i t in a m ie urrin inee ap apol ollip pop opro ro oteein i E heterozygote implications. hete he tero r zy ygotee model: mod odel el: pharmacotherapeutic phhar arma maco coth therapeu uti ticc im impl plic icatio ions ns. Circulation. Circ Ci rcul u at atio ionn. 2004;110:1678-1685. 2 04 20 04;1 110 10:1 :167 6 88-1685 85. 44. Kirii H, N Niwa T, Y Yamada Y, W Wada H, S Saito K, IIwakura Y, As Asano M, M Moriwaki H, S Seishima 44 Ki Kiri riii H iwaa T iw amad am adaa Y adaa H ad aito ai to K waku wa kura ra Y Asan anoo M oriw or iwak akii H eish ei shim imaa M. Lack of interleukin-1beta decreases the severity of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 2003;23:656-660. 45. Lievens D, von Hundelshausen P. Platelets in atherosclerosis. Thromb Haemost. 2011;106:827838. 46. Johansson ME, Zhang XY, Edfeldt K, Lundberg AM, Levin MC, Boren J, Li W, Yuan XM, Folkersen L, Eriksson P, Hedin U, Low H, Sviridov D, Rios FJ, Hansson GK, Yan ZQ. Innate immune receptor NOD2 promotes vascular inflammation and formation of lipid-rich necrotic cores in hypercholesterolemic mice. Eur J Immunol. 2014;44:3081-3092. doi: 10.1002/eji.201444755. 47. Yuan H, Zelka S, Burkatovskaya M, Gupte R, Leeman SE, Amar S. Pivotal role of NOD2 in inflammatory processes affecting atherosclerosis and periodontal bone loss. Proc Natl Acad Sci U S A. 2013;110:E5059-5068.

20 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

Figure Legends:

Figure 1. Both human and mouse platelets express NOD2. A, RT-PCR detection of NOD1, NOD2 (panel A1) and monocyte-specific CD14 (panel A2) in human platelets and peripheral blood mononuclear cells (PBMC). B, Western Blot detection of NOD1 and NOD2 in human platelets and PBMC. CHO cells were used as a negative control. C, RT-PCR detection of NOD1, NOD2 (panel C1), and monocyte-specific CD14 (panel C2) in mouse platelets. 299 bp NOD1 and 273 bp NOD2 are expected. D, Western blot detection of NOD1 and NOD2 in mouse platelets.

Figure 2. NOD2 activation induced by MDP potentiates platelet activation induced by thrombin and collagen. human an nd co coll llag ll ag gen e . A,, MDP MD concentration-dependently concentration-dependentl tlly ppotentiates otentiates hum man pplatelet lattelet la te aggregation and ATP U/mL ȝg/mL. B,, NO NOD2 A TP P release induced in ndu duccedd by b tthrombin hrom hr ombi om binn 0.02 0.022 U /m mL or ccollagen ollage genn 00.3 ge .33 ȝ g/mL. mL B OD2 D ddimerization imer im e iz er i at atiion ion iin n human hu huma mann platelets ma p attel pl elet etss stimulated stiimu imulatted with wit ithh MDP MDP 100 ȝg/mL. ȝg/mL mL.. C, mL C, MDP MDP potentiates pote po tenntia te nt ate tess mouse mous mo usse platelet plat pl attel eleet aaggregation gg gre reggattion tio and ATP rele release induced ȝg/mL. Aspirinated eas a e in indu d ce du cedd by tthrombin hrrom ombi binn 0.02 bi 0 02 U/mL 0. U/m /mL L and and collagen coll co lllag agen en n 0.3 0.3 ȝ g/mL g/ mL.. As mL Aspi piri pi rina ri nate na t d washed te platelets were incubated with MDP for 15 min, and then stimulated with thrombin or collagen. D, Genotyping of NOD2-/- and WT mice. Even number lanes show 1000 bp PCR products of mutant, while odd number lanes show the 370 bp PCR products of wild type.

Figure 3. NOD2 agonist MDP accelerates clot retraction NOD2-dependently. Clot retraction in human or mouse washed platelets was assayed as described in Materials and Methods. A, Results shown are representative of four experiments using platelets from different donors or mice. MDP 10 ȝg/mL was used. B, Quantification of clot retraction at 60 min expressed as mean ± SD, n = 4.

21 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

Figure 4. Impaired thrombus formation and hemostasis in NOD2-deficient mice. A, Impaired thrombus formation in mesenteric arterioles of NOD2-/- mice as compared with WT mice. Thrombosis was induced by FeCl3 injury, and recorded with intravital microscopy. Calcein was used to label platelets. MDP (100 ȝg/100 ȝL per mouse) or saline were injected intraperitoneally 12 hours before FeCl3 injury. B, Statistical analysis of FeCl3-induced thrombus formation. Occlusion time and time to first thrombus (larger than 20 ȝm and stable for more than 2 min) were analyzed. Data are expressed as mean ± SD with 11 WT and 12 NOD2-/- mice. C, MDP injection causes less blood loss in WT but not in NOD2-/- mice. Data are expressed as mean ± SD, n = 8.

Figure 5. Plasma from patients with bacterial infection f enhances platelet platelet aggregation NOD2de epe pend nden nd entl tly. tl y A, Representative Reppresentative platelett aggregation Re aggregaati tion onn tracings. B, Su S mm mar aryy of aggregation dependently. Summary ex experiments xpe periments ex exp expressed presse sedd as as m mean eann ± SD ea D ooff 12 eexperiments. xperriment ntts. P Platelet latele telett poor poor pplasma lasm ma (P (PPP (PPP) PP)) pr PP prepared rep par ared e ed fr rom 1144 patients pati pa tieents ti ents with wit ithh proven prrov oven n bacteremia bac acte terrem te remia mia was wa pooled pooole po oled and and used used d as as septic seept p icc P PP P. PPP PPP pr prep ep parred ffrom room from PPP. prepared he same patients pattieent ntss after afte af t r cure te c ree of cu of infection in nfe fect ctio ct i n was w s used wa u ed as us as healthy heal he alth al thyy PPP. th PPP For For each eac achh experiment, expe ex peri pe rime ri ment me n , PRP the prepared from WT or NOD2-/- mice was preincubated with patient PPP (1:1 vol/vol) for 10 min, then platelet aggregation was induced by 0.02 U/mL thrombin or 0.3 ȝg/mL collagen. Please note that though septic plasma also enhances platelet aggregation of NOD2-/- mice, the potentiating effects are significantly weakened compared with that on WT mice.

Figure 6. RIP2/MAPK mediates the potentiating effects of MDP on platelet activation. A, Platelets express RIP2. B, MDP phosphorylates RIP2, Erk, p38, and JNK in human platelets. Results shown are representative of at least three experiments using platelets from different

22 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

donors or mice. C, Enhanced platelet aggregation is abolished by MAPK inhibitors. Human platelets (aspirinated washed platelets) were pretreated with 10 ȝM PD98059 (ERK1/2 inhibitor), 10 ȝM SB203580 (p38 MAPK inhibitor), or 10 ȝM SP600125 (JNK inhibitor) for 10 min, then treated with 10 ȝg/mL MDP or vehicle as control before stimulation by 0.02 U/mL thrombin or 0.3 ȝg/mL collagen. D, Enhanced clot retraction in human washed platelets is abolished by MAPK inhibitors. Clot retraction was assayed as in Figure 3. The upper panel is the representative of 4 experiments using platelets from different donors, the lower panel is the summary expressed as mean ± SD. MAPK inhibitors and MDP were used at the same concentration as in panel C.

Figure 7. MDP elevates platelet cGMP and NO levels NOD2-dependently. A, MDP concentration-dependently human Human co onc ncen enttrat en trat atio ionn-deepe io pend n ently increases cGMP in hu hum man platelets. Hu Huma an platelets pl were treated 1 with platelets w ith h MDP at 37 37ºC ºC ffor orr 115 5 mi min, n, tthe hee ccGMP GMP M in pl latellets wa wass ddetected ettect cteed ed bby y 125 I ra radioimmunoassay. adi dioi oimm mmun mm u oa un oasssay ssay y. B,

MDP MD DP el eelevates evat ev atees at es ccGMP GMP GM P in pplatelets l teele la lets ts ffrom rom ro m WT bbut ut nnot ot in i N NOD2-/OD2OD 2-//- m mice. icce. e D Data atta ar aree ex eexpressed preesse pr seed as m mean ea ean ± SD using pplatelets late la tele te leets ffrom rom ro m di ddifferent fferren ff entt do ddonors nors no rs oorr mi m mice ce ((n n = 4) 4).. C, C MD M MDP P po pote potentiation tent te ntia nt i tiion ooff pl ia platelet aggregation induced by low concentration of thrombin was abolished by PKG inhibitor KT5823. Human washed platelets were used. Tracing shown are representative of 3 experiments using platelets from different donors. D, MDP elevates NO in human platelets. Platelets preincubated with fluorescent NO sensor DAF-FM DA were stimulated with MDP. Upper panel shows platelet fluorescence under confocal microscope (scale bar = 5 ȝm), bottom panel is the quantification of NO expressed as fluorescence per platelet in arbitrary unit (mean ± SD of 4 experiments). E, MDP elevates platelet NO in WT but not in NOD2-/- mice. NO was assayed as in panel D. Upper panel is representative of 4 experiments using platelets from different mice,

23 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

DOI: 10.1161/CIRCULATIONAHA.114.013743

bottom panel is the quantification of NO expressed as mean ± SD, n = 4. The concentrations for MDP, thrombin and KT5823 were 10 ȝg/mL, 0.02 U/mL and 8 ȝM, respectively. F, MAPK and NO/cGMP/PKG pathways downstream of RIP2 mediate the role of NOD2 in platelets.

Figure 8. MDP stimulates platelet IL-1ȕ maturation. A, MDP time- and concentration-dependently stimulates IL-1ȕ accumulation in human platelets. IL-1ȕ was assayed by flow cytometry as described under Materials and Methods. Results shown are representative of 3 experiments using platelets from different donors. B & C, Caspase-1 inhibitor FMK002 abolishes MDP-induced IL1ȕ maturation in human platelets. Platelets were treated with MDP (100 ȝg/mL) overnight, in the resuspended presence or absence of caspase-1 inhibitor FMK002 (25 ȝM), then platelets were re esuusppen ende dedd in de reducing educing SDS sample buffer and probed for IL-1ȕ by Western blot (B) or flow cytometry (C). Results of 3 (panel B) and Resu Re sult su ltss shown lt show sh ownn are ow arre representative re and nd 4 experimentss (panel (pane neel C1) C1) using platelets from different donors, respectively. Panel the C1 expressed mean di fer diff erent dono ors rs,, re esppecti ectiiveely ly. Pane P ane nell C22 iiss th he ssummary um mmaryy of of ppanel an nel C 1 ex exp preesseed as m eaan ± SD ((n n= 4).. D D,, MD MDP but not NOD2-/mice. Platelets 4) M P stimulates stim st imul ulat a ess IL-1ȕ at IL L-1ȕ aaccumulation L-1 cccum umuulat ulattio on in pplatelets l teeleets la t ffrom ro om WT WT,, bu ut no ot NO NOD2 D2-/ D2 -/-- mi m cee. Plat P lateeleets ets with mature platelet were treated w ithh MD it MDP P (1 ((100 00 ng nng/mL) /mL) /m L)) oovernight, vern ve rnig rn ig ght ht,, ma matu ture tu r IIL-1ȕ re L 1ȕ pprotein Lrote ro t in te n iin n pl plat atel at e ett llysates el ysat ys ates at es was measured by ELISA. Data are expressed as mean ± SD with n = 4.

24 Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

A1

A2

B

PBMC

250bp 500bp 1000bp 1500bp 2000bp

CD14

platelets actin in

NOD1

NOD2

NOD2

11KD

NOD1

95KD

GAPDH P

C1

BM

C at pl

ts e l e

CH HO CHO

C2 C 2

platelets PBMC P BM MC p

D 200 BP 200BP 300BP 300 30 00 BP 400BP 40 00 BP 500 50 B BP 500BP 600BP 600 60 BP 700BP 700 70 0 BP 800BP 900BP

CD14 CD D14 platelets PBMC

platelets

NOD2 N OD2 NOD1 GAPDH PBMC platelets

Figure 1

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

A

collagen

aggregation

thrombin

control MDP1ȝg/mL MDP3ȝg/mL

ATP re ele ease ATPrelease

M D P 1 0 ȝ g/mL MDP10ȝg/mL

DP M

co

nt

ro

l

B

dimer

monomer

Figure 2

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

C WT collagen

thrombin

collagen

aggregation

thrombin

NOD2Ͳ/Ͳ

control

ATPrelease

MD MDP10ȝg/mL

D

NOD2Ͳ/Ͳ N OD2ͲͲ/Ͳ

WT WT

12345678 1 2 3 4 5 6 7 8 1000 750 500 250

NOD2

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015 Figure 2, cont’d

A

humanmouse MDPͲ

WTNOD2Ͳ/Ͳ +Ͳ +Ͳ +

0min

15 min 15min

30 min 30min

45min 45 min

60min 60 min

B

Figure 3

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

WT

A

NOD2 Ͳ/Ͳ

baseline

5min

8min n C

12min in control con ntrol

B

Figure 4

MDP MDP

ccontrol ontro ol

MDP MDP

C

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

A

thrombin

collagen

healthyPPP+NOD2Ͳ/Ͳ healthy PPP + WT W healthyPPP+WT septic PPP + NOD D2Ͳ/Ͳ septicPPP+NOD2Ͳ/Ͳ

septicPPP+WT se septic eptic PPP + WT W

B

Figure 5

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

A

B

RTͲPCR PCR RIP2

human

mouse

PBMCplatelet

PBMC PBMCplatelet platelet

MDP 05101530(min) pͲRIP2 p RIP2 RIP2 pͲErk

A ctin Actin

WB

Erkk Erk pͲp38 p Ͳp p38 8

R IP 2 RIP2

GAPDH

p 38 p38 pͲJNK JNK

Figure 6

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

C thrombin

D

collagen MDP+SB203580 control+SB203580

Time(min)106060

60

60

control

control

MDP MDP

MD MDP+PD98059 DP + PD98059 con ntrol + PD98059 control+PD98059

SB203580 PD98059 SSP600125 P600125

MDP+SP600125 control+SP600125

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015 Figure 6, cont’d

+ + +

A

B

C tthrombin hrombin

KT5823 KT5823+MDP KT control

M MDP(10ȝg/mL)

Figure 7

Downloaded from http://circ.ahajournals.org/ at BIBLIOTHEQUE DE L'UNIV LAVAL on March 3, 2015

MDP

controlMDP

F

MDP NOD2 RIP2

? iNOS/NO/sGC ? MAPK MAPK cGMP

NOD2Ͳ/Ͳ

human

control

E

WT

D

PKG P potentiate activation P

Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.

Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in p...
4MB Sizes 2 Downloads 12 Views