Genetic and Pharmacological Modifications of Thrombin Formation in Apolipoprotein E-deficient Mice Determine Atherosclerosis Severity and Atherothrombosis Onset in a Neutrophil-Dependent Manner

Background Variations in the blood coagulation activity, determined genetically or by medication, may alter atherosclerotic plaque progression, by influencing pleiotropic effects of coagulation proteases. Published experimental studies have yielded contradictory findings on the role of hypercoagulability in atherogenesis. We therefore sought to address this matter by extensively investigating the in vivo significance of genetic alterations and pharmacologic inhibition of thrombin formation for the onset and progression of atherosclerosis, and plaque phenotype determination. Methodology/Principal Findings We generated transgenic atherosclerosis-prone mice with diminished coagulant or hypercoagulable phenotype and employed two distinct models of atherosclerosis. Gene-targeted 50% reduction in prothrombin (FII−/WT:ApoE−/−) was remarkably effective in limiting disease compared to control ApoE−/− mice, associated with significant qualitative benefits, including diminished leukocyte infiltration, altered collagen and vascular smooth muscle cell content. Genetically-imposed hypercoagulability in TMPro/Pro:ApoE−/− mice resulted in severe atherosclerosis, plaque vulnerability and spontaneous atherothrombosis. Hypercoagulability was associated with a pronounced neutrophilia, neutrophil hyper-reactivity, markedly increased oxidative stress, neutrophil intraplaque infiltration and apoptosis. Administration of either the synthetic specific thrombin inhibitor Dabigatran etexilate, or recombinant activated protein C (APC), counteracted the pro-inflammatory and pro-atherogenic phenotype of pro-thrombotic TMPro/Pro:ApoE−/− mice. Conclusions/Significance We provide new evidence highlighting the importance of neutrophils in the coagulation-inflammation interplay during atherogenesis. Our findings reveal that thrombin-mediated proteolysis is an unexpectedly powerful determinant of atherosclerosis in multiple distinct settings. These studies suggest that selective anticoagulants employed to prevent thrombotic events may also be remarkably effective in clinically impeding the onset and progression of cardiovascular disease.


Introduction
Blood coagulation and inflammation are evolutionary coupled host-defense mechanisms, which operate via common molecular and cellular pathways, serve as protection against infections or bleeding, promote wound healing and restore the integrity of injured tissues [1][2][3]. Atherosclerosis is a progressive chronic inflammatory vascular disorder, which can result in atherosclerotic plaque rupture and subsequent superimposed thrombus formation [4][5][6]. Besides the detrimental role of coagulation during the onset of acute atherothrombotic complications, there is evidence that local activation of hemostatic factors within early human atherosclerotic lesions may also be important in atherogenesis [7]. In addition to the overt leukocyte infiltration into the lesions and enhanced cell death, which are considered major markers for plaque instability, today's concept of a vulnerable plaque suggests that repeated plaque microruptures and subclinical microthrombosis are critical processes to plaque growth and subsequent atherothrombosis [8][9][10]. Histopathological reports demonstrate that thrombi may exist prior to rupture [11,12]. Numerous in vitro studies indicate that key clotting proteases such as thrombin can also catalyze a wide range of cellular actions related to cardiovascular function and pathophysiology -e.g. vascular permeability, oxidative stress, migration and proliferation of vascular smooth muscle cells, leukocyte adhesion, chemotaxis, inflammation, and apoptosis [13]. Experimental animal studies demonstrate that administration of direct thrombin inhibitors in ApoE 2/2 mice attenuates atherosclerotic plaque progression and promotes plaque stability of advanced atherosclerotic lesions by reducing the levels of inflammation and the number of macrophages infiltrating the lesions [14][15][16]. In sharp contrast, there is also evidence showing that hypercoagulability in ApoE 2/2 mice carrying prothrombotic mutations promotes atherosclerotic plaque stability via thrombin-mediated impairment of monocyte transendothelial migration [17]. In the near future, millions of patients with arterial vascular disease will be treated with novel, selective anticoagulant agents. Whereas this matter remains of major scientific and clinical significance, there is still limited understanding of the relevance of blood coagulation in atherosclerosis in vivo [18]. In attempting to reconcile these apparently contradictory findings, we extensively investigated the in vivo significance of genetic alterations and pharmacologic inhibition of thrombin formation for the onset and progression of atherosclerosis, but also plaque phenotype determination.

Methods
Animals TM Pro/Pro mice, carrying a thrombomodulin (TM) gene mutation resulting in diminished TM-dependent generation of activated protein C (APC) [19], and prothrombin (FII) heterozygous mice with genetically imposed hypoprothrombinemia [20] were crossed into a pure C57BL/6 background for at least 8 generations and subsequently crossbred to ApoE 2/2 mice (Charles River, Maastricht, The Netherlands), carrying the same background. Only female mice were used throughout the entire study. All animal experimental protocols were carried out in compliance with the Dutch government guidelines and were approved by the Animal Care and Use Committee of Maastricht University (Maastricht, The Netherlands).

Mouse Models of Atherosclerosis
In a spontaneous atherosclerosis model, female TM Pro/ Pro :ApoE 2/2 , FII 2/WT :ApoE 2/2 (age, 8-9 weeks; n = 10 per group) and control ApoE 2/2 mice (age, [8][9] weeks; n = 20) received regular chow diet (Hope Farms, Woerden, The Netherlands) for 35 weeks and were then sacrificed for a detailed analysis. In a separate experimental setup, consisting of identical groups, carotid atherosclerotic plaques were induced via placement of perivascular collars around the common carotid arteries as described before [21]. All animals were fed on a high-fat diet (15% cocoa butter, 1% corn oil, 0.25% cholesterol, 40.5% sucrose, 10% cornstarch, 20% casein, free of cholate, total fat content 16%; Hope Farms, Woerden, The Netherlands) for two weeks before collar placement and for additional six weeks after surgery. Diets and water were provided ad libitum throughout all experiments.

Pharmacological Interventions
Female TM Pro/Pro :ApoE 2/2 mice (n = 10 per treatment group; age, 8-9 weeks) fed on a standard high-fat diet (D12451; Research Diets, NJ, USA) for 2 weeks were subsequently subjected to a surgical implantation of non-constrictive perivascular carotid collars and then assigned to different interventions or placebo for a total of 6 weeks. The study design involved an intervention arm with mice receiving standard D12451 high-fat chow supplemented with oral Dabigatran etexilate (7.5 mg DE/gram chow). In a second intervention arm, TM Pro/Pro :ApoE 2/2 mice were fed on a standard D12451 high-fat diet and received intraperitoneal (i.p.) administration of recombinant murine APC (rmAPC)) in bolus doses of 2.5 mg/kg/per every 5 days. Placebotreated mice received injection of saline and were fed on standard D12451 high-fat chow. rmAPC was produced in the laboratory of Dr. Charles T. Esmon  For an expanded Methods section, please see the online supplement of the article (Methods S1).

Statistical Analysis
All statistics were performed using Prism, version 6.00 (GraphPad Software Inc., San Diego, CA, USA) and IBM SPSS Statistics 20.0 (SPSS Japan Inc., an IBM company, Tokyo, Japan). Data sets were assessed for normality using Kolmogorov-Smirnov test or Bartlett's test for homogeneity of variance. Data were compared using unpaired 2-tailed t test or one-way ANOVA, followed by Newman-Keuls posthoc test for multiple comparisons. In case of non-normal distribution, non-parametric tests such as Mann-Whitney or Kruskall-Wallis test with Dunn's post hoc analysis were used as appropriate. Data are expressed as mean 6 SD, unless otherwise stated. A 2-tailed p,0.05 was considered statistically significant.

Coagulation Phenotype is a Key Factor in Atherosclerotic Plaque Growth and Phenotype Determination
We then assessed the extent, as well as the phenotype of the atherosclerotic plaques formed in the aortic arch in experimental cohorts of mice following 35 weeks on a regular chow diet. FII 2/+ : ApoE 2/2 mice with a genetic deficit in prothrombin exhibited highly attenuated atherosclerotic lesion formation relative to control ApoE 2/2 mice ( Figure 1A,B). Macrophage infiltration (MAC-2 + cells) and a-smooth muscle actin (SMA; a-SMA + cells) content were unaffected in FII 2/+ :ApoE 2/2 mice lesions compared to ApoE 2/2 control mice ( Figure 1A,C,D). However, hypoprothrombinemia was also linked to a significant decrease in neutrophil recruitment ( Figure 1A,E), abundant collagen deposition ( Figure 1A,F), thus showing a more fibrotic appearance, stable plaque phenotype and decreased number of advanced atherosclerotic lesions formed. In sharp contrast, pro-thrombotic TM Pro/Pro :ApoE 2/2 mice displayed severe atherosclerosis development with remarkably increased total plaque area ( Figure 1A,B). TM Pro/Pro :ApoE 2/2 mice showed unstable lesions ( Figure 1A,B), associated with markedly decreased a-SMA and collagen content ( Figure 1A,C,F), and significantly higher neutrophil (Ly6G + cells) infiltration ( Figure 1A,E). These effects were independent of plasma lipid levels (Table S1B) and could not be attributed to an increased uptake of modified lipoproteins by macrophages ( Figure  S1A,B,C,D). Hypercoagulable TM Pro/Pro :ApoE 2/2 mice showed significantly increased spontaneous mortality rates, albeit that the exact cause of death could not be pinpointed ( Figure 1G).
Hence, to further verify the net effects of underlying alterations in clotting potential on plaque phenotype, we also studied the impact of both genetic perturbations on collar-induced carotid artery atherosclerosis [21]. High-fat diet fed FII 2/+ :ApoE 2/2 mice displayed significantly decreased plaque volume, degree of stenosis, intima/media ratio and expansion of the arterial wall, 6 weeks after bilateral perivascular carotid collar placement ( Figure 2A,B,C,F,G). Furthermore, hypocoagulability ameliorated plaque stability, testified by a significantly increased mean fibrous cap thickness (Figure 2A,E). Conversely, TM Pro/Pro :ApoE 2/2 mice lesions were substantially larger, accompanied by significantly increased luminal stenosis, intima/media ratio and outward remodeling (Figure 2A,B,C,F,G). Similar to spontaneous atherosclerosis, collar-induced carotid artery plaques in FII 2/+ :ApoE 2/2 mice presented a stable pro-fibrotic phenotype, whereas TM Pro/Pro : ApoE 2/2 lesions showed pronounced features of plaque vulnerability, including larger necrotic cores ( Figure 2A,D), thin fibrous caps ( Figure 2A,E) and significant decrease in collagen content ( Figure 3A,B). Immunofluorescence microcopy analyses for caspase-3 revealed enhanced apoptosis within the lesions of the procoagulant ApoE 2/2 mice (data not shown). High plaque vulnerability in hypercoagulable mice was also associated with a pro-inflammatory plaque phenotype, signs of intraplaque hemorrhage, plaque dissection, but also spontaneous atherothrombosis ( Figure 4E, Figure 5), whereas fibrin deposits were extensively distributed throughout ruptured TM Pro/Pro :ApoE 2/2 lesions ( Figure 5). Intraplaque accumulation of macrophages and neutrophils was significantly diminished in hypocoagulable mice ( Figure 4A,B,C), indicating that deficiency in prothrombin results in a less inflammatory plaque profile. In contrast, in TM Pro/Pro : ApoE 2/2 advanced carotid artery lesions were abundantly infiltrated with neutrophils when compared to ApoE 2/2 control mice, whereas no changes were observed in terms of macrophage content ( Figure 4A,B,C). A pronounced Ly6G + cell intraplaque recruitment was also observed during early stages of atherosclerosis in TM Pro/Pro :ApoE 2/2 mice ( Figure 4D). To provide further understanding into how alterations in blood coagulation potential affect the thrombogenicity of the arterial vessel wall, we pursued complementary studies of vessel occlusion following ferric chloride injury of healthy arteries. Times to occlusion and cessation of blood flow as a result of thrombus formation were significantly shortened in TM Pro/Pro :ApoE 2/2 mice relative to control ApoE 2/2 mice, whereas occlusion times in FII 2/+ :ApoE 2/2 animals was comparable to control ApoE 2/2 cohorts ( Figure S2).

Hypercoagulability Triggers Initiation and Progression of Atherosclerotic Lesions in a Neutrophil-Dependent Manner
There was a strong positive association between the number of circulating neutrophils in peripheral blood and the extent of atherosclerotic plaque burden ( Figure 4F). No significant correlation was observed between the number of peripheral blood monocytes and plaque size. Because of the increased neutrophil counts observed in hypercoagulable TM Pro/Pro :ApoE 2/2 mice after 35 weeks on a regular chow diet (Table S1C), and the abundant infiltration of neutrophils within vulnerable-appearing atherosclerotic lesions ( Figure 4A,C), we explored the impact of Figure 2. Morphometrical analysis of periadventitial cuff-induced atherosclerosis in mice with genetically imposed alterations in blood coagulation potential. (A) Representative hematoxylin and eosin (H&E)-stained sections of carotid arteries of FII 2/+ :ApoE 2/2 , TM Pro/Pro : ApoE 2/2 and control ApoE 2/2 mice (top row). Necrotic core areas of the atherosclerotic lesions were identified and quantified by using toluidine blue (TB) staining (second and third row). (B, C) Whereas hypocoagulable mice were significantly protected against plaque progression (26.5612.6*10 3 in FII 2/+ :ApoE 2/2 vs. 69.2618.4*10 3 mm 2 in ApoE 2/2 control mice, n = 10 per group, p,0.0001), pro-thrombotic mice developed severe and occlusive atherosclerotic burden (146.4652.7*10 3 in TM Pro/Pro :ApoE 2/2 vs. 53.9627.0*10 3 mm 2 in ApoE 2/2 control mice, n = 10 per group, p = 0.0001). The degree of stenosis in TM Pro/Pro :ApoE 2/2 reached an average of 88.668.1% (vs. 62.2616.1% in ApoE 2/2 mice, n = 10 per group, p = 0.0002), whereas it was substantially lower in FII 2/+ :ApoE 2/2 mice (36.8611.9% vs. 64.969.6% in ApoE 2/2 mice, n = 10 per group, p,0.0001). (A, D) Pearson's chi-squared test (x 2 ) detected a significant difference in the number of advanced atherosclerotic lesions (presence of fibrous cap atheromata [54]) formed between FII 2/+ :ApoE 2/2 (4 out of 10) and TM Pro/Pro :ApoE 2/2 mice (10 out of 10) (n = 10 per group, p = 0.0108). In fact, the necrotic area within the lesions of the hypercoagulable mice was significantly increased: 56.2610.8% of the total plaque area, as compared to 29.0617.7% in the control ApoE 2/2 group (n = 10 per group, p = 0.0024). (E) Hypocoagulable mice showed more stable advanced lesions, as indicated by the significantly thicker fibrous caps in comparison to ApoE 2/2 mice (n = 10 per group, p = 0.0081). (F) Intima/media ratio was significantly increased in TM Pro/Pro :ApoE 2/2 mice, whereas profoundly decreased in FII 2/+ :ApoE 2/2 mice. Of note, the average outer diameter of the common carotid artery is 0.36 mm [21], thus suggesting that TM Pro/Pro :ApoE 2/2 atherosclerotic plaques undergo a dramatic outward remodeling as indicated in panel (G). hypercoagulability on neutrophil function and hematopoiesis in the context of atherosclerosis. Intravital microscopy studies revealed that neutrophils were significantly more adherent to atherosclerotic lesions in the common carotid artery of TM Pro/ Pro :ApoE 2/2 than in ApoE 2/2 control mice after 6 weeks on a high-fat diet ( Figure 4G,H,I,J). These data consolidated our histological findings ( Figure 4A,C,D), suggesting that hypercoagulability can promote initiation and progression of atherosclerotic lesions in a neutrophil-dependent manner.

Hypercoagulability and Its Effects on Systemic Inflammation and Hematopoiesis: Enhanced Accumulation of Reactive Oxygen Species in Neutrophils
Consistent with this view, hypercoagulability promoted a significant increase in plasma CCL2 and CXCL1 levels ( Table  S2). TM Pro/Pro :ApoE 2/2 mice showed significantly higher IL-6 plasma levels after 35 weeks on regular chow diet. Although there were trends toward increased IL-1b, expression of other key proinflammatory cytokines such as TNF-a, IFN-c, IL-5 and IL-12 were not statistically different between hypercoagulable and control ApoE 2/2 mice, indicating that TM Pro/Pro :ApoE 2/2 mice  (Table  S2). In addition, the higher plasma expression levels of granulocyte-colony stimulating factor (G-CSF) in TM Pro/Pro :ApoE 2/2 mice raises the possibility that the loss of TM function not only impacts on thrombin activity but also affects granulopoiesis in the bone marrow. However, we did not detect major changes in hematopoiesis between hypercoagulable and control ApoE 2/2 mice after 10 weeks on regular chow diet ( Figure S3), including any preferential differentiation towards granulocytic-type colonies. Despite a minor but significant increase in the common myeloid progenitor (CMP) cells, lineage-negative (LK, LS, LSK), granulocyte-macrophage progenitor (GMP) and erythroid/mega-karyocyte progenitor (EMP) populations in TM Pro/Pro :ApoE 2/2 mice remained unaffected ( Figure S3). Nevertheless, the relative percentage of mature granulocytes in bone marrow, as well as of pro-atherogenic Ly6C high monocytes [22] as measured by flow cytometry, was significantly increased in the pro-thrombotic mice ( Figure 6A,B,C). Of interest, another consequence of chronic hypercoagulability was enhanced accumulation of reactive oxygen species in neutrophils and not monocytes, as assessed by DHR fluorescence, the latter considered a measure of neutrophil senescence ( Figure 6D,E). The panel represents an atherosclerotic dissection with superimposed thrombus formation in a TM Pro/Pro :ApoE 2/2 mouse at 6 weeks after carotid collar placement. Using Perl's Prussian blue stain (blue color), we detected free ferric ions deposited within the sites of plaque dissection, indicating the areas of intraplaque hemorrhage. Whereas the carotid lesions in 5 out of 10 TM Pro/Pro :ApoE 2/2 mice were associated with either rupture, dissection or intraplaque hemorrhage, none of the control ApoE 2/2 mice plaques had any signs of severe plaque vulnerability (Pearson's chi-squared test (x 2 ), n = 10 per group, p = 0.0325). Statistical analysis including all experimental groups indicated that the number of circulating neutrophils in peripheral blood was strongly correlated to the extent of atherosclerotic plaque burden (F). Using intravital microscopy, we confirmed that the relative percentage of circulating neutrophils in TM Pro/Pro :ApoE 2/2 mice was found significantly higher than ApoE 2/2 control mice (n = 6 per group, p = 0.0107). Whereas there were no differences found in the general leukocyte rolling and arrest between TM Pro/Pro :ApoE 2/2 and ApoE 2/2 mice after 6 weeks on a high-fat diet (Rhodamine-labeled leukocytes) (n = 6 per group, p = 0.2886), Ly-6G + neutrophils in TM Pro/Pro :ApoE 2/2 mice were significantly more adherent to atherosclerotic lesions in the common carotid artery than in ApoE 2/2 control mice (n = 6 per group, p = 0.0139). Bar represents 100 mm.

Administration of Direct Thrombin Inhibitor Dabigatran Etexilate (DE) or rmAPC Substantially Decreases Systemic Inflammation, Aborts Atherosclerosis, Promotes Plaque Stability And Prevents Against Atherothrombosis in Hypercoagulable Mice
To study the role of thrombin in modulating atherogenesis in vivo, we administered either the specific oral thrombin inhibitor DE or a recombinant form of the natural anti-coagulant APC for 6 weeks after carotid collar placement in hypercoagulable TM Pro/ Pro :ApoE 2/2 mice on high-fat diet. Remarkably, both interventions completely rescued plaque formation ( Figure 7A,B), as also evident by the decreased degree of stenosis, intima/media ratio and positive outward remodeling (Figure 7C,F,G). Whereas in the placebo group 5 out of 10 animals had plaques with overt signs of plaque vulnerability (defined as, i.e. plaque dissection, intraplaque hemorrhage or superimposed thrombus formation), oral DE or rmAPC treatments limited the occurrence of plaque destabiliza-tion and atherothrombotic phenomena, and resulted in substantially reduced leukocyte recruitment and enhanced plaque stability ( Figure 7A,H,I; Figure 8). In addition, both interventions led to a pronounced decrease in thrombin generation, suggesting that even ApoE 2/2 mice exerted a low-grade hypercoagulable state ( Table  S1A, Table S3A). DE and rmAPC therapies significantly limited systemic inflammation (Table S3C), as further exemplified by decreased neutrophil and lymphocyte counts and cytokine and chemokine profiles that show a shift to an anti-inflammatory state ( Table S4).

Major Findings
These studies provide strong evidence directly documenting that thrombin and other hemostatic system components are powerful determinants of inflammatory vessel wall disease, and even capable of superseding other pro-atherosclerotic insults. We here Here we present a new hypercoagulable atherosclerosis model, which closely mimics the composition and events leading to plaque destabilization, as normally observed in human atherothrombosis. In a series of sections, demonstrating carotid atherosclerotic plaques, obtained from TM Pro/Pro :ApoE 2/2 mice at 6 weeks after collar placement on high-fat diet regimen, we show multiple signs of plaque vulnerability. (A) A non-occlusive but rapidly progressing atherosclerotic lesion, characterized by abundant infiltration of leukocytes. (B) TM Pro/Pro :ApoE 2/2 mice plaques tend to rupture and dissect (upper arrow) even during the non-occlusive phase, accompanied by ''silent'' intraluminal thrombosis (lower arrows). Despite the detrimental pathologic characteristics of those lesions, these data confirm the hypothesis that arterial thrombosis might exist long before a fatal event takes place. This is further consolidated by the presence of so called ''buried fibrous caps'' (indicated by the arrows) in TM Pro/Pro :ApoE 2/2 mice plaques [55], considered a marker of healed plaque ruptures, and also observed in human atherosclerosis. Blue color denotes a massive intraplaque hemorrhage (iron ions deposition) (C). Hypercoagulability induces a severe inflammatory and pro-necrotic intraplaque environment, leading to the formation of enormous necrotic core, thin fibrous caps, further plaque destabilization (D) and atherothrombosis (occlusive intraluminal thrombosis/abundant fibrin(ogen) deposition (indicated by the arrows)) (E). Thrombi undergo fibrotic organization involving vascular smooth muscle cells and fibroblasts ingrowth, and are then partially recanalized by newly formed vessels (arrows, blue color -iron deposition/presence of erythrocytes)(F). doi:10.1371/journal.pone.0055784.g005 demonstrate that thrombin activity can influence onset, progression and qualitative properties of atherosclerotic plaques. In two distinct experimental setups (spontaneous and collar-induced atherosclerosis), we show that genetically-imposed 50% reduction in prothrombin (FII 2/+ ) in atherosclerosis-prone ApoE 2/2 mice remarkably diminishes lesion formation and promotes plaque stability. In contrast, mice with genetically impaired anticoagulant function of TM, crossed on ApoE 2/2 background, develop severe atherosclerotic disease. We here for the first time demonstrate the importance of neutrophils in the coagulation-inflammation interplay during atherogenesis. The principal finding of this study is that hypercoagulability induces enhanced mobilization of neutrophils from the bone marrow into the circulation, accompanied with neutrophil hyper-reactivity, increased oxidative stress, apoptosis and abundant intraplaque neutrophil infiltration, thus promoting unstable atherosclerotic plaque phenotype and spontaneous atherothrombosis. Administration of either the synthetic specific thrombin inhibitor DE or a recombinant form of the natural anticoagulant APC, counteract the pro-inflammatory, proatherogenic and pro-thrombotic phenotype of hypercoagulable TM Pro/Pro :ApoE 2/2 mice, resulting in plaque stability and preventing atherothrombosis.

Coagulation and Inflammation in Atherosclerosis
Given the multifactorial nature of atherosclerosis and the wellknown capacity of coagulation proteases and their receptors (protease-activated receptors, PARs) and substrates to control inflammatory and reparative processes [18], one would anticipate that hemostatic factors might contribute, at least incrementally, to plaque development. In fact, various pro-thrombotic states have been associated with enhanced atherosclerosis progression in mice in vivo [23][24][25][26][27][28][29]. Nevertheless, the mechanisms through which clotting contributes to atherosclerosis progression remain unclear to date. Thrombin is a central coagulation protease, which through the activation of PAR-1 is known to promote numerous pro-atherogenic actions in vitro such as endothelial permeability, migration and proliferation of VSMC, platelet activation, leukocyte adhesion and recruitment, cytokine and chemokine production, vascular calcification, angiogenesis and apoptosis [13]. Similar effects can also be triggered via both PAR-1 and PAR-2 by either TF-FVIIa complex or FXa [18]. In contrast, APC counteracts inflammation through PAR-1 signaling at multiple levels such as enhancing the endothelial barrier integrity, attenuating TF and TNF-a release by monocytes, inhibiting cytokine production, leukocyte endothelial transmigration and NF-kB pathways [30,31]. Our data strongly suggests that increased thrombin generation due to diminished APC production in TM Pro/Pro :ApoE 2/2 mice may be mechanistically-coupled to the pro-atherosclerotic phenotype. Previous studies have indicated that hypercoagulability can have beneficial effects on plaque stenosis during the intermediate phases of progression by promoting positive vascular remodeling [17]. Importantly, our data shows that plaques of prothrombotic mice had profound composition changes, with overt features of plaque vulnerability at later stages of disease development (at 35 weeks on regular chow diet), characterized by the presence of large necrotic cores, thin fibrous caps, significantly increased neutrophil intraplaque infiltration and apoptosis, decreased collagen and VSMC content, occlusive stenosis and spontaneous atherothrombosis. Despite that we show a significant association between hypercoagulability and high rate of spontaneous mortality in TM Pro/Pro :ApoE 2/2 mice, further studies are needed to precisely determine the underlying cause of this observation.
There are numerous pathways, which have been implicated to play a role in the complex interplay between coagulation and inflammation in various pathologic conditions [30]. Here, for the first time, we show the strong potential of the coagulation system (in particular thrombin) to regulate inflammation in atherosclerosis as highlighted by the effects of direct thrombin inhibition therapy on leukocyte counts, chemokine and cytokine levels. Neutrophils represent another intriguing cellular interface between blood coagulation and inflammation [3,32]. Although the importance of neutrophils in atherosclerosis remains to be defined in detail, several studies have highlighted their pro-atherogenic potential and proposed role in atherosclerotic plaque destabilization [18,[33][34][35][36][37][38]. Our data demonstrate that hypercoagulability promotes enhanced accumulation of reactive oxygen species in neutrophils and, thus triggering enhanced neutrophil senescence. Systemic inflammation involving activated neutrophils is associated with unstable coronary artery disease and considered an independent predictor for cardiovascular outcome in patients [39]. Our data indicate increased neutrophil rolling and arrest on early carotid atherosclerotic plaques in TM PRO/PRO :ApoE 2/2 mice. In addition, the significantly higher neutrophil intraplaque infiltration, consolidated by striking correlations between neutrophil counts and the amount of plaque burden, suggest a key role for neutrophils in this coagulation-inflammation interplay in atherosclerosis. The increased number of circulating pro-atherogenic neutrophils in TM Pro/Pro :ApoE 2/2 mice can be in part explained by enhanced mobilization from the bone marrow as a result of exuberant plasma G-CSF, CCL-2 and CXCL-1 expression. G-CSF is an essential regulator of the neutrophil mobilization from the bone marrow. Numerous experimental and human studies have shown an association between higher G-CSF plasma levels, neutrophil activation, endothelial dysfunction, enhanced oxidative stress, hypercoagulability and platelet aggregation [40][41][42]. CCL-2 and CXCL-1 are chemokines recognized for their potent neutrophil chemoattractant activity and capacity to promote vascular inflammation [32], but also known to be critical players in recruitment of monocytes and neutrophils to sites of chronic inflammation [39,43,44]. Thrombin acts as a secretagogue, promotes endothelial dysfunction and induces the release of Pselectin, which is stored in the Weibel-Palade bodies [45]. Pselectin is a powerful mediator of neutrophil adhesion to the endothelium, but also plays an important role in atherogenesis [29,45]. Thrombin is among one of the most potent platelet activators [18]. One may also assume that part of the proatherogenic effects observed in the hypercoagulable mice can be also mediated via platelet activation, known for their crucial role in atherosclerosis progression [46,47]. Platelets interact with a variety of cellular partners such as monocytes, neutrophils, endothelial cells, endothelial progenitor cells, and others, thus induce key inflammatory responses including leukocyte adhesion, migration, proteolysis, thrombosis, but also facilitate the differentiation of macrophages to foam cells [48]. Although we did neither detect any significant correlation between the number of peripheral monocytes and atherosclerotic plaque burden, nor changes in the number of macrophages infiltrating the lesions in TM PRO/PRO : ApoE 2/2 versus control mice, this also does not rule out a role of monocytes in atherogenesis. Neutrophils can rapidly undergo apoptosis as a result of the enhanced oxidative stress. The abundant number of pro-apoptotic leukocytes within the lesions of TM PRO/PRO :ApoE 2/2 mice can overload the phagocytic clearance capacity of the macrophages, thus promoting enhanced macrophage death and subsequent plaque necrosis [49]. In fact, the necrotic core areas and apoptotic indices within the lesions of hypercoagulable mice were substantially increased.

Conclusions and Potential Clinical Implications
Steadily increasing thrombin-antithrombin plasma levels, considered a sensitive marker of thrombin formation in vivo, were independently associated with the presence and severity of coronary atherosclerotic plaques, as defined by coronary computed tomographic angiography (CCTA) [50]. In conclusion, we here provide substantial new evidence showing that controlling coagulation via thrombin inhibition is a potential new therapeutic target to treat atherosclerosis. Given the promising safety profile and significant clinical benefits, which selective anticoagulants may offer over traditional anticoagulant therapy [51,52], including the reduction of risk of stroke and all-cause mortality after acute coronary syndromes, the potential clinical importance of these findings allows the unique opportunity to study if and how administration of novel classes of anticoagulants modifies atherosclerosis phenotype in patients. Nevertheless, due to the large heterogeneity in humans, the multifactorial nature of atherosclerosis, the dual-faceted character that many coagulation factors can exert, and their beneficial roles under normal physiological conditions, one should consider long-term specific coagulation inhibition with caution [53]. There is an urgent need of clinical trials to fully assess the overall benefit and risk balance of longterm therapy with novel oral anticoagulant agents. Figure S1 Hypercoagulability in TM Pro/Pro :ApoE 2/2 mice does not alter lipid uptake in bone marrow-derived macrophages (BMM). (A) There were no significant differences found in the lipid uptake in BMM derived from TM Pro/Pro : ApoE 2/2 and control ApoE 2/2 mice, as determined by flow cytometry analysis. (B, C, D) In addition, we also used high performance thin layer chromatography to test the free cholesterol, cholesterol esters and triglycerides accumulation in BMM in response to LDL and oxidized LDL loading and there were no significant differences detected between BMM obtained from TM Pro/Pro :ApoE 2/2 and control ApoE 2/2 mice. Error bars represent mean 6 SD. Abbreviations: HP-TLC -high performance thin layer chromatography; BMM -Bone marrow-derived macrophages; LDL -low-density lipoprotein; oxLDL -oxidized low-density lipoprotein. (DOC) Figure S2 20% FeCl 3 -induced arterial injury in hyperand hypocoagulable atherosclerosis-prone mice. Time to occlusion (TTO) and closing times (CT) were established. TTO is defined as the time after FeCl 3 application required for the blood flow to decline to 90%, whereas CT represents the time from the start of flow reduction to a complete occlusion of the carotid artery. (A, B) Both TTO and CT were significantly shortened in TM Pro/Pro :ApoE 2/2 as compared to ApoE 2/2 control mice (TTO: 4.460.9 vs. 14.1611.1 min., respectively; n = 10 per group, p = 0.0010) (CT: 1.260.8 vs. 11.3613.0 min., respectively; n = 10 per group, p = 0.0010), suggesting for a pro-thrombotic arterial vessel wall phenotype. In contrast, hypocoagulability in FII 2/+ :ApoE 2/2 mice had no effect on thrombus formation during FeCl 3 -induced arterial injury. Of note, all 10 out of 10 of the TM Pro/Pro :ApoE 2/2 mice formed an occlusive thrombus (animals depicted at 30 min. represent all mice, which did not induce occlusive thrombus formation, indicated by an arrow). *p,0.05; **p,0.01; ***p,0.001. Dotted lines represent mean. (DOC) Figure S3 The effects of hypercoagulability on hematopoiesis. Using a CFU-C (colony forming unit in culture) assay, we established that there were no significant differences in the amount of total colonies produced by TM Pro/Pro :ApoE 2/2 as compared to ApoE 2/2 control mice after 8 weeks on a regular chow diet (A). Furthermore, we could not find any changes in the composition, as determined by the CFU subset analysis, indicating that hypercoagulability does not affect hematopoiesis in the bone marrow compartment (B). FACS analysis of the bone marrow consolidated the results of the CFU-C assay (C, D, E). The amount of LSK (Lin2/Sca-1+/c-Kit+) cells showed a tendency towards an increase in the TM Pro/Pro :ApoE 2/2 compared to ApoE 2/2 control mice (4.260.8% vs. 3.760.7%; n = 12 per group, p = 0.0529) (F). The amount of CMP (common myeloid progenitor) cells was significantly increased in the TM Pro/Pro : ApoE 2/2 mice compared to the controls (15.163.3% vs. 12.762.3%; n = 12 per group, p = 0.0402). (G). In addition, EMP and GMP populations in the bone marrow remained unaffected by the hypercoagulable state in TM Pro/Pro :ApoE 2/2 mice (H, I). *p,0.05; **p,0.01; ***p,0.001. Error bars represent mean 6 SD. Abbreviations: CFU -colony forming unit; GMgranulocyte-macrophage progenitor; G -granulocyte progenitor; M -macrophage progenitor; LK -cells positive for LIN 2 c-Kit + Sca-1 2 lineage markers; LSK -cells positive for LIN 2 c-Kit + Sca-1 + lineage markers; CMPs -common myeloid progenitors; GMPgranulocyte/macrophage progenitors; EMP -erythroid/megakaryocyte progenitors.  on high-fat diet and treated with placebo, oral Dabigatran etexilate or mouse recombinant APC (n = 10 per group). *p,0.05; **p,0.01; ***p,0.001 (Intervention groups compared to placebo group). Data are presented as mean 6 SD. Abbreviations: IL -interleukin; TNF-a -tumor necrosis factor-alpha; IFN-c -Interferon-gamma; G-CSF -Granulocyte colony-stimulating factor; MCP-1 -monocyte chemotactic protein-1; MIP-1a -Macrophage inflammatory protein-1a; MIP-1b -Macrophage inflammatory protein-1b; RANTES -Regulated upon Activation, Normal T-cell Expressed, and Secreted; KC -keratinocyte chemoattractant; APC -Activated Protein C. (DOC)