Involvement of the Antimicrobial Peptide LL-37 inHuman Atherosclerosis

Kristina Edfeldt, Birgitta Agerberth, Martin E. Rottenberg, Gudmundur H. Gudmundsson,Xiong-Biao Wang, Kaushik Mandal, Qingbo Xu, Zhong-qun Yan

Objective—Antimicrobial peptides are effector molecules of the innate immune system. To understand the function ofvascular innate immunity in atherosclerosis, we investigated the role of LL-37, a cathelicidin antimicrobial peptide, inthe disease process.

Methods and Results—Using real-time polymerase chain reaction, we found a 6-fold increase in human cationic antimicrobialprotein 18/LL-37 transcript in human atherosclerotic lesions compared with normal arteries. Immunohistochemical analysisof atherosclerotic plaques showed that LL-37 was expressed mainly by macrophages and some endothelial cells. Western blotdemonstrated existence of active LL-37 peptide and abundant proprotein in atheroma specimens. To understand the functionalimplication of LL-37 production in atherosclerosis, the transcription profile was assessed in endothelial cells treated withLL-37. Our data show that LL-37 induces expression of the adhesion molecule intercellular adhesion molecule-1 and thechemokine monocyte chemoattractant protein 1 in endothelial cells. Intriguingly, Chlamydia pneumoniae withstood theantimicrobial activity of LL-37 in vitro, although inflammatory response was induced on infection.

Conclusion—LL-37 is produced in atherosclerotic lesions, where it may function as an immune modulator by activatingadhesion molecule and chemokine expression, thus enhancing innate immunity in atherosclerosis. (Arterioscler ThrombVasc Biol. 2006;26:1551-1557.)

Key Words: atherosclerosis � antimicrobial peptide � immune system � inflammation � infection

Antimicrobial peptides, identified by their ability to killbacteria, fungi, and certain viruses, are conserved in

species as diverse as plants, insects, and mammals and aremain components of the innate immune system.1 Catheli-cidins comprise a family of antimicrobial peptides charac-terized by a conserved cathelin-like domain and a variableC-terminal region with antimicrobial properties.2 Humancathelicidin or human cationic antimicrobial protein 18(hCAP18)/LL-37 is the only cathelicidin member identi-fied in humans so far. From hCAP18/LL-37, a 37-aapeptide, LL-37, is generated after proteolytic cleavage.3

hCAP18/LL-37 was initially isolated from neutrophils andsubsequently shown to be expressed by epithelial cellslining the most common entry sites of bacteria, namely theintestine, skin, and the airways.3–7 Recently, LL-37 hasbeen identified in inflammatory cells such as monocytes,macrophages, T cells, and mast cells, and its expression isinduced in some inflamed tissues.7–12 Intriguingly, some micro-organisms induce the expression of LL-37, whereas otherssuppress it, probably as an immune escape mechanism.13,14

LL-37 has been shown to be bactericidal against a wide variety

of microorganisms, including both Gram-positive and Gram-negative bacteria.15

See coverIn addition to antimicrobial effects, LL-37 has many other

biological functions. For instance, LL-37 is chemotactic for mastcells, neutrophils, monocytes, and T cells.8,16,17 It can inducemast cell degranulation and subsequent histamine release.18

LL-37 plays a role in wound healing by promoting neovascu-larization and re-epithelialization of healing skin.10,19 LL-37 alsoregulates differentiation and maturation of dendritic cells.20

Thus, there is mounting evidence that LL-37 constitutes animportant regulator and effector molecule of innate immunity.

Because LL-37 plays a role in host defense and ininflammation elicited by inflammatory diseases of the skinand airways such as psoriasis7,21 and sarcoidosis,22 and isupregulated in cutaneous injury10 and pulmonary infections,23

we hypothesized that this peptide might contribute to theinnate immunity involved in atherosclerosis.

Infection with the obligate intracellular Gram-negativebacterium Chlamydia pneumoniae is recognized as a major

Original received July 11, 2005; final version accepted April 3, 2006.From the Cardiovascular Research Unit, Center for Molecular Medicine, Department of Medicine, Karolinska University Hospital (K.E., Z.-q.Y.), the

Immunological Research Unit, Center for Molecular Medicine, Department of Medicine, Karolinska University Hospital (X.-B.W.), the Department ofMedical Biochemistry and Biophysics (B.A.), the Microbiology and Tumorbiology Centre (M.E.R.), Karolinska Institute, Stockholm, Sweden; theInstitute of Biology, University of Iceland, Reykjavik, Iceland (G.H.G.); the Department of Cardiothoracic Surgery, St. George’s Hospital and MedicalSchool, London, UK (K.M., Q.X.); and College of Pharmacy, Dalian Medical University, China (Z.-q.Y.)

Correspondence to Zhonq-qun Yan, Cardiovascular Research Unit, Center for Molecular Medicine, CMM L8:03, Karolinska University Hospital, 17176 Stockholm, Sweden. E-mail Zhong-qun.Yan@cmm.ki.se

© 2006 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000223901.08459.57

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cause of sinusitis, pharingitis, bronchitis, and pneumonia.Seroepidemiological studies indicate that C pneumoniae rep-resents by far the most prevalent chlamydial infection,affecting �50% of the population worldwide. Coronary heartdisease and myocardial infarction have been associated to Cpneumoniae infection by seroprevalence studies24,25 and bythe direct detection of the organism within atheroscleroticplaques26,27; nevertheless, precise effects of vascular innateimmune system against C pneumoniae infection are largelyunknown.

In this study, we show that LL-37 is expressed in humanatherosclerotic lesions and that it induces inflammatory re-sponses in human endothelial cells. C pneumoniae is notsensitive to LL-37 and can withstand high levels of theantimicrobial peptide.

MethodsDetailed Materials and Methods are provided in the expandedmethods, available online at http://atvb.ahajournals.org.

Specimen CollectionAtherosclerotic plaque samples were obtained from patients under-going carotid endarterectomy. Healthy vessels were represented byinternal mammary and renal arteries obtained from patients under-going coronary artery bypass surgery and nephrectomy, respectively.All human specimens were collected with the informed consent ofthe patients after permission by the local ethics committee wasgranted. In total, 25 atherosclerotic lesions (mRNA analysis n�13;protein detection n�12) and 19 healthy vessels (mRNA analysisn�12; protein detection n�7) were included in the present study.

Cell CultureHuman umbilical vein endothelial cells (HUVECs; Clonetics, Bio-Whittaker) maintained at 37°C, 5% CO2 in endothelial growthmedium-2 ([EGM-2] Clonetics; Bio Whittaker) were stimulated inEGM-2 with different concentrations of synthetic LL-37 for 6 hoursand 24 hours. Human monocytes were isolated from peripheral bloodfrom 2 donors, cultured in DMEM containing 10% FCS, 2 mmol/LL-glutamine, and 1 mmol/L sodium puryvate, and maintained at37°C, 5% CO2. Human macrophages were generated by stimulatingmonocytes with 500 U/mL granulocyte/macrophage colony-stimulating factor (GM-CSF; Peprotech EC) for 7 days.

C pneumoniae Infection and Infectivity AssayC pneumoniae isolate Kajaani 628 was used in the present study. Theinfectivity, as measured by inclusion forming units (IFU) of bacterialpreparation, was determined in HL cells (a human epithelial cell lineconventionally used in C pneumoniae cultivation). Aliquots ofbacteria were incubated with different concentrations of LL-37 forvarious times and diluted 10- to 200-fold before they were used toinfect overnight duplicate cultures of confluent HL cells. Alterna-tively, infected HL cells were coincubated with different concentra-tions of LL-37. Cells were incubated at 35°C for 72 hours in 5%CO2, thereafter stained with a fluorescein isothiocyanate–conjugatedChlamydia-specific monoclonal antibody (Bio-Rad), and IFU of Cpneumoniae were quantified by fluorescence microscopy.

C pneumoniae Infection of Human Monocytesand MacrophagesMonocytes and macrophage cultures were infected with C pneu-moniae by centrifugation for 1 hour, 500g at 35°C. A multiplicity ofinfection of 3 was used. At different time points after infection, cellswere washed with PBS and then lysed in RNA extraction buffer.

Real-Time RT-PCRTotal RNA isolated from human specimens and cells was reverse-transcribed to cDNA before real-time PCR using primers and probesfor human LL-37,29 intercellular adhesion molecule-1 (ICAM-1),monocyte chemoattractant protein 1 (MCP-1), cyclooxygenase 2,cyclophilin A, and GAPDH.

cDNA Array AnalysisA focused cDNA array (GEArray Q Series Human CardiovascularDisease Gene Array II; Atherosclerosis, SuperArray Inc.) was usedto analyze the effect of LL-37 on 96 atherosclerosis-related genes inhuman endothelial cells.

Immunohistochemistry and ImmunofluorescenceAcetone-fixed cryostat sections were preincubated for 30 minuteswith 5% normal serum. After incubation with primary antibody at4°C overnight, sections were incubated with biotinylated goat-antirabbit 7.5 �g/mL or horse anti-mouse IgG 7.5 �g/mL, followed byavidin-biotin peroxidase complex and developed with diaminoben-zidine (all from Vector Laboratories).

Double-staining was performed using immunofluorescence. Afterpreincubation with 5% normal serum in 50 mmol/L NH4Cl for 30minutes, sections were incubated with primary antibodies overnightat 4°C, followed by goat anti-rabbit biotin or goat anti-mouse TexasRed 10 �g/mL. Subsequently, 1 �g/mL Oregon green streptavidin(Molecular Probes) was incubated for 30 minutes at room tempera-ture when appropriate. 4,6-diamidino-2-phenylindole dihydrochlo-ride (Sigma) was used to stain cell nuclei in the tissue.

Western Blot AnalysisTissue homogenates or cell lysates were separated by SDS-PAGEusing 10% to 20% Tris-Tricine Ready Gels (Bio-Rad Laboratories)

Figure 1. Increased expression of LL-37 in atheroscleroticlesion. A, Real-time RT-PCR for human hCAP18/LL-37 tran-script in normal vessels (n�12) and in atherosclerotic lesions(n�13). Dot plots show hCAP18/LL-37 normalized to cyclophilinA (arbitrary units). The bar indicates the median value for eachgroup. ***P�0.0001 using nonparametric Mann–Whitney test. B,Immunohistochemical analysis of LL-37 protein expression innormal vessel (Ba) and atherosclerotic (Bb and Bc) lesions. Con-secutive section to (Bc) blocked with excess LL-37 (Bd). Browncells are positive for LL-37. Cell nuclei are stained blue withhematoxylin. Original magnification �100.

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for LL-37 and 7.5% Tris-HCl gel for ICAM-1, and transferred topolyvinylidene difluoride membranes as described previously.30

Immunoreactivity was detected using rabbit anti-human LL-37followed by goat anti-rabbit conjugated with horseradish peroxidase(DakoCytomation) or mouse anti-human ICAM-1 (Santa Cruz Bio-technology) followed by rabbit anti-mouse conjugated with horse-radish peroxidase (DakoCytomation).

Enzyme-Linked Immunosorbent AssayConcentrations of human MCP-1 were determined in supernatantsfrom HUVECs stimulated with LL-37 for 24 hours by DuoSetELISA (R & D Systems Inc.) following manufacturer instructions.

Statistical AnalysisResults are reported as mean�SEM using Student t test to assessdifferences between treatments in the in vitro cell culture experi-ments. The Mann–Whitney nonparametric test was used for com-paring mRNA levels of LL-37 in human specimens because the datadid not seem normally distributed. Differences were consideredsignificant at P�0.05.

ResultsHigh Levels of hCAP18/LL-37 mRNA Expressionin Human Atherosclerotic LesionsmRNA levels of hCAP18/LL-37 were detected in 12 normalarteries and 13 atherosclerotic lesions by real-time RT-PCR.Despite modestly elevated LL-37 mRNA detected in 2normal arterial samples, there was a 6-fold increase in themRNA expression in atherosclerotic specimens comparedwith the normal vessels (Figure 1A).

LL-37 Is Present in Inflammatory Cells WithinHuman Atherosclerotic LesionsCellular localization of LL-37 in lesions was investigated byimmunohistochemical staining using a peptide-specific anti-

body. LL-37 was detected in the intima of all atheroscleroticlesions examined (n�12). In contrast, few LL-37–positivecells could be found in the wall of normal vessels of internalmammary arteries (Figure 1Ba). By double-label immuno-fluorescence, LL-37 colocalized predominantly with cellsexpressing CD163 (macrophages) but only occasionally withcells expressing CD3 (T lymphocytes; Figure 2). Interest-ingly, LL-37 was also found in some endothelial cells in 4 of12 examined endarterectomy specimens (Figures 1B and 2).Furthermore, �-smooth muscle actin–positive cells stainedfor LL-37 neither in atherosclerotic nor in normal vessels(data not shown).

Both the Proform and Mature Form of LL-37Exist Inside Atherosclerotic LesionshCAP18/LL-37 is an 18-kDa protein stored as an inactiveproform. LL-37, the bioactive peptide, is cleaved from itsC-terminal segment by proteinase 3.31 To assess the presenceof processed LL-37 in atheroma, 6 lesions and 4 normalarteries were analyzed by Western blot using a specificantibody against LL-37.3 Both the active peptide LL-37 (4.5kDa) and the proprotein hCAP18 (18 kDa) were detected inall examined atheromatous specimens (Figure 3). ThehCAP18 proprotein was also found in 2 of 4 normal arteries,but only trace amounts of LL-37 were identified in 1 of 4 suchspecimens, the other being negative (Figure 3).

LL-37 Induces MCP-1 and ICAM-1 mRNAExpression in HUVECsThe proposed function of LL-37 as an inflammatory stimulusled us to investigate the gene expression profile of HUVECsstimulated with 2 �mol/L LL-37. The transcription profile 6

Figure 2. Cellular localization of LL-37 in athero-sclerotic lesion. Immunofluorescent staining forLL-37 (green) and cell markers (red) on athero-sclerotic lesions. Double-positive cells are yellowin the overlay panel. Cell nuclei are stained bluewith 4,6-diamidino-2-phenylindole dihydrochlo-ride (DAPI). Original magnification �200 and�400. CD163 (macrophages), von Willebrandfactor (vWF; endothelial cells) and CD3 (T cells).

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hours after LL-37 stimulation was screened using an athero-sclerosis-specific cDNA array containing 96 atherosclerosis-related genes. By comparing LL-37–stimulated HUVECswith untreated control cells, we found that LL-37 induced alimited set of inflammatory genes, including MCP-1,ICAM-1, and cyclooxygenase 2 (data not shown). To validatethese findings, expression of MCP-1 and ICAM-1 was furtheranalyzed by real-time RT-PCR. Stimulation of HUVECs withLL-37 for 24 hours resulted in a dose-dependent induction ofMCP-1 and ICAM-1 in HUVECs (Figures 4A and 5A).MCP-1 was also measured in culture supernatants of LL-37–stimulated endothelial cells by ELISA. Incubation of endo-thelial cells with LL-37 leads to a dose-dependent increase inMCP-1 secretion (Figure 4B). By means of Western blot, wecould demonstrate that LL-37 also induces ICAM-1 proteinin a dose-dependent manner (Figure 5B).

C pneumoniae Infection and Its Effect onLL-37 LevelsBy means of real-time RT-PCR, hCAP18/LL-37 mRNAexpression was studied in human peripheral blood mono-cytes. The present results suggest that primary monocytesexpressed very low levels of hCAP18/LL-37 in vitro. Incomparison, GM-CSF–differentiated macrophages displayeda 10- and 24-fold increase in level of the gene transcripts(Figure 6A). Certain microorganisms influence the expres-sion of hCAP18/LL-37, possibly as part of a virulencestrategy.14,32,33 Because C pneumoniae may be present in theatherosclerotic lesion, we investigated whether C pneumoniaeinfection affects the expression of the antimicrobial peptide.Assessment of hCAP18/LL-37 mRNA revealed a transientdownregulation in human monocytes and macrophages afterthe infection (Figure 6B and 6C).

C pneumoniae Is Resistant to LL-37The effect of LL-37 on the infectivity and replication of Cpneumoniae was determined by directly incubating the pep-tide with C pneumoniae for 2.5 hours and 24 hours. LL-37did not alter the infectivity of the bacteria in HL cells in thedoses between 2.5 and 40 �mol/L (data not shown). Toinvestigate whether LL-37 could influence the replicatingform of C pneumoniae, already infected cells were incubatedwith different doses of LL-37 for 72 hours. Our resultsdemonstrated no effect of LL-37 on C pneumoniae replica-tion by doses up to 10 �mol/L (data not shown). In controlexperiments, LL-37 exhibited strong antimicrobial activity

against Neisseria gonorrhoeae with a minimal bactericidalconcentration of 0.8 �mol/L.33

DiscussionThe present data show, for the first time, that LL-37, anantimicrobial cathelicidin, is produced in the human athero-sclerotic lesion. Cathelicidins are key components of innateimmunity, and LL-37 was found in infiltrating macrophagesas well as in some endothelial cells of the lesions, suggestingthat it is upregulated as part of the inflammatory process ofatherosclerosis. Both mRNA and protein for its proform, aswell as active LL-37 peptide, were detected in surgicalspecimens, indicating that it is produced by lesion cells.

Apart from its antimicrobial effect, LL-37 has also beenshown to induce the expression of some inflammatorygenes.34 In agreement with this, we show that LL-37 is aninducer of inflammation when acting on endothelial cells. Adose-dependent upregulation of ICAM-1 and MCP-1 tran-scripts was observed in endothelial cells after stimulationwith LL-37 for 24 hours. This tendency was confirmed forMCP-1 when determining protein levels in supernatants fromstimulated endothelial cells and for ICAM-1 in cell lysates byWestern blot analysis. The stimulatory effects of LL-37 onendothelial cells occur within the range of concentration (2 to15 �mol/L) detected in the bronchoalveolar lavage fluid frominfants with systemic or pulmonary inflammation.23 Our data

Figure 4. Expression of MCP-1 in endothelial cells on LL-37stimulation. A, Real-time RT-PCR for human MCP-1 in HUVECsstimulated with different doses of LL-37 for 24 hours. Data arepresented as mean�SEM of 3 independent experiments. Foldchange�MCP-1/cyclophilin ALL-37 stimulated/MCP-1/cyclophilinAcontrol in arbitrary units. *P�0.05 vs control; **P�0.01 vs control;***P�0.001 vs control using Student t test. B, Concentrations ofMCP-1 in supernatants from HUVECs stimulated with differentdoses of LL-37 for 24 hours measured by ELISA. Data are pre-sented as mean�SEM of 3 independent experiments. *P�0.05vs control;**P�0.01 vs control; ***P�0.001 vs control using Stu-dent t test.

Figure 3. Presence of both proform and active form of LL-37 inatherosclerosis. Western blot analysis with rabbit LL-37 anti-body on protein extracts from normal arteries (n�4) and athero-sclerotic lesions (n�4). Synthetic LL-37 (0.5 ng) with a molecularweight of 4.5 kDa was loaded as positive control. The band at18 kDa corresponds to the proform of LL-37, and bands at 14kDa and 6 kDa are LL-37 oligomers or LL-37 peptidecomplexes.

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suggest that LL-37 on its own is a modest inducer ofinflammation in endothelial cells. It is possible that LL-37, inconjunction with other inflammatory stimuli such as cyto-kines, may amplify the immune response of the diseasedvessel (eg, by inducing the expression of adhesion moleculesand chemokines). These results suggest that besides itsantimicrobial activities, LL-37 may act as an immune mod-ulator in atherosclerosis.

Infectious agents including C pneumoniae, herpes simplexvirus, cytomegalovirus, and Helicobacter pylori have beenassociated with cardiovascular disease.35,36 Despite somepatients with atherosclerosis being seropositive for H pylo-ri,37–39 this bacterium has not been isolated from any athero-sclerotic plaques. Given the presence of abundant LL-37 inatheroma lesions and its strong bactericidal effect against thisbacterium,40 it seems plausible that H pylori may not be ableto persist in atherosclerotic lesions. Unlike H pylori, Cpneumoniae has been isolated from lesions.41,42 Similar torecent findings,43 data of the present study show that LL-37did not alter either infectivity or replication of C pneumoniae.Thus, our finding may reveal a new paradigm of C pneu-moniae infection; by resistance to LL-37 (and perhaps otherinnate immune effectors), C pneumoniae has found a way toescape attack by innate immunity. Therefore, it may residepersistently in the atherosclerotic lesion.

LL-37 is primarily produced by macrophages in athero-sclerotic lesions. Whereas some bacteria influence the expres-

sion of LL-37,13,14,32,33,40 C pneumoniae infection does notinduce the gene in human monocytes and macrophages. Onthe other hand, expression of LL-37 is evidently upregulatedin the GM-CSF–primed human monocytes. Thus, cell differ-entiation is likely an essential mechanism regulating LL-37expression in macrophages.

LL-37 may also have an angiogenic role by acting directlyon endothelial cells to induce neovascularization.44 Neovas-cularization can promote atherosclerosis and lead to lesiongrowth through mechanisms that are not fully resolved butmight involve increased recruitment of leukocytes into thelesion.45 However, we did not see LL-37 staining in mi-crovessels of the atherosclerotic lesions, and it remainsuncertain whether LL-37 may contribute to new vesselformation at these sites.

The presence of LL-37 in human atherosclerotic lesionstogether with previous findings locating �-defensins in theplaque46 indicates that antimicrobial peptides may play a rolein the pathogenesis of the disease. It is possible that LL-37,either directly or by inducing cell surface adhesion molecules

Figure 5. LL-37 induces expression of ICAM-1 in endothelialcells. A, Real-time RT-PCR for human ICAM-1 in HUVECs stim-ulated with different doses of LL-37 for 24 hours. Data are pre-sented as mean�SEM of 3 independent experiments. Foldchange�ICAM-1/cyclophilin ALL-37 stimulated/ICAM-1/cyclophilinAcontrol in arbitrary units. *P�0.05 vs control; **P�0.01 vs control;***P�0.001 vs control using Student t test. B, Western blotanalysis with mouse ICAM-1 antibody on protein extracts fromHUVECs stimulated with different doses of LL-37 orinterleukin-1� (IL-1�; 50 ng/mL) for 24 hours. �-Actin was usedas loading control.

Figure 6. Induction of LL-37 expression in human monocytesand macrophages. A, Real-time RT-PCR for human hCAP18/LL-37 transcript normalized to cyclophilin A in human peripheralblood-derived monocytes and macrophages from 2 donors (bargraph shows pooled duplicates or triplicates from donors 1 and2, respectively). Real-time RT-PCR for human hCAP18/LL-37transcript normalized to cyclophilin A in human monocytes (B),or human macrophages from 2 donors infected with C pneu-moniae for 6, 24, and 48 hours (C). Fold change�hCAP18/LL-37/cyclophilin AC pneumoniae infected/ hCAP18/LL-37/cyclophilin Acontrol

in arbitrary units.

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and chemokines, promotes the recruitment of leukocytes tothe site of infection in the vessel wall, thus enhancing innateimmunity and modulating the local inflammatory response ofdiseased vessels.

Together, our data demonstrate that the human antimicro-bial peptide LL-37 is expressed in atherosclerotic lesionsmainly in macrophages but may also be expressed in endo-thelial cells and other immune cells such as T cells. LL-37functions as an immune modulator inducing expression ofinflammatory genes including adhesion molecules and chemo-kines in human endothelial cells. C pneumoniae, a bacteriumthat has been found in atherosclerotic plaques withstands theantimicrobial activity of LL-37. Our findings offer new insightsinto the function of innate immunity in atherosclerosis.

AcknowledgmentsThe authors thank Ingrid Tornberg and Monica Lindh for excellenttechnical assistance. We also thank Goran K. Hansson for review ofthis manuscript and helpful discussion.

Sources of FundingThis work was supported by grants from the Swedish ResearchCouncil (to B.A., M.E.R., and Z.-q.Y.), the Swedish Heart-LungFoundation, the AFA Foundation, the Swedish Foundation forInternational Cooperation in Research and Higher Education(STINT), the European Community QLK2-CT-2002-00846 grant,and Karolinska Institute.

DisclosuresNone.

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Edfeldt et al LL-37 in Atherosclerosis 1557

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Xiong-Biao Wang, Kaushik Mandal, Qingbo Xu and Zhong-qun YanKristina Edfeldt, Birgitta Agerberth, Martin E. Rottenberg, Gudmundur H. Gudmundsson,

Involvement of the Antimicrobial Peptide LL-37 in Human Atherosclerosis

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2006 American Heart Association, Inc. All rights reserved.

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doi: 10.1161/01.ATV.0000223901.08459.572006;26:1551-1557; originally published online April 27, 2006;Arterioscler Thromb Vasc Biol. 

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