Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E

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1 ARTICES Circulting ctivted pltelets excerte therosclerosis in mice deficient in polipoprotein E YUQING HUO 1, ANDREAS SCHOBER 2, S. BRADEY FOROW 1, DAVID F. SMITH 1, MATTHEW CRAIG HYMAN 1, STEFFEN JUNG 3, DAN R. ITTMAN 4, CHRISTIAN WEBER 2 AND KAUS EY 1 1 Deprtment of Biomedicl Engineering nd Crdiovsculr Reserch Center, University of Virgini, Helth Science Center, Chrlottesville, Virgini, USA 2 Deprtment of Moleculr Crdiovsculr Reserch, University Hospitl Achen, Achen, Germny 3 Skirll Institute of Biomoleculr Medicine nd 4 Howrd Hughes Medicl Institute, New York, New York, USA Correspondence should e ddressed to K..; e-mil: klusley@virgini.edu Pulished online 16 Decemer 22, doi:1.138/nm81 We studied whether circulting ctivted pltelets nd pltelet leukocyte ggregtes cuse the development of therosclerotic lesions in polipoprotein-e deficient (Apoe / ) mice. Circulting ctivted pltelets ound to leukocytes, preferentilly monocytes, to form pltelet monocyte/leukocyte ggregtes. Activted pltelets nd pltelet leukocyte ggregtes intercted with therosclerotic lesions. The interctions of ctivted pltelets with monocytes nd therosclerotic rteries led to delivery of the pltelet-derived chemokines CC5 (regulted on ctivtion, norml T cell expressed nd secreted, RANTES) nd CXC4 (pltelet fctor 4) to the monocyte surfce nd endothelium of therosclerotic rteries. The presence of ctivted pltelets promoted leukocyte inding of vsculr cell dhesion molecule-1 (VCAM-1) nd incresed their dhesiveness to inflmed or therosclerotic endothelium. Injection of ctivted wild-type, ut not P-selectin deficient, pltelets incresed monocyte rrest on the surfce of therosclerotic lesions nd the size of therosclerotic lesions in Apoe / mice. Our results indicte tht circulting ctivted pltelets nd pltelet leukocyte/monocyte ggregtes promote formtion of therosclerotic lesions. This role of ctivted pltelets in therosclerosis is ttriuted to pltelet P-selectin medited delivery of pltelet-derived proinflmmtory fctors to monocytes/leukocytes nd the vessel wll. Atherosclerosis is multi-fctoril vsculr disese involving endothelil cells, vsculr smooth muscle cells, mononucler cells, pltelets, growth fctors nd cytokines 1. The response-to-injury hypothesis of therogenesis 2 hs een modified drmticlly over the pst three decdes. The originl version of this hypothesis proposed tht the first step in therosclerosis ws endothelil denudtion nd the key events in the development of therosclerosis were the relese of growth fctors from deposited pltelets nd consequent smooth-muscle prolifertion 2,3. In ddition, pltelets intercting with monocyte-like cells hve lso een shown to contriute to fom cell formtion 4. Atherogenesis is chronic inflmmtory process in which monocytes nd T cells interct with structurlly intct ut dysfunctionl endothelium of rteries 1. It ws further demonstrted tht interventions to reduce mononucler cell recruitment to vessels, key step in the initition of therosclerosis, were le to protect nimls from therosclerosis 5 8. This inflmmtion hypothesis of therosclerosis, emphsizing the role of mononucler cells in the development of therosclerosis, mde questionle the involvement of pltelets in the development of spontneous therosclerotic lesions. Activted pltelets re present in the circulting lood of therosclerotic individuls. The presence of circulting ctivted pltelets ws found in the circulting lood of ptients with unstle therosclerosis 9 12, stle coronry disese 13 nd hypercholesterolemi 14,15. Activted pltelets in lood re prone to ind leukocytes, preferentilly monocytes, to form pltelet leukocyte ggregtes 13,16,17. Therefore, pltelet ctivtion is one of the mjor chrcteristics present throughout the therosclerotic process. Circulting ctivted pltelets might ffect endothelil inflmmtion nd leukocyte endothelil interctions, which re crucil events in therosclerosis. P-selectin expressed on ctivted pltelets increses monocytoid cell dhesion to endothelil cells in n in vitro ssy 18. Activted pltelets ind to circulting lymphocytes nd my support lymphocyte homing to lymph nodes 19. Activted pltelets lso relese proinflmmtory cytokines (for exmple, CD4 2 nd interleukin (I)-1β 21 ), resulting in endothelil ctivtion. We hve previously found tht the CC chemokine CC5 (regulted on ctivtion, norml T cell expressed nd secreted, RANTES) secreted y stimulted pltelets is immoilized on microvsculr or ortic endothelium nd triggers monocyte rrest 22. Although these oservtions re suggestive, the role of circulting ctivted pltelets nd pltelet leukocyte ggregtes in the formtion of therosclerotic lesions in vivo hs not een tested so fr. Here, we investigted the interctions of circulting ctivted pltelets with monocytes/leukocytes nd therosclerotic crotid rteries of Apoe / mice in vivo. esion formtion in Apoe / mice ws studied following repeted injections of ctivted pltelets. NATURE MEDICINE VOUME 9 NUMBER 1 JANUARY 23 61

2 ARTICES 5 3 shoulders, ut not in the centrl regions, of estlished therosclerotic lesions Fig. 1 Interctions of ctivted pltelets with leukocytes in vivo. Injection of ctivted wild-type pltelets into wild-type mice cused sequestrtion of neutrophils for 8 min nd monocytes for 18 min. Top right corner, minutes fter injection of pltelets. R 1, grnulocytes; R 2, monocytes; R 3, lymphocytes. Activted pltelets interct with leukocytes nd endothelium Activted, fluorescently leled pltelets infused into C57B/6 mice through jugulr vein were le to ind to leukocytes immeditely. Monocytes nd neutrophils (Gr-1 nd Mc-1 positive cells), ut not CD3 + lymphocytes, ound ctivted ut not resting or P-selectin deficient pltelets (dt not shown). In norml C57B/6 mice, circulting pltelet leukocyte ggregtes were no longer detectle t 3 to 4 hours fter single injection of ctivted pltelets. Perfusion of ctivted pltelets cused leukocytes nd preferentilly monocytes to e removed from the circultion. Following infusion of ctivted pltelets into C57B/6 mice, the monocyte popultion disppered from the circultion lmost immeditely nd returned t 3 to 4 hours, when most of the pltelet leukocyte ggregtes were disengged (Fig. 1). Most neutrophils lso disppered, ut returned ck to the circultion much sooner thn monocytes, t 3 to 8 minutes. Infusion of GFP-expressing neutrophils demonstrted tht the recovery of neutrophils ws due to leukocytes returning to the circultion rther thn relese from one mrrow. eukocytes returning to the circultion were no longer decorted with pltelets (dt not shown). In n in vitro prllel plte flow chmer ssy, ctivted, ut not resting, wild-type pltelets were le to interct with I- 1β ctivted humn ortic endothelil cells (HAECs). The interctions were minly chrcterized y trnsient tethering nd rolling, wheres firm dhesion only rrely occurred. Activted pltelets without P-selectin (Selp / ) showed ttenuted interctions with inflmed endothelium (Fig. 2). Similr to these in vitro dt, fluorescently leled, ctivted wild-type pltelets, ut not Selp / pltelets, intercted with therosclerotic crotid rteries of Apoe / mice, ut not crotid rteries of ge-mtched C57B/6 mice (Fig. 2). Fluorescently leled pltelets not only tethered nd rolled ut lso rrested on therosclerotic endothelium (Fig. 2c). However, ctivted pltelets dherent on therosclerotic rteries often detched to re-enter the flowing lood within short time. These interctions minly occurred on the erly therosclerotic lesions nd on the Deposition of pltelet-derived chemokines To determine whether deposition of plteletderived proinflmmtory fctors on endothelium ws ssocited with pltelet endothelil interctions, we perfused ctivted pltelets on I-1β treted ortic endothelil cells under sher flow. A sustntil grnulr deposition of RANTES (Fig. 3) nd immoiliztion of pltelet fctor-4 (PF-4) in liner pttern (dt not shown) were detected on the surfce of inflmed HAEC following perfusion of ctivted humn or mouse wild-type pltelets. In contrst, the perfusion of ctivted Selp / pltelets resulted in few interctions with endothelium nd reduced immoiliztion of RANTES (Fig. 3). Feeding Apoe / mice with western diet for 6 weeks induced n inflmmtory, therosclerotic phenotype of the endothelium of crotid rteries without visile therosclerotic lesions 23. Injected ctivted wild-type pltelets, ut not Selp / pltelets, showed roust interctions with these crotid rteries. Consistent with this oservtion, en fce immunostining showed tht much higher levels of RANTES (Fig. 3) nd PF-4 (dt not shown) were present on the crotid rteril endothelium of Apoe / mice receiving ctivted wild-type pltelets thn those perfused with ctivted Selp / pltelets. c Pltelets/mm 2 /min tethering rolling firm dhesion Pltelets/mm 2 /min tethering rolling firm dhesion Flow Fig. 2 Interctions of ctivted pltelets with therosclerotic rteries., Activted wild-type ( ) ut not Selp / (P / ) pltelets ( ) or resting pltelets ( ) intercted with I-1β treted ortic endothelil cells under physiologicl sher stress in vitro. Dt re expressed s interctions per mm 2 per minute. n = 4. P <.1 compred with Selp / pltelets or resting pltelets., eled pltelets intercted with therosclerotic mouse crotid rteries in vivo following perfusion of ctivted wild-type ut not Selp / pltelets or resting pltelets. n = 6. P <.1 compred with Selp / pltelets or resting pltelets perfusion groups. c, Interctions of ctivted pltelets with n therosclerotic rtery in vivo. A clcein-leled pltelet (lck circle) tethers, rolls nd rrests on endothelium of therosclerotic crotid rtery, nd detches ck to lood flow. The lesion () is outlined y roken lines. 62 NATURE MEDICINE VOUME 9 NUMBER 1 JANUARY 23

3 ARTICES control IgG Fig. 3 Deposition of pltelet-derived proinflmmtory fctors on therosclerotic endothelium., Perfusion of ctivted wild-type ut not Selp / (P / ) pltelets over ctivted ortic endothelil cells deposited RANTES., En fce preprtion of crotid rteries of Apoe / mice fed with western diet for 6 weeks. Injection of ctivted wild-type ut not Selp / pltelets deposited RANTES on the therosclerotic endothelium of mouse crotid rteries. c, Men fluorescence intensity determined y flow cytometry of ctivted ortic endothelil cells stined for RANTES. d, RANTES deposition on en fce preprtion of crotid rteries from Apoe / mice mesured y imge processing. n=4, p<.1. ct. P / ct. wt c Men fluorescence intensity IgG ct. P / Activted pltelets were lso le to deposit proinflmmtory fctors on monocytes. Using confocl microscopy, we found tht pltelets dherent on the monocyte surfce stined positively for RANTES nd PF-4. We found tht RANTES ws distriuted diffusely on the monocyte memrne res where pltelets were ound (Fig. 4). No stining of RANTES or PF4 ws found on monocytes not ound with pltelets. The very lte ntigen-4 (VA-4) nd vsculr cell dhesion molecule-1 (VCAM-1) pthwy is known to e crucil for monocyte dhesion to the vessel wll to initite therosclerosis 6. To test whether ctivted pltelets could ctivte monocytes nd increse the ffinity of their VA-4 integrins for lignd inding, we mesured VCAM-1 IgG inding to Mono Mc 6 (MM6) cells, monocytic cell line expressing phenotypic nd functionl fetures of mture monocytes 24. Using flow cytometry, we show tht VA-4 of MM6 cells incuted with ctivted pltelets ws le to ind more VCAM-1 IgG (Fig. 4). This result is consistent with chnge in VA-4 ffinity. ct. wt d Percent positive pixels IgG ct. P / ct. wt nti-p-selectin nti-rantes Overly Pretretment of monocytes with pertussis toxin (PTX) or pltelets with P-selectin locking ntiody inhiits inding of VCAM-1 IgG with VA-4 on monocyte pltelet ggregtes (Fig. 4). This finding indictes tht VA-4 ctivtion requires dhesive contct etween pltelets nd monocytes nd proceeds through PTX-sensitive G-protein coupled receptors. The RANTES receptors CCR1, 3 nd 5 re known to couple through PTX-sensitive G proteins. Activted pltelets promote leukocyte dhesion Under the epifluoresence intrvitl microscope, interctions of leukocytes in vivo leled with rhodmine 6G with therosclerotic crotid rteries were rre, consistent with the chronic nture of therosclerosis. Following perfusion of ctivted ut not resting wild-type pltelets or the superntnt of ctivted wild-type pltelets, sustntil interctions of rhodmine 6G leled leukocytes with therosclerotic crotid rteries occurred immeditely nd persisted throughout the experiment (1 2 hours; Fig. 5). To investigte whether ctivted pltelets ffect monocyte rrest on therosclerotic rteries, EGFP-expressing monocytes isolted from CX3CR1 EGFP mice 25 were injected into Apoe / mice vi til veins. Few GFP-expressing monocytes intercted with therosclerotic crotid rteries. However, n increse in monocyte rrest on therosclerotic endothelium ppered fter perfusion of ctivted wild-type ut not Selp / - pltelets. Similr to the ehvior of ctivted pltelets, monocytes lso minly intercted with the erly therosclerotic lesions nd the edges of dvnced lesions (Fig. 5). To investigte the ssocitions etween pltelets nd leukocytes intercting with therosclerotic lesions in vivo, we used scnning electron microscopy. Consistent with the intrvitl study, we found mny more leukocytes dherent on therosclerotic crotid rteries of Apoe / mice treted with ctivted Men fluorescence intensity Act. plt + + +/G MM6 cells /PTX + +/HP1/2 VCAM-1-IgG Fig. 4 Deposition of pltelet-derived proinflmmtory meditors on the surfce of monocytes., Activted pltelets (leled with FITC-conjugted nti-p-selectin) relesed RANTES (leled with PE-conjugted nti-rantes) to the monocyte (MM6 cell) surfce. Relese cn e complete (middle pltelet) or incomplete., Whole lood flow cytometry shows tht monocytes (MM6 cells) incuted with ctivted pltelets ind more VCAM-1 IgG. VCAM-1 IgG inding ws significntly inhiited on monocytes pretreted with pertussis toxin or in the presence of P-selectin locking ntiody (G1). Pretretment of MM6 cells with HP1/2, VA-4 locking ntiody, completely rogted VCAM-1 IgG inding. n = 4. P <.1, compred with no tretment. NATURE MEDICINE VOUME 9 NUMBER 1 JANUARY 23 63

4 ARTICES Act. wt. Res. wt. Supernt. of ct. wt. Buffer eukocyte tethering (cells/min) rolling (cells/min) dhesion (cells/5 mins) c resting wt ct. P / ct. wt Fig. 5 Monocyte/leukocyte endothelil interctions in the 6 presence of ctivted pltelets., Rhodmine 6G leled 5 leukocyte tethering, rolling nd dhesion on therosclerotic 4 crotid rteries of Apoe / mice in vivo were incresed following perfusion of ctivted wild-type pltelets ut not resting pltelets or the superntnt of ctivted wild-type pltelets. n = 9. P <.1., EGFP-expressing mouse monocytes isolted from CX3CR1-EGFP mice sustntilly intercted with Pre-perfusion Buffer supernt. ct. wt. pit. the edge of mouse crotid therosclerotic lesions 2 min following injection of ctivted wild-type ut not Selp / or resting MM6 cells wild-type pltelets. The lesions () re indicted y roken lines. c, eukocyte ccumultion on n therosclerotic lesion t 3 min fter ctivted pltelet perfusion s seen y scnning EM t low (top) nd high (ottom, r, 1 µm) mgnifiction. Pltelets (rrows) ssocited with leukocytes dherent on therosclerotic lesions of Apoe / mice following perfusion of ctivted wild-type pltelets (left) ut not ctivted pltelets lcking P-selectin (right). wild-type pltelets, ut not with resting pltelets or ctivted Selp / pltelets. Pltelets were present on therosclerotic crotid rteries s pltelet leukocyte ggregtes, ut rrely s individul pltelets directly dherent on therosclerotic endothelium (Fig. 5c). This indictes tht ctivted pltelets ound to leukocytes rpidly promote leukocyte rrest. To investigte the role of endothelium pre-perfused with ctivted pltelets in the susequent rrest of monocytes, we used n in vitro prllel plte flow system. Pre-perfusion of ctivted wild-type pltelets, ut not resting pltelets, ctivted Selp / pltelets or the superntnt of ctivted wild-type pltelets through cultured inflmed endothelil monolyer incresed the rrest of Mono Mc 6 cells (Fig. 5d). Perfusion of ctivted humn pltelets into Apoe / mice lso cused n increse in leukocyte interctions with therosclerotic crotid rteries. Pretretment of Apoe / mice with monoclonl ntiody RB4.34, n ntiody ginst mouse P-selectin, locked the mouse endothelil P-selectin function, ut not P-selectin on perfused humn ctivted pltelets. In contrst to the crucil role of endothelil P-selectin in monocyte rrest in the sence of pltelets in n ex vivo model 23, d Monocytic cells/mm2 ct. P / e endothelil P-selectin lockde only prtilly inhiited incresed leukocyte dhesion (y 45 ± 5%; Fig. 5e) due to the presence of ctivted pltelets, indicting significnt contriution of pltelet P-selectin. Activted pltelets ccelerte therosclerosis To investigte whether circulting ctivted pltelets eventully eukocyte dhesion (%) Act. humn + + RB4.34 ma + d, Arrest of MM6 cells on I-1 stimulted ortic endothelil cells ws incresed when ctivted wild-type, ut not Selp /, pltelets or the superntnt of ctivted wild-type pltelets were pre-perfused over the endothelil monolyer. n = 4. P <.1. e, Blocking mouse endothelil P-selectin with RB4.34 only prtilly inhiited leukocyte dhesion on therosclerotic endothelium in vivo in the presence of ctivted humn pltelets. n = 4. P <.1. superntnt of ct. wt treted mice ct. P / treted mice ct. wt treted mice Surfce re stined with oil red O (% of ortic re) supernt. ct. P / ct. wt Fig. 6 Repeted injections of wild-type ut not Selp / ctivted pltelets or the superntnt of ctivted wild-type pltelets excerte therosclerosis in Apoe / mice. The 8-week-old mle Apoe / mice fed with western diet were injected with ctivted wild-type pltelets, Selp / ctivted pltelets or the superntnt of ctivted pltelets vi the til vein every 5 d for 12 weeks. En fce nlysis showed tht lesion sizes of mice treted with ctivted wild-type pltelets were incresed y 39 ± 6% compred with those treted with ctivted Selp / pltelets or superntnt of ctivted wild-type pltelets., Representtive oil red O- stined orts from Apoe / mice with different tretment., Quntittive dt on lesion size from the whole ort; ech dt point represents vlue from single mouse. n = 1. P < NATURE MEDICINE VOUME 9 NUMBER 1 JANUARY 23

5 ARTICES contriute to the formtion of therosclerotic lesions, we injected ctivted wild type or Selp / pltelet suspensions into the til veins of Apoe / mice. Ech mouse received n injection of ctivted mouse pltelets t per 2-g mouse weight every 5 dys for 12 weeks. The numer of injected ctivted pltelets corresponds to 5 7% of totl pltelets in the mouse, which would increse y two- to three-fold the numer of ctivted circulting pltelets in Apoe / mice (dt not shown). This numer of ctivted wild-type pltelets cused 1 15% of mouse leukocytes to e decorted with pltelets s determined y flow cytometry (dt not shown). Perfusion of ctivted pltelets did not cuse difference in the numer of leukocytes nd profiles of lood cholesterol t the time when mice were scrificed. However, therosclerotic lesions in Apoe / mice injected with ctivted wild-type pltelets were 39 ± 6% lrger thn those of Apoe / mice treted with ctivted Selp / pltelets. Injection of the superntnt of ctivted wild-type pltelets in similr wy did not increse the size of therosclerotic lesions of Apoe / mice (Fig. 6). Discussion We hve shown tht circulting ctivted pltelets promote monocyte recruitment to therosclerotic rteries nd ccelerte the formtion of therosclerotic lesions in Apoe / mice. Activted pltelets interct with monocytes nd the endothelium of the vessel wll, depositing chemokines on the cell surfce. These processes re likely to occur in ptients with therosclerosis, in whom ctivted pltelets re commonly oserved We infused ctivted pltelets to illustrte the disese process in compressed time frme. A vriety of pthwys re involved in the interction of pltelets with endothelium nd leukocytes. Endothelil P-selectin 26, von Willernd fctor 27,28, pltelet glycoproteins (GP) I 29,3 nd II/III 3,31 hve importnt roles in pltelet endothelil interctions in different models. Pltelet P-selectin is required for pltelet interction with leukocytes 16,32. Here, we show tht pltelet P-selectin is indispensle for interctions of ctivted pltelets with therosclerotic rteries nd leukocytes/monocytes in vivo. The interctions of ctivted pltelets with vessel wlls occur in trnsient wy, resulting in little pltelet ccumultion on the endothelil surfce of therosclerotic lesions. This my e one of the resons why the involvement of pltelets in the formtion of therosclerotic lesions ws not pprecited in histologicl studies. P-selectin on ctivted pltelets, required to initite pltelet leukocyte interctions, is lso crucil to mintin the ggregtes etween leukocytes nd ctivted pltelets 33. These ggregtes likely cuse monocytes nd neutrophils to dispper from the circultion. Neutrophils return to the circultion within 6 8 minutes following single injection of ctivted pltelets, wheres monocyte numers do not recover until minutes. During the time when monocytes dispper from the circultion, incresed monocyte dhesion ws oserved on therosclerotic lesions in crotid rteries. This indictes tht pltelet monocyte ggregtion is one of the wys in which circulting ctivted pltelets my prticipte in the formtion of therosclerotic lesions. Our study shows tht ctivted pltelets cn deliver the chemokines RANTES nd PF4 to endothelium nd leukocytes/monocytes. This is consistent with the oservtion tht pltelets present proinflmmtory meditors on their memrne surfce upon degrnultion 34. When direct interctions of pltelets with endothelium nd leukocytes re rogted, chemokine delivery is olished, ecuse proinflmmtory fctors secreted y ctivted pltelets my e rpidly diluted in the loodstrem. It is lso possile tht P-selectin enggement is required for chemokine relese. Alterntively, or in ddition, pltelet-derived sustnces my e ctively clered from circulting lood. For exmple, Duffy ntigen/receptor, promiscuous chemokine receptor expressed on the surfce of erythrocytes 35, inds CXC nd CC chemokines, including RANTES 36. Pltelet contct-medited deposition my lso e relevnt for the deposition of other pltelet-derived meditors contriuting to therosclerosis. Epiderml growth fctor, pltelet-derived growth fctor, β-thromogloulin nd products of the lipoxygense pthwy, oth mitogenic nd chemotctic 37, re cndidtes tht my induce monocyte recruitment nd/or smooth muscle cell prolifertion. Consistent with this ide, rogtion of pltelet interctions with endothelium nd monocytes/leukocytes y removl of pltelet P-selectin is prticulrly effective t delying the onset of therosclerotic disese 38 nd reducing neointim formtion fter vsculr injury (D.R. Mnk, mnuscript sumitted) in Apoe / mice. Deposited pltelet-derived meditors ctivte monocytes nd cuse monocyte recruitment. Consistent with previous in vitro studies 22,39, pltelet-derived meditors deposited on the endothelium cuse incresed monocyte rrest, process medited y monocyte integrin ctivtion induced y endothelium-ssocited proinflmmtory meditors. In this study, we show tht ctivtion of monocyte integrins cn lso e triggered y pltelet-derived meditors deposited on the monocyte surfce. An upregultion of VCAM-1 IgG inding to monocytes ws demonstrted, indicting incresed ffinity of VA-4 integrin for lignd In ddition, pltelet interction with monocytes my lso increse monocyte integrin vidity s result of clustering, which ws not mesured in our study. Other integrins 43 my lso prticipte in this pltelet-medited incresed monocyte rrest. Our dt provide the first direct evidence for n ctive contriution of circulting ctivted pltelets in the formtion of therosclerotic lesions. Pltelet P-selectin medited interctions led to deposition of pltelet-derived proinflmmtory fctors to the vessel wll nd monocytes, resulting in ctivtion of monocyte integrins, incresed monocyte recruitment nd excertion of therosclerosis. Prevention of pltelet ctivtion nd/or rogtion of pltelet interctions with leukocytes/monocytes nd the vessel wll, nd neutrliztion of pltelet-derived proinflmmtory fctors my ecome interesting mens for therpeutic or preventive interventions in therosclerosis. Methods Mice. Mle Apoe / mice nd Selp tm1by/tm1by mice were otined either from The Jckson ortory (Br Hror, Mine) or s gift from A. Beudet (Bylor University, Houston, Texs). Wild-type C57B/6 mice were from Hilltop Frms (Scottsdle, Pennsylvni). CX3CR1-EGFP mice were gift from D.R. ittmn (Howrd Hughes Medicl Institute) nd mintined s heterozygous reeding colony t the University of Virgini. Antiodies nd regents. The monoclonl ntiodies G1 (locking) nd S12 (non-locking) ginst humn P-selectin were provided y R. McEver (University of Oklhom, Oklhom). Thromin-receptor ctivting peptide (TRAP, SFRNP) ws otined from Peninsul ortories Inc. (Sn Crlos, Cliforni), recominnt I-1β from PeproTech, Inc. (Rocky Hill, New Jersey), nd humn thromin nd hirudin from Sigm Chemicl Chemicl (St. ouis, Missouri). Rt ntiodies ginst mouse CD11 (clone M1/7), mouse P-selectin (IgG1; RB4.34) nd control rt IgG1 were purchsed from PhrMingen (Sn Diego, Cliforni), polyclonl rit ntiodies ginst humn RANTES nd humn PF-4 from Snt Cruz Biotechnology (Snt Cruz, Cliforni), nd ntiodies ginst humn IgG NATURE MEDICINE VOUME 9 NUMBER 1 JANUARY 23 65

6 ARTICES conjugted PE nd rit ntiodies conjugted to Texs red from Vector ortories (Burlingme, Cliforni). Humn VCAM-1 IgG ws from R&D Systems Inc. (Minnepolis, Minnesot). Cell culture, pltelet isoltion nd ctivtion. Humn ortic endothelil cells (HAECs) (Clonetics, Sn Diego, Cliforni) nd humn monocytic Mono Mc 6 cells, provided y P.C. Weer (Munich, Germny) were cultured s descried 22. Humn nd mouse pltelets were isolted y gel-filtrtion 44. Pltelet ctivtion ws chieved y treting humn pltelets with TRAP for 1 minutes t 2 µm nd mouse pltelets with thromin for 15 min t. 5U/ml, followed y neutrliztion with equimolr dose of hirudin. Interctions of pltelets nd Mono Mc 6 cells with cultured endothelil cells in prllel plte flow chmer ssys. minr flow ssys were crried out s descried 22. Confluent HAECs grown in petri dishes (for monocyte rrest) or on glss coverslips (for immunostining) were ctivted with I-1β (1 ng/ml) for 12 h nd ssemled s the lower wll of flow chmer. Activted pltelets (1 8 pltelets/ml) were perfused t wll sher stress of 1.5 dyne/cm 2 for 2 min t 37 C. Mono Mc 6 cells (1 6 cells/ml) were perfused for 5 min. The interctions of clcein-leled pltelets nd Mono Mc 6 cells with endothelil cells were quntified in multiple fields. Immunostining ws crried out on fixed HAECs treted with n ntiody ginst RANTES or rit polyclonl PF-4 ntiody nd TRITC- or Texs red-conjugted secondry ntiody. Imges were recorded with fluorescence microscope ( 1 oil immersion ojective). Endothelil cells were detched mechniclly nd RANTES deposition ws mesured y flow cytometry. Flow cytometry. Whole lood drwn from mouse crotid rteries ws heprinized nd fixed with PFA t 1% for 6 min. Fixtion ws stopped nd red lood cells were lysed y Tris solution nd Tyrode s uffer. Smples were incuted with monoclonl ntiodies ginst Mc-1, Gr-1 or CD3 conjugted with PE for 3 min nd nlyzed y flow cytometry on FAC- Scn (Becton Dickinson; Plo Alto, Cliforni). Intrvitl microscopy of Apoe / mouse crotid rteries. Apoe / mice were nesthetized, followed y cnnultion of the trche nd right jugulr vein. The peri-dventitil tissues round the left crotid rteries were crefully seprted from the vessel. Most of the common crotid rtery, externl ifurction nd externl rnch were exposed nd left intct. Following perfusion of clcein AM leled pltelets, the interctions of pltelets nd pltelet leukocyte ggregtes with therosclerotic crotid rteries were oserved y intrvitl microscopy (Axioskop FS; Crl Zeiss, Thornwood, New York) with sline immersion ojective (SW 2,.5 numericl perture) nd strooscopic epifluoresence illumintion (6 s 1 ; Stroex, Chdwick- Helmuth, Mountin View, Cliforni). Rhodmine 6G (1 mg/m, Moleculr Proes, Inc., Eugene, Oregon) ws injected I.V. to lel leukocytes in vivo. Interctions etween leukocytes or pltelets nd endothelium lsting less thn 1 s were defined s tethering, more thn 1 s s rolling. eukocytes or pltelets not moving for more thn 3 s were defined s dhered. VCAM-1 IgG inding ssy. We suspended 1 6 MM6 cells with or without pertussis toxin (25 ng/ml for 3 h t 37 o C) or monoclonl ntiody HP1/2 (1 µg/ml for 2 min t 37 o C) tretment in 1 ml of whole lood (uffy cot removed) contining 2 µg VCAM-1-IgG nd plced them in 24-well pltes rotted t rte of 6 rpm. A volume of 5 µl (5 1 6 ) ctivted pltelet suspension ws dded to ech well for 3 min nd fixed y dding.5 ml of 4% prformldehyde t 22 C. Red lood cells were lysed with Tris:glycine solution (25 mm Tris, 5 mm glycine). Binding of VCAM-1 IgG ws detected with PE-conjugted got nti-humn IgG y flow cytometry. Deposition of pltelet-derived proinflmmtory fctors on therosclerotic endothelium. Aorts of Apoe / mice were hrvested nd fixed with 4% PFA/PBS. Immunostining ws crried out using primry ntiodies ginst RANTES, PF4 nd secondry got nti-rit conjugted with Texs red. Sytox green (Moleculr Proes, Eugene, Oregon) ws used to lel nuclei of endothelil cells. Imges of the endothelil cell monolyer were otined y using Bio-Rd MRC-124ES confocl microscope equipped with krypton/rgon lser nd numericl perture ojective (Nikon). Mesurement of therosclerotic lesion size of Apoe / mice. The orts of Apoe / mice were collected nd stined with oil red O s descried 45. Imges were scnned into Mcintosh computer nd the percent surfce res occupied y oil red O stined lesions were determined using imge nlysis softwre (NIH Imge). All niml experiments nd cre were pproved y the University of Virgini Animl Cre & Use Committee, in ccordnce with AAAAC guidelines. Sttisticl nlysis. Dt re represented s the men ± s.e.m. of t lest 4 independent experiments nd were compred using two-tiled Student s t-test. The null hypothesis ws rejected t P <.5. 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