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OBJECTIVE Aberrant neutrophil activation occurs during the advanced stages of atherosclerosis. Once primed, neutrophils can undergo apoptosis or release neutrophil extracellular traps (NETs). This extracellular DNA exerts potent pro-inflammatory, prothrombotic and cytotoxic properties. The goal of this study was to examine the relationships between extracellular DNA formation, coronary atherosclerosis and the presence of a prothrombotic state. APPROACH AND RESULTS In a prospective, observational, cross-sectional cohort of 282 individuals with suspected coronary artery disease (CAD), we examined the severity, extent, and phenotype of coronary atherosclerosis by using coronary computed tomographic angiography (CCTA). Double-stranded DNA, nucleosomes, citrullinated histone H4 and myeloperoxidase (MPO)-DNA complexes, considered in vivo markers of cell death and NETosis, respectively, were established. We further measured various plasma markers of coagulation activation and inflammation. Plasma double-stranded DNA, nucleosomes and MPO-DNA complexes were positively associated with thrombin generation and significantly elevated in patients with severe coronary atherosclerosis or extremely calcified coronary arteries. Multinomial regression analysis, adjusted for confounding factors, identified high plasma nucleosome levels as an independent risk factor of severe coronary stenosis (OR: 2.14, 95% CI 1.26-3.63; p=0.005). Markers of NETs, such as MPO-DNA complexes, predicted the number of atherosclerotic coronary vessels and the occurrence of major adverse cardiac events. CONCLUSIONS Our report provides evidence demonstrating that markers of cell death and NET formation are independently associated with CAD, prothrombotic state and occurrence of adverse cardiac events. These biomarkers could potentially aid in the prediction of cardiovascular risk in patients with chest discomfort.

• PAD4-mediated chromatin decondensation and release by neutrophils exacerbate injury after MI/R. • Combining reduction of neutrophil recruitment with extracellular DNA cleavage could be a new approach to reduce cardiac damage after MI.Innate immune cells play a major role in the early response to myocardial ischemia/ reperfusion (MI/R) injury. Recombinant human ADAMTS13 (rhADAMTS13), cleaving von Willebrand factor (VWF), reduces leukocyte recruitment in mice. Death of cardiomyocytes and the possible formation of neutrophil extracellular traps (NETs) may result in chromatin release that is prothrombotic and cytotoxic. We investigated the pathophysiological role of extracellular chromatin during MI/R to evaluate the therapeutic potential of targeting extracellular DNA and VWF by using DNase I with/without rhADAMTS13. Finally, we examined the impact of histone citrullination and NETosis by peptidylarginine deiminase 4 (PAD4) on MI/R. We used a 24-hour MI/R mouse surgical model. MI/R injury caused an increase in plasma nucleosomes, abundant neutrophil infiltration, and the presence of citrullinated histone H3 at the site of injury. Both monotherapies and coadministration of DNase I and rhADAMTS13 revealed a cardioprotective effect, resulting in subsequent improvement of cardiac contractile function. PAD4 2/2 mice, which do not produce NETs, were also significantly protected from MI/R and DNase I treatment had no further beneficial effect. We demonstrate that extracellular chromatin released through NETosis exacerbates MI/R injury. Targeting both VWF-mediated leukocyte recruitment and chromatin removal may be a new therapeutic strategy to reduce ischemia-related cardiac damage. (Blood. 2014;123(1):141-148)

Background A growing health problem, venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), requires refined diagnostic and therapeutic approaches. Neutrophils contribute to thrombus initiation and development in experimental DVT. Recent animal studies recognized neutrophil extracellular traps (NETs) as an important scaffold supporting thrombus stability. However, the hypothesis that human venous thrombi involve NETs has not undergone rigorous testing. Objective To explore the cellular composition and the presence of NETs within human venous thrombi at different stages of development. Patients and Methods We examined sixteen thrombi obtained from 11 patients during surgery or at autopsy using histomorphological, immunohistochemical and immunofluorescence analyses. Results We classified thrombus regions as unorganized, organizing, and organized according to their morphological characteristics. We then evaluated them focusing on neutrophil and platelet deposition as well as micro-vascularization of the thrombus body. We observed evidence of NET accumulation, including the presence of citrullinated histone H3 (H3Cit)-positive cells. NETs, defined as extracellular diffuse H3Cit areas associated with myeloperoxidase and DNA, localized predominantly during the phase of organization in human venous thrombi. Conclusions NETs are present in organizing thrombi in patients with VTE. They are associated with thrombus maturation in humans. Dissolution of NETs might thus facilitate thrombolysis. This finding provides new insights into the clinical development and pathology of thrombosis and provides new perspectives for therapeutic advances.

In addition to its established roles in the haemostatic system, thrombin is an intriguing coagulation protease demonstrating an array of effects on endothelial cells, vascular smooth muscle cells (VSMC), monocytes, and platelets, all of which are involved in the pathophysiology of atherosclerosis. There is mounting evidence that thrombin acts as a powerful modulator of many processes like regulation of vascular tone, permeability, migration and proliferation of VSMC, recruitment of monocytes into the atherosclerotic lesions, induction of diverse pro-inflammatory markers, and all of these are related to the progression of cardiovascular disease. Recent studies in transgenic mice models indicate that the deletion of the natural thrombin inhibitor heparin cofactor II promotes an accelerated atherogenic state. Moreover, the reduction of thrombin activity levels in apolipoprotein E-deficient mice, because of the administration of the direct thrombin inhibitor melagatran, attenuates plaque progression and promotes stability in advanced atherosclerotic lesions. The combined evidence points to thrombin as a pivotal contributor to vascular pathophysiology. Considering the clinical development of selective anticoagulants including direct thrombin inhibitors, it is a relevant moment to review the different thrombin-induced mechanisms that contribute to the initiation, formation, progression, and destabilization of atherosclerotic plaques.

Background-Thrombin generation in vivo may be important in regulating atherosclerotic progression. In the present study, we examined for the first time the activity and presence of relevant coagulation proteins in relation to the progression of atherosclerosis. Methods and Results-Both early and stable advanced atherosclerotic lesions were collected pairwise from each individual (nϭ27) during autopsy. Tissue homogenates were prepared from both total plaques and isolated plaque layers, in which the activity of factors (F) II, X, and XII and tissue factor was determined. Microarray analysis was implemented to elucidate local messenger RNA synthesis of coagulation proteins. Part of each specimen was paraffin embedded, and histological sections were immunohistochemically stained for multiple coagulation markers with the use of commercial antibodies. Data are expressed as median (interquartile range [IQR] Key Words: atherosclerosis Ⅲ hypercoagulability Ⅲ immunohistochemistry Ⅲ plaque Ⅲ thrombosis A therosclerosis is widely recognized as a chronic inflammatory disease. 1 Rupture of an atherosclerotic plaque is considered the predominant underlying cause of acute atherothrombotic events such as myocardial infarction, ischemic stroke, and vascular death. A close relation between blood coagulation and atherosclerosis 2,3 is supported by studies revealing the presence of specific coagulation proteins within an atherosclerotic lesion. Tissue factor (TF) and factor (F) VII, of which the complex is the principal initiator of coagulation in vivo, are expressed on macrophages and vascular smooth muscle cells (SMC) within the arterial vessel wall and atherosclerotic lesion. 4,5 Both proteins potentially participate in multiple proatherogenic processes such as migration and proliferation of SMC, 6 inflammation, and angiogenesis. 7 In addition to the single effects of each protein, the local interaction between macrophage/SMC-derived TF and FVII may provide a catalytic complex for subsequent generation of thrombin and fibrin, of which the latter is also detectable in atherosclerotic lesions. 8,9 The procoagulant condition of the atherosclerotic lesion may be further enhanced by the presence of various proinflammatory cytokines (eg, tumor necrosis factor-␣, interleukin-1 10 ), which may downregulate local expression of anticoagulant proteins such as thrombomodulin and the endothelial protein C receptor on endothelial cells. 11 Received September 4, 2009; accepted June 28, 2010 Clinical Perspective on p 830Thrombin, a key enzyme in blood coagulation, may also play a critical role in many processes related to the development, progression, and atherothrombotic potential of atherosclerotic plaques. 12 Direct evidence for the role of thrombin in the atherogenic process comes from experiments showing reduced progression of atherosclerosis in apolipoprotein E Ϫ/Ϫ mice on pharmacological inhibition of thrombin. 13 Moreover, decreased expression of TF pathway inhibitor (TFPI) on an apolipoprotein E Ϫ/Ϫ background increased the atheroscle...

BackgroundVariations 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 FindingsWe 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/SignificanceWe 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.

Recent data indicate an important contribution of coagulation factor (F)XII to in vivo thrombus formation. Because fibrin structure plays a key role in clot stability and thrombosis, we hypothesized that FXII(a) interacts with fibrin(ogen) and thereby regulates clot structure and function. In plasma and purified system, we observed a dose-dependent increase in fibrin fiber density and decrease in turbidity, reflecting a denser structure, and a nonlinear increase in clot stiffness with FXIIa. In plasma, this increase was partly independent of thrombin generation, as shown in clots made in prothrombindeficient plasma initiated with snake venom enzyme and in clots made from plasma deficient in FXII and prothrombin. Purified FXII and ␣-FXIIa, but not ␤-FXIIa, bound to purified fibrinogen and fibrin with nanomolar affinity. Immunostaining of human carotid artery thrombi showed that FXII colocalized with areas of dense fibrin deposition, providing evidence for the in vivo modulation of fibrin structure by FXIIa. These data demonstrate that IntroductionBlood coagulation culminates in the formation of fibrin, which binds platelets and forms a clot. Fibrin is formed from fibrinogen via cleavage of 2 fibrinopeptides from the A␣-and B␤-chains N-termini, located in the E-region, by thrombin. 1 Fibrinopeptide cleavage exposes binding sites for complementary sites in the D-region, triggering polymerization and the production of protofibrils. Protofibrils aggregate laterally to form fibers, which branch out and form a 3-dimensional network. 2 There is increasing evidence that the structure of fibrin regulates thrombosis. Dense fibrin clots with small pores and increased fiber density are more resistant to lysis. 3 Structural characteristics affect the mechanical properties of fibrin. 4 Both venous and arterial thrombosis has been associated with the formation of an altered fibrin network. [5][6][7][8][9][10] The role of factor (F)XII in hemostasis has long been contested because deficiency in FXII, unlike deficiencies of other coagulation factors, does not lead to bleeding diathesis in humans 11 or in mice. 12 However, recent in vivo data show that FXII deficiency or inhibition in rodent models reduces thrombus formation while maintaining normal hemostasis. [12][13][14][15] These findings indicate the existence of FXII-related mechanisms that are preferentially involved in thrombosis but not hemostasis.Contact activation is triggered by the binding of FXII (80 kDa) to a negatively charged surface and involves the formation of ␣-FXIIa via autocatalysis. Bound ␣-FXIIa converts prekallikrein into kallikrein. Kallikrein can further convert ␣-FXIIa to ␤-FXIIa by an additional cleavage at R334-N335. ␣-FXIIa consists of a heavy and light chain that are disulphide linked (80 kDa), whereas ␤-FXIIa (28 kDa) lacks the heavy chain and loses its capacity to bind to negatively charged surfaces. 16 The N-terminal region of FXII (␣-FXIIa heavy chain) shows strong homology with tissuetype plasminogen activator (tPA), with the presence of fibr...