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review article mechanisms of disease the hemostatic system as a modulator of atherosclerosis julian ilcheff borissoff m.d henri m.h spronk ph.d and hugo ten cate m.d ph.d from the laboratory for clinical thrombosis and hemostasis departments of internal medicine and biochemistry cardiovascular research institute of maastricht maastricht university medical center maastricht the netherlands address reprint requests to dr ten cate at the laboratory for clinical thrombosis and hemostasis departments of internal medicine and biochemistry cardiovascular research institute of maastricht maastricht university medical center universiteitsingel 50 p.o box 616 box 8 maastricht 6200 md the netherlands or at h.tencate@maastrichtuniversity.nl n engl j med 2011;364:1746-60 copyright © 2011 massachusetts medical society ardiovascular disease is one of the leading causes of death and complications worldwide the classic concept of atherosclerosis assigns a pivotal role to inflammation in the onset and progression of this disease.1,2 various inflammatory cell types e.g macrophages neutrophils and lymphocytes play crucial roles in the destabilization and subsequent rupture or erosion of an atherosclerotic plaque ultimately resulting in atherothrombosis.3 inflammation is closely linked to coagulation in several pathologic conditions.4 intriguingly extensive bidirectional cross-talk between the two systems has been established in many complex diseases,5,6 including atherosclerosis although there is no clinical evidence of a role for the hemostatic system in the progression of atherosclerosis ample experimental data indicate that platelets and the coagulation system are important determinants of both atherogenesis and atherothrombosis in numerous clinical trials the administration of antiplatelet or anticoagulant therapy has not been associated with attenuation or regression of plaque growth nevertheless the hemostatic system is well known for its capacity to exert a multitude of actions on the vasculature which may influence the molecular and cellular composition of the arterial wall and presumably of the atherosclerotic plaque this review covers recent advances in this field and discusses mechanisms of hemostasis as potential modulators of plaque phenotype c cross ta l k mech a nisms l ink ing the hemos tat ic s ys tem w i th atheroscl erosis hemostasis hemostasis is accomplished through a network of processes that include the platelet system coagulation and anticoagulant and fibrinolytic pathways which all support the dynamic equilibrium that provides proper blood flow.7,8 such processes evolved to maintain the blood in a fluid state under physiologic conditions and to arrest bleeding after vascular injury9-15 fig 1a and 1b disruption of this well-regulated balance leads to pathologic conditions such as thrombosis and bleeding molecular and cellular responses in the vasculature the targeting of genes that encode distinct hemostatic factors and their effect on arterial thrombosis in vivo has been extensively studied see table 1 in the supplementary appendix available with the full text of this article at nejm.org abundant experimental data suggest a role for various constituents of the platelet membrane and coagulation system in the regulation of atherosclerosis progression beyond their traditional hemostatic functions platelets are considered important in proinflammatory conditions such as atherosclerosis.16 in addition numerous coagula1746 n engl j med 364;18 nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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mechanisms of disease tion proteins have been implicated in processes such as the disruption of the endothelial barrier oxidative stress leukocyte recruitment inflammation migration and proliferation of vascular smooth-muscle cells vsmcs immune responses apoptosis of platelets and other cell types and angiogenesis.17,18 some of these actions mostly mediated by the complex of tissue factor and factor viia tf­fviia factor xa and thrombin involve the activation of g-protein­coupled protease-activated receptors pars 1 2 3 and 4 pars are widely distributed on vascular cells under normal conditions and are overexpressed during atherogenesis.19 platelets the cellular interface between hemostasis and atherosclerosis pioneering studies have documented a prominent role of platelets in experimental studies of atherogenesis.20,21 platelets exert a plethora of proatherogenic activities and create an interface between hemostasis innate immunity and inflammation in atherosclerosis.16 a systemic inflammatory environment independent of vesselwall injury induces a phenotypic switch to a proatherogenic endothelium this results in enhanced expression of cell-adhesion molecules such as p-selectin and e-selectin the primary adhesion of platelets on a compromised vascular endothelial surface is accomplished through the binding of platelet glycoprotein ib receptors to von willebrand factor whereas firm adhesion is mediated through 3 integrins once adherent platelets also secrete atherogenic mediators such as cytokines chemokines growth factors adhesion molecules and coagulation factors the upregulation of p-selectin expression on the surfaces of both platelets and endothelial cells potentiates the interactions with p-selectin glycoprotein ligand 1 which is expressed on leukocyte membranes the binding between platelets and circulating leukocytes monocytes and neutrophils dendritic cells and progenitor cells produces coaggregates that support further leukocyte activation adhesion and transmigration processes considered to be critical for plaque formation and progression22-29 fig 2 gests an active cell-based coagulation network within human atherosclerotic lesions the role of these coagulation proteins in atherogenesis is indicated by increased thrombin-generating activity in early atherosclerotic lesions as compared with that in stable advanced lesions.30 these findings are supported by experimental data31 and a clinical study showing that increased plaque echogenicity more fibrous structure rather than plaque echolucency lipid-rich higher content of inflammatory cells and thinner fibrous caps is associated with thrombin generation in plasma from patients with carotid-artery stenosis.32 the abundance of coagulation factors within early atherosclerotic vessels and local generation of thrombin or fibrin may be attributable to primary protective mechanisms against vascular injury however the persistent inflammatory environment within the arterial wall supported in part by coagulation-mediated actions may maintain local thrombin generation which will eventually turn into a vicious cycle contributing to the formation of intraplaque thrombi33,34 and thus ultimately leading to plaque instability t issue fac t or e x t r insic path wa y tissue factor is a transmembrane class ii cytokine receptor which is considered the primary physiologic trigger of the coagulation cascade.8 tissue factor is also physiologically essential for vascular development in mice tissue factor deficiency is associated with a high rate of embryonic death and impaired vascular integrity tissue factor is differentially distributed among the various cell types of the vessel wall under physiologic conditions in normal blood vessels the inner endothelial lining does not express tissue factor whereas the surrounding layers consisting of vsmcs adventitial fibroblasts and pericytes show abundant synthesis of tissue factor this specific vascular localization of tissue factor is generally attributed to its role in the prevention of bleeding after injury also referred to as a hemostatic envelope.35 within the atherosclerotic lesion tissue factor is predominantly localized on macrophages vsmcs and foam-cell­derived debris within the coagulation system during atherosclerotic necrotic core.30,36-38 tissue factor activity is sigplaque progression nificantly higher in lesions obtained from pawe have found a local synthesis of several func tients with unstable angina or myocardial intionally active coagulation proteins which sug farction than in those from patients with a stable 1747 n engl j med 364;18 nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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the newe ng landjour na l of m e dic i n e figure 1 facing page platelets and coagulation factors in the regulation of thrombus formation panel a shows platelet adhesion and aggregation in which atherothrombosis begins with an endothelial injury or rupture of an atherosclerotic plaque this process triggers transient neurohumoral vasoconstrictor mechanisms which are reinforced by the release of endothelium-derived factors such as endothelin the platelet membrane receptors glycoprotein ib/ix/v and glycoprotein vi elicit platelet tethering to the exposed thrombogenic subendothelial proteins von willebrand factor and collagen in addition glycoprotein vi generates intracellular signals to mediate platelet adhesion and aggregation through the activation of integrin receptors such as glycoprotein ia/iia and glycoprotein iib/iiia with the latter also serving as a receptor for fibrinogen these molecular events ultimately contribute to the formation of the primary hemostatic plug.10 panel b shows the tissue factor extrinsic pathway in which tissue factor the major trigger of coagulation is exposed at the site of plaque erosion or rupture tissue factor forms a catalytic complex with factor viia that leads to the subsequent activation of factors ix and x in a so-called prothrombinase complex activated factor x together with activated factor v promotes a downstream enzymatic cleavage of prothrombin which yields small amounts of thrombin.11 thrombin is a pleiotropic central coagulation enzyme12 that not only converts fibrinogen into fibrin but also has a substantial role in the activation of platelets and activates factor xiii to induce fibrin polymerization a fundamental process for the formation of a stable clot or thrombus furthermore by supporting positive-feedback activation of the upstream factors v viii and xi thrombin plays a crucial part in the amplification and propagation phases of coagulation the activated platelet surface is also a critical catalyst for the coagulation cascade platelets actively participate in the clotting process by introducing extra amounts of tissue factor factor v fibrinogen and factor xiii into the system derived from various local sources fibrinogen and factors v and xiii stored in granules 13 and facilitating the direct activation of factor xi by thrombin and the subsequent activation of factor ix on the platelet surface factor ixa forms the so-called tenase complex together with factor viiia thereby igniting a burst of additional thrombin generation which is essential in forming sufficient fibrin and sealing the defect panel c shows the contact activation intrinsic pathway which is not considered to be essential for protection against bleeding in vivo even though its components may be involved in the pathogenesis of arterial thrombosis.14 the exposure of plasma prekallikrein high-molecular-weight kininogen and factors xi and xii to anionic surfaces15 results in the conversion of prekallikrein to kallikrein which activates factor xii into factor xiia but also cleaves high-molecular-weight kininogen leading to the release of the inflammatory mediator and vasodilator bradykinin factor xiia activates factor xi and favors the conversion of more prekallikrein to kallikrein thereby reciprocally amplifying the cascade this sequence of proteolytic reactions leads to the activation of factor ix which ultimately cleaves factor x into its active form and culminates in the convergence of both coagulation pathways gray circles indicate the inactive form of a coagulation protein and green circles indicate the active form form of cardiovascular disease,39-41 suggesting a role of this coagulation protein in plaque thrombogenicity factor vii is also extrahepatically expressed within both normal and atherosclerotic vessels and colocalizes with tissue factor on macrophages and vsmcs.30 apart from its coagulation properties the tf­fviia complex is multifunctional with a capacity to promote cell signaling gene transcription and subsequent protein synthesis par-2 activation is essential in the mediation of tf­fviia­induced signaling the latter may engage several proatherogenic processes such as monocyte and fibroblast chemotaxis inflammation vsmc migration and proliferation vascular remodeling angiogenesis contributing to plaque destabilization induction of oxidative stress in macrophages and apoptosis42 fig 3 surprisingly reduced vascular expression of tissue factor does not affect atherosclerosis progression in transgenic mice.43 there are few clinical data regarding the role of tf­fviia on atherosclerosis progression levels of plasma tissue factor antigen modulated by known polymorphisms of the tissue factor gene are positively associated with both an increased risk of death from cardiovascular causes44 and an increased carotid intima­media thickness,45 which is considered a marker of subclinical atherosclerosis a similar relation between factor vii and increased intima­media thickness has been documented both in healthy young adults and in patients with peripheral arterial disease.46,47 c om mon c oagul at ion path wa y pleiotropic factor x a once activated factor xa initiates intracellular signaling in various cell types of the cardiovascular system preferentially mediated by par-2 or when in ternary complex with tf­fviia through both par-1 and par-2.17 par-1 par-2 or both are present in abundance on endothelial cells leuko 1748 n engl j med 364;18 nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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mechanisms of disease a c xi xiia prekallikrein kallikrein glycoprotein iib iiia fibrinogen glycoprotein ib ix v xia xii anionic surfaces platelet glycoprotein vi high-molecularweight kininogen von willebrand factor collagen bradykinin b prothrombinase complex x xa va prothrombin stable clot xiii xiiia fibrin vii tissue factor viia tissue factor positive thrombin feedback ix ixa v thrombin va xa fibrinogen thrombin viii thrombin tenase complex viiia ixa prothrombin x xi additional amount of factor provided by platelets thrombin xiia ix xia viia color figure cytes vsmcs fibroblasts and dendritic cells factor xa­dependent par-mediated signaling contributes to the production of proinflammatory cytokines including interleukin-6 interleukin-8 and chemokine c-c motif ligand 2 ccl2 and to the expression of cell-adhesion molecules including e-selectin intracellular adhesion molecule 1 icam-1 and vascular-cell adhesion molecule 1 vcam-1 along with tissue factor up-regulation vsmc proliferation and the release of growth factors vascular endothelial growth factor plateletderived growth factor and transforming growth factor 17 all these may contribute to the progression of atherosclerotic plaque involving in draft 4 flammation leukocyte transmigration 4/11/11 restenoauthor sis and angiogenesis fig borissoff vascular 3 1of note fig remodeling and neointimal formation were title reduced on targeted delivery of nonspecific facme de longo tor xa inhibitors heparin and low-molecularartist knoper weight heparins coupled to author please note antian antifibrin 48 figure has been redrawn and type has been reset body please check carefully issue date 5/05/11 thrombin thrombin is a unique serine protease that is pivotal to coagulation and that may also display various actions toward other systems e.g immune nervous gastrointestinal and musculoskeletal systems governed by the interaction and proteo n engl j med 364;18 nejm.org may 5 2011 1749 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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the newe ng landjour na l of m e dic i n e homeostasis resting platelets adp thrombin plasmin platelet activating factor thromboxane a2 epinephrine serotonin atp adenosine adp cd39 cd73 healthy endothelium nitric oxide prostacyclin shear stress imbalance platelet activation hemoglobin­haptoglobin complex clearance further activation cd163 platelet factor 4 monocyte-tomacrophage differentiation collagen impaired endothelium atherosclerotic plaque development adhesion to endothelial cells and monocytes p-selectin leukocyte recruitment adam15 ccl2 and 3 p-selectin icam-1 vcam-1 inflammation interleukin-1 platelet factor 4 neutrophil-activating peptide 2 rantes cd40l tnf interleukin-8 platelet monocyte differentiation to foam cells platelet factor 4 thrombosis tissue factor plaque destabilization matrix metalloproteinases figure 2 platelets in atherogenesis color figure draft 6 intact endothelium normally expresses cd39 ecto-atpase and cd73 ecto-5-nucleotidase which act in tandem to induce the 4/19/11 breakdown of the prothrombotic adenosine 5-triphosphate atp and adenosine diphosphate adp into the largelyauthor borissoff antiinflammatory ade2 fig nosine thus preventing platelet activation and aggregation healthy endothelium also secretes vasodilators such as prostacyclin and nititle tric oxide which have potent antiadhesive and antiaggregating effects at the time of activation platelets undergo a substantial change me in shape and promptly release a variety of autocrine and paracrine mediators such as adp epinephrine and thromboxane a 2 studies de longo investigating how platelets orchestrate these widely differing atherogenic actions have provided an increased understanding of the artist knoper mechanisms involved much attention has focused on cytokine-like and chemokine systems such as the cd40­cd40l dyad ccl5 author please note figure has been redrawn and the been reset rantes and platelet factor 4.23,24 platelet factor 4 supports monocyte differentiation into macrophages and down-regulatestype has atheplease check carefully roprotective receptor cd163 which accounts for the clearance of hemoglobin­haptoglobin complexes transgenicdate 5/05/11 mice lacking platelet issue factor 4 have diminished progression of atherosclerosis furthermore cd40 and its ligand cd40l which belongs to the superfamily of tumor necrosis factor receptor and ligand is widely expressed in the vessel wall e.g in endothelial cells vascular smooth-muscle cells and fibroblasts and several immune constituents monocytes or macrophages neutrophils mast cells t and b cells and dendritic cells 25 the complex array of proinflammatory immune-modulating effects and prothrombotic features26 assert an integral role for cd40­cd40l in atherogenesis overall these findings support the hypothesis that platelets are important proinflammatory players that elicit multifaceted cellular interactions and are directly involved in the early development of atherosclerotic lesions platelets are primary mediators in both adaptive and innate immunity.27 hence the targeting of platelet chemokines appears to be therapeutically unsuitable in the context of atherosclerosis because of the severe impairment of multiple systemic immune responses which may also result in carcinogenesis.28,29 adam15 denotes adam metallopeptidase domain­containing protein 15 ccl2/3 chemokine c-c motif ligand 2/3 icam-1 intercellular cell-adhesion molecule 1 tnf tumor necrosis factor and vcam-1 vascular-cell adhesion molecule 1 lytic activation of its direct cellular targets par-1 3 and 4 49,50 thrombin is entwined with the regulation of vascular physiology and pathophysiology51 fig 3 thrombin is an example of a multifaceted molecule with broad physiologic properties by binding to thrombomodulin throm1750 n engl j med 364;18 bin favors the transformation of protein c into activated protein c a potent anticoagulant and antiinflammatory molecule moreover thrombin can diminish the release of interleukin-12 and promote the up-regulation of interleukin-10 in monocytes thus inducing immunosuppressive nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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mechanisms of disease x vii tissue factor viia tissue factor xa prothrombin fibrinogen endothelial permeability monocyte transmigration ldl accumulation platelet reactivity inflammation fibrinogen thrombin indicates activation of corresponding protease-activated receptor below fibrin protease-activated receptor 1 endothelial cells leukocytes vascular smoothmuscle cells fibroblasts dendritic cells platelets protease-activated receptor 2 endothelial cells leukocytes vascular smoothmuscle cells dendritic cells protease-activated receptor 3 endothelial cells fibroblasts dendritic cells protease-activated receptor 4 endothelial cells leukocytes vascular smoothmuscle cells platelets proatherogenic cellular responses endothelial dysfunction endothelial permeability platelet activation monocyte recruitment vascular remodeling stable plaque angiogenesis oxidative stress apoptosis proteolysis inflammation unstable plaque figure 3 nonhemostatic actions triggered by the tissue factor and common activation pathways in the phenotypic modulation of the arterial wall color figure thrombin factor xa and the tissue factor­factor viia complex can activate protease-activated receptors which are draft 4 4/04/11 widely expressed on endothelial cells leukocytes vascular smooth-muscle cells fibroblasts dendritic cells and author borissoff platelets resulting in a plethora of proatherogenic actions gray circles indicate the inactive form of a coagulation 3 fig protein and green circles indicate the active form ldl denotes low-density lipoprotein title me and antiinflammatory actions thrombin may also play a role in normal vasomotor regulation.18 the endothelial decay of thrombomodulin during atherogenesis may allow thrombin to potentiate atherogenic processes such as endothelial dysfunction and barrier disruption oxidative stress apoptosis inflammation overexpression of cytokines or chemokines activation of platelets and leukocytes leukocyte recruitment migration and proliferation of vsmcs and angio longo genesis which suggests an important role in the artist knoper author please note pathogenesis of cardiovascular disease.18 thromfigure has been redrawn and type has been reset bin factor xa factor xia factorplease check carefully plasixa and issue date 5/05/11 min also show enzymatic activity for cleavage of complement proteins c3 and c5 into their active forms.52 proteins c3 and c5 are known to induce inflammation and chemotaxis of inflammatory cells human coronary atherosclerotic lesions overexpress anaphylatoxin receptors c3ar and c5ar as compared with healthy vessels de n engl j med 364;18 nejm.org may 5 2011 1751 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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the newe ng landjour na l of m e dic i n e primarily localized on macrophages but also on endothelial cells intimal vsmcs t cells and mast cells overall these data establish a new interface between coagulation and inflammation in atherosclerosis the administration of thrombin-specific inhibitors reduces restenosis in rabbits with atherosclerosis after angioplasty.53,54 another piece of evidence for the in vivo relevance of these effects comes from a study showing that the direct thrombin inhibitor melagatran reduces atherosclerosis progression in apolipoprotein e­knockout mice and promotes plaque stability by inhibiting proinflammatory transcription factors and attenuating the synthesis of matrix metalloproteinases.55 furthermore mice with combined deficiency of factor viii and apolipoprotein e had significantly less development of atherosclerotic lesions than control mice despite having more pronounced hyperlipidemia.56 in contrast hypercoagulability has been linked with atherosclerosis progression in murine studies showing that homozygosity for factor v leiden a known prothrombotic mutation promotes atherogenesis.57 however a recent study showed an increase in the size of atherosclerotic plaques in procoagulant mice indicating that a hypercoagulable state contributes to a more stable plaque phenotype.31 overall these findings suggest that hemostasis exerts various effects on the vasculature and by the action of distinct regulators may ultimately contribute to determining the plaque phenotype the clinical evidence in this regard remains inconsistent despite the fact that prothrombotic genetic variants have not been consistently linked to the progression of cardiovascular disease in patients,44 clinical data show a positive association between markers of thrombin generation and the atherosclerotic plaque burden.58,59 low levels of factor viii have not shown atheroprotective effects in patients with hemophilia,44 whereas there is clinical evidence that elevated levels of factor viii promote cardiovascular disease.60 in plasma factor viii circulates in a complex with von willebrand factor which modulates factor viii activity in the circulation since mice that are deficient in von willebrand factor have significantly fewer atherosclerotic plaques than control mice von willebrand factor may also play a role in atherosclerosis.61 like the data regarding factor viii and other coagulation proteases clinical data on the association between von wille1752 n engl j med 364;18 brand factor and cardiovascular disease have been inconsistent.44,60 more experimental and clinical data are needed to clarify these relationships fibrinogen fibrin and factor xiii in clinical studies there have been strong associations between increased plasma fibrinogen levels and the risk of cardiovascular disease which suggests hyperfibrinogenemia as an independent predictor of vascular events.62 furthermore the distribution of fibrinogen and fibrin degradation products in atherosclerotic lesions during progression has been clearly documented.63,64 elevated levels of plasma fibrinogen a major determinant of the amount of thrombin that is formed,65 are closely related to an enhanced rate of coronary-artery calcification and increased intima­media thickness both measures of premature atherosclerosis.66 from a cellular and molecular perspective fibrinogen may affect the plaque phenotype through several distinct mechanisms favoring the permeability of endothelial cells extracellular accumulation of low-density lipoprotein ldl cholesterol and the formation of foam cells inducing the migration of monocytes and vsmcs increasing platelet reactivity or aggregation and enhancing inflammation67 fig 3 studies in animals have shown distinct results on the role of fibrinogen in atherosclerosis with some studies indicating that fibrinogen deficiency in transgenic mice is associated with accelerated atherogenesis in a thrombin-dependent manner,68 and others showing that fibrinogen deficiency is not a prerequisite for the development of advanced atherosclerotic plaque.69 increased plasma levels of d-dimer fragments are also associated with enhanced inflammation and an increased incidence of cardiovascular disease and are considered a biomarker of atherothrombosis.70 however the effect of fibrin degradation products on the vascular-wall phenotype is less clear although the results of one study suggested that d-dimers promote a proatherogenic phenotype in human monocytes,71 other studies have shown that both fragments d and e may prevent the proliferation of vsmcs in vitro.72 finally blood coagulation factor xiii may also be related to atherogenesis factor xiii not only cross-links fibrin chains to fibrin on activation which contributes to clot stability but also appears to facilitate the formation of hyperactive dimers of angiotensin ii type 1 receptor thus nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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mechanisms of disease leading to chronic sensitization of circulating monocytes and exacerbating atherosclerosis.73 c on tac t ac t i vat ion in t r insic path wa y the contact activation pathway is considered nonessential for hemostasis in vivo fig 1c and fig 4 however it may be involved in the pathogenesis of arterial thrombosis.14 although experimental data have clearly shown that mice deficient in factor xii are protected against arterial thrombosis and stroke,14 in several epidemiologic studies data on the association between factor xii and the risk of cardiovascular disease in humans are inconsistent.74-76 although additional research is needed in this field the pharmacologic inhibition of factor xii activation represents a potential therapeutic target,77,78 considering that hereditary deficiency of factor xii is not associated with bleeding disorders or other pathologic conditions at a molecular level factor xii influences distinct processes mostly through the plasma kallikrein­kinin system.79 factor xii­mediated bradykinin formation not only regulates vasodilatation and vascular permeability but also induces activation of the complement and fibrinolytic systems by activating components c3 and c5 and facilitating the synthesis of tissue-type plasminogen activator from endothelial cells whereas kallikrein activates urokinase-type plasminogen activator and plasminogen platelet-derived inorganic polyphosphates80 and misfolded proteins which are found abundantly in atherosclerotic arteries,81 can also activate factor xii leading to kallikrein formation without triggering coagulation.82 levels of tissue kallikrein and plasma prekallikrein are associated with the severity of cardiovascular disease83,84 and have been found to be critical in the process of vascular repair.85 given the proangiogenic and proinflammatory nature of factor xii86 and the plasma kallikrein­ kinin system chronic stimulation of these responses may promote a proatherogenic intraarterial environment over time misfolded proteins platelet polyphosphates anionic surfaces complement activation epidermal growth factor receptor urokinase receptor xii xiia angiogenesis prekallikrein kallikrein complement activation high-molecularweight kininogen bradykinin vascular permeability inflammation color figure 4 contact activation pathway and its proinflammatory figure and proangiogenic properties draft 3 4/04/11 author borissoff the contact system plays a role in various physiologic processes such as 4 fig blood-pressure regulation coagulation fibrinolysis angiogenesis and intitle flammation it consists of factor xii prekallikrein and high-molecular-weight kininogen the activation of the proinflammatoryme kallikrein­kinin and comde longo plement systems is triggered by the proteolytic cleavage of factor xii autoartist knoper activation in reaction to contact with negatively charged artificial or bioauthor please note figure has been redrawn and type has been reset logic surfaces the gray circle indicates the inactive form of acheck carefully coagulation please protein and green circles indicate the active form green5/05/11 with plus circles issue date signs indicate either positive-feedback reactions or induction of a process fig 5a although tfpi is expressed on endothelial cells vsmcs and macrophages in the fibrous cap and shoulder areas of the plaques it also colocalizes with tissue factor and attenuates its activity within atherosclerotic lesions.30,96,97 this finding suggests a role for tfpi not only in the regulation of tissue factor procoagulant aca n t ic oagul a n t path wa ys tivity but also in the control of tissue factor­ in va scul a r infl a m m at ion induced proatherogenic signaling the administissue factor pathway inhibitor tfpi which is tration of recombinant tfpi has reduced the rates widely distributed in healthy arterial vessels tends of inflammation and death in an animal model to be overexpressed in atherosclerotic lesions87 by decreasing the expression of tumor necrosis n engl j med 364;18 nejm.org may 5 2011 1753 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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the newe ng landjour na l of m e dic i n e figure 5 facing page anticoagulant pathways and their nonhemostatic features the regulation of coagulation operates at three levels inhibition of thrombin factor xa and factor ixa by antithrombin inhibition of factor xa the tissue factor­factor viia tf­fviia complex and hence thrombin formation by tissue factor pathway inhibitor and proteolytic inactivation of factor v and factor viii by activated protein c as shown in panel a antithrombin is a serine protease that inhibits key coagulation enzymes such as thrombin factor xa and factor ixa its action is amplified by as much as 4000 times in the presence of heparin or heparin-like substances such as heparan sulfate proteoglycan antithrombin has apparent antiinflammatory effects,88 as seen in an increase in the release of prostacyclin and a decrease in nuclear factor b signaling which is known to have multiple proinflammatory responses similar effects have been found after the administration of synthetic direct thrombin inhibitors which has contributed to plaque stability in vivo.55 antithrombin attenuates leukocyte recruitment during inflammation which hints at another potential atheroprotective role heparin also stimulates the release from endothelial cells of tissue factor pathway inhibitor which then binds to factor xa and the tf­fviia complex to form an inactive quaternary complex thus showing a multitude of antiatherogenic functions like antithrombin heparin cofactor ii has the ability to inactivate thrombin factor xa and factor ixa whereas the plasma form of heparin cofactor ii is an inefficient inhibitor in the absence of glycosaminoglycans e.g heparan sulfate and dermatan sulfate heparin cofactor ii is implicated both in vascular remodeling and in atherogenesis mice that are deficient in heparin cofactor ii have enhanced intimal hyperplasia after vascular injury 89 such mice have increased neointima formation and enhanced atherogenesis as compared with control mice however the findings in clinical studies have been inconsistent with some indicating that heparin cofactor ii is a strong predictive marker against atherosclerosis90,91 and one indicating that its presence is not predictive.92 protein z is a cofactor of another protein named protein z­related protease inhibitor which inhibits factor xa and factor xia in the coagulation cascade although the roles of protein z and protein z­related protease inhibitor in inflammation and the onset of atherosclerosis are poorly understood a few clinical trials have shown a significant inverse relationship between levels of these proteins and the clinical severity of atherosclerosis.93-95 as shown in panel b thrombin also behaves as an anticoagulant molecule physiologically binding to the endothelial protein c receptor protein c is transformed into activated protein c by an activation complex established between thrombin and thrombomodulin this process is followed by dissociation of activated protein c from the endothelial protein c receptor and the formation of a complex between activated protein c and protein s the latter allows the inactivation of factor va and factor viiia and thus limits further thrombin generation gray circles indicate the inactive form of a coagulation protein and green circles indicate the active form factor tnf chemokines and myeloperoxidase.88 moreover tfpi is a potent inhibitor of matrix metalloproteinases which are considered key players in plaque destabilization and atherothrombotic complications decreased tfpi expression has been associated with up-regulation of the synthesis of matrix metalloproteinases in plaques with a vulnerable phenotype in addition tfpi has inhibited endothelial migration and angiogenesis in mice several studies in animals have shown that tfpi attenuates neointimal hyperplasia and stenosis but also suppresses the release of proatherogenic platelet-derived growth factor bb ccl2 and matrix metalloproteinase 2.98-101 in agreement with these findings tfpi-deficient mice have significantly more atherosclerotic plaques than control mice,102 whereas vasculardirected tfpi overexpression appears to regulate lipoprotein clearance and temporarily lowers plasma cholesterol levels also reducing atherosclerotic plaque development.103 clinical data suggest that plasma tfpi is a marker of endothelial dysfunction high levels of both free and total tfpi levels are associated with an increased atherosclerotic burden and coronary-artery calcifica1754 tion,104,105 whereas low levels of total tfpi are associated with an increased risk of atherothrombosis.106,107 in addition to its anticoagulant properties the protein c pathway is known for its protective effects on vascular gene-expression profiles involving antiapoptotic and antiinflammatory responses as well as its stabilizing effect on the endothelial barrier fig 5b 108 studies of atherosclerosis have shown a substantial downregulation of the local expression of endothelial protein c receptor and thrombomodulin within atherosclerotic vessels suggesting impaired activation of protein c and hence a reduced antiatherogenic response several mechanisms such as enhanced shedding of thrombomodulin from dysfunctional endothelium an abundance of ldlcholesterol deposits and local inflammation within the arterial wall may account for the attenuation of the anticoagulant activities of protein c within the atherosclerotic plaque overexpression of thrombomodulin has been shown to limit neointimal formation in rabbits,109 whereas a genetic impairment of the protein c­activating cofactor function of thrombomodulin resulting n engl j med 364;18 nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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mechanisms of disease a xii xiia tissue factor pathway inhibitor thrombin x heparin stimulates release from endothelial cells viii angiogenesis vascular remodeling tissue factor viia xa tissue factor inactive quaternary complex inflammation vii lipoprotein clearance antithrombin action is amplified 4000 times in the presence of heparin or heparin-like substances e.g heparan sulfate or dermatan sulfate inflammation v xiii thrombin heparin cofactor ii plasma form requires glycosaminoglycans e.g heparan sulfate or dermatan sulfate to function as an efficient inhibitor vascular remodeling xi protein z­related protease inhibitor and protein z ix xia viiia ixa antithrombin and heparin cofactor ii tissue factor pathway inhibitor thrombin protein z­related protease inhibitor and protein z prothrombin va antithrombin and heparin cofactor ii fibrinogen fibrin xiiia b thrombin thrombomodulin protein c activated protein c antiinflammatory activity antiapoptotic activity gene-expression regulation endothelial barrier protection activated protein c endothelial protein receptor c protein s inactivation of va and viiia thrombin generation vascular endothelium color figure in diminished formation of activated protein c is associated with an increased atherosclerotic burden in mice.31 the determinants of soluble levels of thrombomodulin in patients with atherosclerosis are poorly understood the results of various clinical studies that have examined the relationship between thrombomodulin and the extent of atherosclerotic burden have been inconsistent.110-114 in monkeys progressive atherosclerosis is associated with impaired formation of activated protein c whereas dietary regression of atherosclerosis was found to enhance the anticoagulant response.115 mice with a heterozygous deficiency in protein c have enhanced focal arterial inflammation and thrombosis leading to increased neointima formation and localized thrombosis.116 in agreement with these findings several clinin engl j med 364;18 cal studies have confirmed a significant associaauthor borissoff 5 fig tion between circulating low levels of activated title protein c and a greater extent or severity of athme erosclerosis.117-119 de longo furthermore protein s which has been deartist knoper author please scribed as linking hemostasis inflammation andbeen redrawn andnote been reset figure has type has apoptosis forms a complex with the complement please check carefully issue date 5/05/11 system regulator c4b-binding protein c4bp a major inhibitor of the classical complement pathway localizing it on the surface of apoptotic cells120 and thus promoting phagocytic activity by macrophages.121 intriguingly protein s significantly inhibits the expression of macrophage scavenger receptor a and diminishes the uptake of acetylated ldl cholesterol mediated by this receptor resulting in a decreased intracellular lipid content in macrophages.122 these actions are mostly attributable to the ability of protein s to nejm.org may 5 2011 draft 6 4/19/11 1755 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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the newe ng landjour na l of m e dic i n e bind to and induce phosphorylation of the mer receptor tyrosine kinase in addition protein s plays a role in the protection of the integrity of the blood­brain barrier.123 the expression of protein s is reduced within atherosclerotic plaques obtained from patients with unstable angina as compared with specimens from patients with stable angina.124 hereditary deficiency of both proteins c and s has been associated with an increased incidence in arterial thromboembolic events125 and peripheral-artery disease fig 5b 89-95,126 f u t ur e per spec tives hemostasis is anatomically and functionally entwined with the vasculature besides its essential roles in protecting vascular integrity and maintaining normal blood flow accumulating data suggest an intimate cross-talk between hemostasis and inflammation underscoring the role of both systems in many complex diseases including atherothrombosis intriguingly numerous studies in animals have also documented that hemostasis is closely linked to the pathophysiology of atherogenesis is this association mostly based on experimental data corroborated by clinical data as well the current concept of a vulnerable plaque suggests that repeated plaque microruptures followed by subclinical thrombosis are critical for plaque growth and vulnerability.127-129 in agreement with these findings histopathological studies showed that two thirds of coronary thrombi obtained from patients who died suddenly from cardiovascular causes were in later stages of maturation suggesting that thrombi may exist long before a rupture occurs.33,34 in addition the contemporary understanding of atherothrombosis has evolved substantially establishing new roles for the hemostatic system beyond thrombosis we have summarized the potential array of actions of hemostasis in relation to the phenotype of the atherosclerotic vascular wall presumably linked to plaque stability but is all of this clinically relevant antithrombotic therapy with the use of antiplatelet or anticoagulant agents is the key to atherothrombosis prevention in various clinical situations.130-133 the role of antiplatelet therapy in secondary prevention is no longer questioned given the strong overall effect of drugs such as aspirin.134 a meta-analysis of primary-prevention 1756 n engl j med 364;18 trials has indicated that the use of aspirin is associated with a reduction of approximately 30 in the risk of myocardial infarction with more limited effects on the risk of stroke.135 in addition to aspirin s antiplatelet actions the efficacy of this drug may be due in part to its antiinflammatory actions.136-138 it is difficult to dissect the contribution of platelets in any of these antiinflammatory effects of aspirin also for more selective antiplatelet drugs including clopidogrel prasugrel and ticagrelor which target platelet receptors resulting in impaired platelet activation antiinflammatory and atherosclerosis-delaying effects have been reported.139 however clinical trials of platelet inhibitors for the prevention of atherosclerosis progression have not shown diminished development of plaque with any consistency.140 for many years oral anticoagulants have been used for short and long-term indications studies of heparin and vitamin k antagonists have shown that short-term use of these drugs is not likely to have a major effect on chronic disorders such as atherosclerosis.141,142 despite the fact that longterm administration of vitamin k antagonists did not have any visible effects on angiographic progression in patients who had undergone coronary-artery bypass grafting an additional followup assessment 3 years after discontinuation of therapy showed a significant 35 reduction in overall mortality in the warfarin group.143 given the powerful effects on risk reduction in thrombotic cardiovascular outcomes one might speculate that this effect was at least partially mediated by effects of vitamin k antagonists on plaque phenotype rather than plaque size at the same time the principal vascular side effect of the long-term administration of these drugs is accelerated calcification this effect is mainly due to direct inhibition of other vitamin k­dependent proteins in the vessel wall including matrix gla protein it is not known whether any additional influence of inhibition of thrombin formation may occur.144,145 the role of the hemostatic system in atherosclerosis in humans requires further investigation only a handful of molecules relevant to hemostasis are targeted by existing medications as more specific interventions are developed new therapeutic avenues and research approaches may open up with the introduction of new oral anticoagulants e.g direct inhibitors of factor xa and thrombin 146,147 which are small nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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mechanisms of disease molecules that can access the vessel wall it will be possible to document the effects of these drugs on plaque formation and especially on plaque stability since both thrombin inhibition55 and a prothrombotic state31 have been suggested as promoters of plaque stability in atherogenic mice the net effects in humans if any are unpredictable in conclusion given the potential of hemostasis to influence molecular and cellular responses in the vasculature new scientific approaches are required notably the majority of experimental data are entirely based on quantification of plaque burden rather than on extensive phenotyping of the lesions this is a major drawback in vascular medicine furthermore most clinical studies predominantly focus on establishing the thrombotic and mortality outcomes whereas few investigate plaque progression during the past decade ulreferences 1 ross r atherosclerosis an inflammatory disease n engl j med 1999;340 115-26 2 libby p inflammation in atherosclerosis nature 2002;420:868-74 3 hansson gk inflammation atherosclerosis and coronary artery disease n engl j med 2005;352:1685-95 4 levi m ten cate h disseminated intravascular coagulation n engl j med 1999;341:586-92 5 levi m van der poll t buller hr bidirectional relation between inflammation and coagulation circulation 2004 109:2698-704 6 esmon ct the interactions between inflammation and coagulation br j haematol 2005;131:417-30 7 davì g patrono c platelet activation and atherothrombosis n engl j med 2007 357:2482-94 8 furie b furie bc mechanisms of thrombus formation n engl j med 2008 359:938-49 9 rosenberg rd aird wc vascularbed­specific hemostasis and hypercoagulable states n engl j med 1999;340:155564 10 ruggeri zm platelets in atherothrombosis nat med 2002;8:1227-34 11 monroe dm hoffman m roberts hr platelets and thrombin generation arterioscler thromb vasc biol 2002;22 1381-9 12 crawley jt zanardelli s chion ck lane da the central role of thrombin in hemostasis j thromb haemost 2007;5 suppl 1:95-101 13 mackman n tilley re key ns role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis arterioscler thromb vasc biol 2007;27:1687-93 trasonography has been a major tool in vascular imaging unfortunately this approach is characterized by poor tissue penetration providing no information on plaque characteristics and is subject to intraobserver and interobserver variability with the development of high-resolution magnetic resonance imaging the assessment of plaque characteristics will improve vessel-wall phenotyping as a means of addressing the role of the hemostatic system in atherosclerosis supported by a marie curie fellowship mest-ct-2005-020706 from the european commission to dr borissoff disclosure forms provided by the authors are available with the full text of this article at nejm.org we thank jo g.r de mey of the department of pharmacology and toxicology tilman m hackeng of the department of biochemistry and mat j.a.p daemen of the cardiovascular research institute maastricht all at maastricht university medical center for their helpful suggestions during the preparation of the manuscript 14 gailani d renne t intrinsic pathway of coagulation and arterial thrombosis arterioscler thromb vasc biol 2007;27 2507-13 15 van der meijden pe munnix ic auger jm et al dual role of collagen in factor xii-dependent thrombus formation blood 2009;114:881-90 16 gawaz m langer h may ae platelets in inflammation and atherogenesis j clin invest 2005;115:3378-84 17 borensztajn k peppelenbosch mp spek ca factor xa at the crossroads between coagulation and signaling in physiology and disease trends mol med 2008 14:429-40 18 borissoff ji spronk hm heeneman s ten cate h is thrombin a key player in the coagulation-atherogenesis maze cardiovasc res 2009;82:392-403 19 ossovskaya vs bunnett nw protease-activated receptors contribution to physiology and disease physiol rev 2004 84:579-621 20 massberg s brand k grüner s et al a critical role of platelet adhesion in the initiation of atherosclerotic lesion formation j exp med 2002;196:887-96 21 huo y schober a forlow sb et al circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein e nat med 2003;9:61-7 22 weber c platelets and chemokines in atherosclerosis partners in crime circ res 2005;96:612-6 23 gleissner ca von hundelshausen p ley k platelet chemokines in vascular disease arterioscler thromb vasc biol 2008;28:1920-7 24 rizvi m pathak d freedman je chakrabarti s cd40-cd40 ligand interactions in oxidative stress inflammation and vascular disease trends mol med 2008;14:530-8 25 lievens d eijgelaar wj biessen ea daemen mj lutgens e the multi-functionality of cd40l and its receptor cd40 in atherosclerosis thromb haemost 2009 102:206-14 26 antoniades c bakogiannis c tousoulis d antonopoulos as stefanadis c the cd40/cd40 ligand system linking inflammation with atherothrombosis j am coll cardiol 2009;54:669-77 27 semple jw freedman j platelets and innate immunity cell mol life sci 2010 67:499-511 28 tyner jw uchida o kajiwara n et al ccl5-ccr5 interaction provides antiapoptotic signals for macrophage survival during viral infection nat med 2005;11 1180-7 29 chiodoni c iezzi m guiducci c et al triggering cd40 on endothelial cells contributes to tumor growth j exp med 2006 203:2441-50 30 borissoff ji heeneman s kilinc e et al early atherosclerosis exhibits an enhanced procoagulant state circulation 2010;122:821-30 31 seehaus s shahzad k kashif m et al hypercoagulability inhibits monocyte transendothelial migration through protease-activated receptor-1 phospholipasecbeta phosphoinositide 3-kinase and nitric oxide-dependent signaling in monocytes and promotes plaque stability circulation 2009;120:774-84 32 with notø at mathiesen eb Østerud b amiral j vissac am hansen jb increased thrombin generation in persons with echogenic carotid plaques thromb haemost 2008;99:602-8 33 kramer mc rittersma sz de winter n engl j med 364;18 nejm.org may 5 2011 1757 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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the newe ng landjour na l of m e dic i n e sharrett ar chambless le prospective study of hemostatic factors and incidence of coronary heart disease the atherosclerosis risk in communities aric study circulation 1997;96:1102-8 61 methia n andre p denis cv economopoulos m wagner dd localized reduction of atherosclerosis in von willebrand factor-deficient mice blood 2001 98:1424-8 62 danesh j lewington s thompson sg et al plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality an individual participant meta-analysis jama 2005;294 1799-809 63 bini a fenoglio jj jr mesa-tejada r kudryk b kaplan kl identification and distribution of fibrinogen fibrin and fibrinogen degradation products in atherosclerosis use of monoclonal antibodies arteriosclerosis 1989;9:109-21 64 lepedda aj cigliano a cherchi gm et al a proteomic approach to differentiate histologically classified stable and unstable plaques from human carotid arteries atherosclerosis 2009;203:112-8 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administration alone potential implications for dosing of antithrombin agents circulation 1998 98:2301-6 55 bea f kreuzer j preusch m et al melagatran reduces advanced atherosclerotic lesion size and may promote plaque stability in apolipoprotein e-deficient mice arterioscler thromb vasc biol 2006 26:2787-92 56 khallou-laschet j caligiuri g tupin e et al role of the intrinsic coagulation pathway in atherogenesis assessed in hemophilic apolipoprotein e knockout mice arterioscler thromb vasc biol 2005 25:e123-e126 57 eitzman dt westrick rj shen y et al homozygosity for factor v leiden leads to enhanced thrombosis and atherosclerosis in mice circulation 2005;111 1822-5 58 di tullio mr homma s jin z sacco rl aortic atherosclerosis hypercoagulability and stroke the apris aortic plaque and risk of ischemic stroke study j am coll cardiol 2008;52:855-61 59 páramo ja orbe j beloqui o et al prothrombin fragment 1+2 is associated with carotid intima-media thickness in subjects free of clinical cardiovascular disease stroke 2004;35:1085-9 60 folsom ar wu kk rosamond wd 1758 n engl j med 364;18 nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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mechanisms of disease amawy y quitterer u factor xiiia transglutaminase crosslinks at1 receptor dimers of monocytes at the onset of atherosclerosis cell 2004;119:343-54 74 zito f lowe gd rumley a mcmahon ad humphries se association of the factor xii 46c>t polymorphism with risk of coronary heart disease chd in the woscops study atherosclerosis 2002 165:153-8 75 govers-riemslag jw smid m cooper ja et al the plasma kallikrein-kinin system and risk of cardiovascular disease in men j thromb haemost 2007;5:1896-903 76 siegerink b govers-riemslag jw rosendaal fr ten cate h algra a intrinsic coagulation activation and the risk of arterial thrombosis in young women results from the risk of arterial thrombosis in relation to oral contraceptives ratio case-control study circulation 2010;122:1854-61 77 hagedorn i schmidbauer s pleines i et al factor xiia inhibitor recombinant human albumin infestin-4 abolishes occlusive arterial thrombus formation without affecting bleeding circulation 2010 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atherosclerosis arterioscler thromb vasc biol 2004 24:1104-10 85 stone oa richer c emanueli c et al critical role of tissue kallikrein in vessel formation and maturation implications for therapeutic revascularization arterioscler thromb vasc biol 2009;29:657-64 86 larusch ga mahdi f shariat-madar z et al factor xii stimulates erk1/2 and akt through upar integrins and the egfr to initiate angiogenesis blood 2010 115:5111-20 87 crawley j lupu f westmuckett ad severs nj kakkar vv lupu c expression localization and activity of tissue factor pathway inhibitor in normal and atherosclerotic human vessels arterioscler thromb vasc biol 2000;20:1362-73 88 okajima k regulation of inflammatory responses by natural anticoagulants immunol rev 2001;184:258-74 89 aihara k azuma h akaike m et al strain-dependent embryonic lethality and exaggerated vascular remodeling in heparin cofactor ii-deficient mice j clin invest 2007;117:1514-26 90 takamori n azuma h kato m et al high plasma heparin cofactor ii activity is associated with reduced incidence of in-stent restenosis after percutaneous coronary intervention circulation 2004;109 481-6 91 aihara k azuma h takamori n et al heparin cofactor ii is a novel protective factor against carotid atherosclerosis in elderly individuals circulation 2004 109:2761-5 92 giri tk ahn cw wu kk tollefsen dm heparin cofactor ii levels do not predict the development of coronary heart disease the atherosclerosis risk in communities aric study arterioscler thromb vasc biol 2005;25:2689-90 93 sofi f cesari f pratesi g et al low protein z levels in patients with peripheral arterial disease thromb haemost 2007;98:1114-7 94 pardos-gea j ordi-ros j serrano s balada e nicolau i vilardell m protein z levels and anti-protein z antibodies in patients with arterial and venous thrombosis thromb res 2008;121:727-34 95 sofi f cesari f tu y et al protein zdependent protease inhibitor and protein z in peripheral arterial disease patients j thromb haemost 2009;7:731-5 96 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inhibitor regulates intimal hyperplasia in atherosclerotic arteries proc natl acad sci u s a 2001;98:4078-83 101 kopp cw hölzenbein t steiner s et al inhibition of restenosis by tissue factor pathway inhibitor in vivo and in vitro evidence for suppressed monocyte chemoattraction and reduced gelatinolytic activity blood 2004;103:1653-61 102 westrick rj bodary pf xu z shen yc broze gj eitzman dt deficiency of tissue factor pathway inhibitor promotes atherosclerosis and thrombosis in mice circulation 2001;103:3044-6 103 pan s white ta witt ta chiriac a mueske cs simari rd vascular-directed tissue factor pathway inhibitor overexpression regulates plasma cholesterol and reduces atherosclerotic plaque development circ res 2009;105:713-20 104 sakata t mannami t baba s et al potential of free-form tfpi and pai-1 to be useful markers of early atherosclerosis in a japanese general population the suita study association with the intimalmedial thickness of carotid arteries atherosclerosis 2004;176:355-60 105 mitchell ct kamineni a palmas w cushman m tissue factor pathway inhibitor vascular risk factors and subclinical atherosclerosis the multi-ethnic study of atherosclerosis atherosclerosis 2009 207:277-83 106 blann ad amiral j mccollum cn lip gy differences in free and total tissue factor pathway inhibitor and tissue factor in peripheral artery disease compared to healthy controls atherosclerosis 2000;152:29-34 107 massberg s grahl l von bruehl ml et al reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases nat med 2010;16:887-96 108 mosnier lo zlokovic bv griffin jh the cytoprotective protein c pathway blood 2007;109:3161-72 109 waugh jm li-hawkins j yuksel e et al thrombomodulin overexpression to limit neointima formation circulation 2000;102:332-7 110 gerdes ve kremer hovinga ja ten cate h brandjes dp büller hr soluble thrombomodulin in patients with established atherosclerosis j thromb haemost 2004;2:200-1 111 peter k nawroth p conradt c et al circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule-1 e-selectin p-selectin and thrombomodulin arterioscler thromb vasc biol 1997;17 505-12 112 salomaa v matei c aleksic n et al soluble thrombomodulin as a predictor of incident coronary heart disease and n engl j med 364;18 nejm.org may 5 2011 1759 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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mechanisms of disease symptomless carotid artery atherosclerosis in the atherosclerosis risk in communities aric study a case-cohort study lancet 1999;353:1729-34 113 wu kk aleksic n ballantyne cm ahn c juneja h boerwinkle e interaction between soluble thrombomodulin and intercellular adhesion molecule-1 in predicting risk of coronary heart disease circulation 2003;107:1729-32 114 aleksic n wang yw ahn c juneja hs folsom ar wu kk assessment of coronary heart disease risk by combined analysis of coagulation factors atherosclerosis 2008;198:294-300 115 lentz sr miller fj jr piegors dj et al anticoagulant responses to thrombin are enhanced during regression of atherosclerosis in monkeys circulation 2002 106:842-6 116 castellino fj ganopolsky jg noria f sandoval-cooper mj ploplis va focal arterial inflammation is augmented in mice with a deficiency of the protein c gene thromb haemost 2006;96:794-801 117 salomaa v matei c aleksic n et al cross-sectional association of soluble thrombomodulin with mild peripheral artery disease the aric study atherosclerosis 2001;157:309-14 118 zorio e navarro s medina p et al circulating activated protein c is reduced in young survivors of myocardial infarction and inversely correlates with the severity of coronary lesions j thromb haemost 2006;4:1530-6 119 matsumoto k yano y gabazza ec et al inverse correlation between activated protein c generation and carotid atherosclerosis in type 2 diabetic patients diabet med 2007;24:1322-8 120 webb jh blom am dahlbäck b vitamin k-dependent protein s localizing complement regulator c4b-binding protein to the surface of apoptotic cells j immunol 2002;169:2580-6 121 anderson ha maylock ca williams ja paweletz cp shu h shacter e serum-derived protein s binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells nat immunol 2003;4:87-91 122 liao d wang x li m lin ph yao q chen c human protein s inhibits the uptake of acldl and expression of sr-a through mer receptor 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kolodgie fd farb a et al healed plaque ruptures and sudden coronary death evidence that subclinical rupture has a role in plaque progression circulation 2001;103:934-40 129 finn av nakano m narula j kolodgie fd virmani r concept of vulnerable unstable plaque arterioscler thromb vasc biol 2010;30:1282-92 130 holmes dr jr kereiakes dj kleiman ns moliterno dj patti g grines cl combining antiplatelet and anticoagulant therapies j am coll cardiol 2009;54:95109 131 bhatt dl fox ka hacke w et al clopidogrel and aspirin versus aspirin alone for the prevention of atherothrombotic events n engl j med 2006;354:170617 132 the warfarin antiplatelet vascular evaluation trial investigators oral anticoagulant and antiplatelet therapy and peripheral arterial disease n engl j med 2007;357:217-27 133 schulman s care of patients receiving long-term anticoagulant therapy n engl j med 2003;349:675-83 134 antithrombotic trialists collaboration collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death myocardial infarction and stroke in high risk patients bmj 2002;324:71-86 [erratum bmj 2002;324 141 135 patrono c garcía rodríguez la landolfi r baigent c low-dose aspirin for the prevention of atherothrombosis n engl j med 2005;353:2373-83 136 ferroni p martini f cardarello cm gazzaniga pp davi g basili s enhanced interleukin-1beta in hypercholesterolemia effects of simvastatin and low-dose aspirin circulation 2003;108:1673-5 137 chiang n hurwitz s ridker pm serhan cn aspirin has a gender-dependent impact on antiinflammatory 15-epilipoxin a4 formation a randomized human trial arterioscler thromb vasc biol 2006;262 e14-e17 138 morris t stables m hobbs a et al effects of low-dose aspirin on acute inflammatory responses in humans j immunol 2009;183:2089-96 139 muhlestein jb effect of antiplatelet therapy on inflammatory markers in atherothrombotic patients thromb haemost 2010;103:71-82 140 dieker hj french jk joziasse ic et al antiplatelet therapy and progression of coronary artery disease a placebo-controlled trial with angiographic and clinical follow-up after myocardial infarction am heart j 2007;1531 66.e1-66.e8 141 the post coronary artery bypass graft trial investigators the effect of aggressive lowering of low-density lipoprotein cholesterol levels and low-dose anticoagulation on obstructive changes in saphenous-vein coronary-artery bypass grafts n engl j med 1997;336:153-62 [erratum n engl j med 1997;337:1859 142 byington rp evans gw espeland ma et al effects of lovastatin and warfarin on early carotid atherosclerosis sexspecific analyses circulation 1999;1003 e14-e17 143 knatterud gl rosenberg y campeau l et al long-term effects on clinical outcomes of aggressive lowering of low-density lipoprotein cholesterol levels and low-dose anticoagulation in the post coronary artery bypass graft trial circulation 2000;102:157-65 144 spronk hm soute ba schurgers lj thijssen hh de mey jg vermeer c tissue-specific utilization of menaquinone-4 results in the prevention of arterial calcification in warfarin-treated rats j vasc res 2003;40:531-7 145 rennenberg rj van varik bj schurgers lj et al chronic coumarin treatment is associated with increased extracoronary arterial calcification in humans blood 2010;115:5121-3 146 schulman s kearon c kakkar ak et al dabigatran versus warfarin in the treatment of acute venous thromboembolism n engl j med 2009;361:2342-52 147 connolly sj ezekowitz md yusuf s et al dabigatran versus warfarin in patients with atrial fibrillation n engl j med 2009;361:1139-51 copyright © 2011 massachusetts medical society 1760 n engl j med 364;18 nejm.org may 5 2011 the new england journal of medicine downloaded from nejm.org on may 5 2011 for personal use only no other uses without permission copyright © 2011 massachusetts medical society all rights reserved.

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