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 $TM^{Pro/Pro}:ApoE^{−/−}$ 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 $TM^{Pro/Pro}:ApoE^{−/−}$ 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

Anticoagulant therapy in critical organ ischaemia/reperfusion injury

Ischaemia/reperfusion (I/R) injury is central to a number of pathologies including myocardial infarction and stroke. Several cellular processes are involved in the progress of I/R injury, involving complex interactions between coagulation and inflammatory or apoptotic processes. Besides for their anti-coagulant function, anticoagulant proteins such as activated protein C (APC), active site inhibited factor Vila (ASIS), tissue factor pathway inhibitor (TFPI), and antithrombin (AT) are also known for their anti-inflammatory or cell protective effects. This review gives an overview of the application of these anti-coagulants in several animal models of I/R injury in critical organs and describes the effects of these proteins on cellular processes including inflammation and apoptosis. The future testing of mutant forms of some of these inhibitors including APC in a clinical setting should be actively explored

The structure-function relationship of activated protein C Lessons from natural and engineered mutations

Protein C is the central enzyme of the natural anticoagulant pathway and its activated form APC (activated protein C) is able to proteolyse non-active as well as active coagulation factors V and VIII. Proteolysis renders these cofactors inactive, resulting in an attenuation of thrombin formation and overall down-regulation of coagulation. Presences of the APC cofactor, protein S, thrombomodulin, endothelial protein C receptor and a phospholipid surface are important for the expression of anticoagulant APC activity. Notably, APC also has direct cytoprotective effects on cells: APC is able to protect the endothelial barrier function and expresses anti-inflammatory and anti-apoptotic activities. Exact molecular mechanisms have thus far not been completely described but it has been shown that both the protease activated receptor 1 and EPCR are essential for the cytoprotective activity of APC. Recently it was shown that also other receptors like sphingosine 1 phosphate receptor 1, Cd11b/CD18 and tyrosine kinase with immunoglobulin-like and EGF-like domains 2 are likewise important for APC signalling. Mutagenesis studies are being performed to map the various APC functions and interactions onto its 3D structure and to dissect anticoagulant and cytoprotective properties. The results of these studies have provided a wealth of structure-function information. With this review we describe the state-of-the-art of the intricate structure-function relationships of APC, a protein that harbours several important functions for the maintenance of both humoral and tissue homeostasi

Contribution of platelet glycoprotein VI to the thrombogenic effect of collagens in fibrous atherosclerotic lesions.

Collagens (types I and III) are among the strongest thrombus-forming components of the vascular subendothelium. We compared the thrombogenic effects of four collagen-containing advanced atherosclerotic lesions with those of purified types I and III collagen fibers. Cell-free homogenates from the human plaques effectively promoted platelet adhesion and aggregate formation under high-shear flow conditions, as well as exposure of procoagulant phosphatidylserine (PS) on platelets. With all plaques, blocking of the glycoprotein VI (GPVI) receptor for collagen abolished aggregation and PS exposure. Blocking of platelet ADP receptors resulted in similar, but less complete inhibitory effects. Type I collagen was more potent than type III collagen in inducing aggregation and PS exposure under flow, via stimulation of GPVI and ADP receptors. Type I collagen also more strongly enhanced thrombin generation with platelets and tissue factor, again via GPVI activation and PS exposure. The plaque material enhanced thrombin generation, partly due to the presence of tissue factor and partly via GPVI and ADP receptors. Together, these results indicate that in advanced plaques collagen type I is a major trigger of thrombus formation and PS exposure, acting via GPVI and ADP release, while tissue factor directly enhances coagulation

The effects of variations in coagulation potential on atherogenesis in a spontaneous atherosclerosis model at 35 weeks on a regular chow diet.

<p>(A) Top row represents images of the aortic arch and its main branches, stained with hematoxylin and eosin (H&E), used to analyze the extent of atherosclerotic plaque burden. To determine plaque phenotype characteristics, sections were stained against α-smooth muscle actin (vascular smooth muscle cell content – second row), MAC-2⁺ (macrophage infiltration – third row), Ly-6G (neutrophil recruitment – fourth row) and with Sirius red (collagen – bottom row). (B) Hypocoagulability in FII^−/+:ApoE^−/− significantly attenuated atherosclerosis plaque development (90.6±35.1*10³ µm² total plaque burden) when compared to normal ApoE^−/− mice (160.6±65.9*10³ µm²)(n = 10 per group, p = 0.0084). Total plaque area in TM^Pro/Pro:ApoE^−/− mice was established 389.1±158.4*10³ vs. 187.0±35.1*10³ µm² in the corresponding control ApoE^−/− group (n = 10 per group, p = 0.0010). (C) TM^Pro/Pro:ApoE^−/− mice atherosclerotic plaques demonstrated a significant decrease in intimal vascular smooth muscle cell content (2.2±1.3% of plaque area) compared to ApoE^−/− mice (8.7±2.9% of plaque area)(n = 10 per group, p = 0.0016). Recruitment of macrophages within the lesions did not differ between all experimental groups (D). Neutrophil infiltration was significantly diminished in the lesions of hypocoagulable FII^−/+:ApoE^−/− mice (n = 10 per group, p = 0.0092 vs. ApoE^−/− mice), and substantially increased in the TM^Pro/Pro:ApoE^−/− intima (n = 10 per group, p = 0.0094 vs. ApoE^−/− mice) (E). A similar trend was observed with regard to collagen deposition within the atherosclerotic plaques. In FII^−/+:ApoE^−/− mice, 29.3±3.6% of the plaque area stained collagen-positive (n = 10 per group, p = 0.0002 vs. ApoE^−/− mice). In contrast, Sirius red staining showed only 4.1±3.0% positivity for collagen in the TM^Pro/Pro:ApoE^−/− lesions (n = 10 per group, p = 0.0002 vs. ApoE^−/− mice) (F). By 35 weeks (established duration of the experiment), we recorded the following fatal events: 6 of 16 TM^Pro/Pro:ApoE^−/−, 1 of 11 FII^−/+:ApoE^−/− and 0 of 20 ApoE^−/− control mice. Dead mice were not included from the study analyses. The exact cause of death remained unclear. Kaplan-Meier analysis of the survival data comparing TM^Pro/Pro:ApoE^−/− vs. ApoE^−/− mice, as determined by the Gehan-Breslow-Wilcoxon test, indicated that hypercoagulability is linked to significantly higher spontaneous mortality rates (p = 0.0165) (G). No significant difference was found between FII^−/+:ApoE^−/− and ApoE^−/− control mice (p = 0.3173) (data not shown).*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. Error bars represent mean ± SD. Arrows indicate examples of positive staining. Abbreviations: H&E – hematoxylin and eosin; α-SMA - α-smooth muscle actin; SR – Sirius red.</p

Inhibition of thrombin activity by administration of direct thrombin inhibitor Dabigatran etexilate or recombinant murine APC substantially attenuates leukocyte recruitment and prevents against severe atherosclerosis progression and atherothrombosis.

<p>(A) Representative hematoxylin and eosin (H&E)-stained sections of atherosclerotic lesions formed in carotid arteries of TM^Pro/Pro:ApoE^−/− mice, which were assigned to different intervention arms (oral Dabigatran etexilate - 7.5 mg DE/gram chow; i.p. administered bolus doses of recombinant murine APC - 2.5 mg/kg/per every 5 days; or placebo) for a total of 6 weeks after cuff placement around the common carotid arteries (top row). Toluidine blue (TB) stainings were used to quantify the size of necrotic core areas (second and third row). Whereas placebo treated TM^Pro/Pro:ApoE^−/− mice all developed advanced lesions (identified by the presence of necrotic core and fibrous cap formation), Dabigatran etexilate- (3 out of 10, Pearson's chi-squared test (<i>χ</i>²), n = 10 per group, p = 0.0031 vs. placebo) and rAPC-treated mice (5 out of 10, Pearson's chi-squared test (<i>χ</i>²), n = 10 per group, p = 0.0325 vs. placebo) had significantly reduced atheromata formed. A total of 5 out 10 animals in the placebo group showed signs of severe plaque vulnerability, whereas none were observed in the intervention arms. Atherosclerotic plaques were further analyzed for the presence of macrophages (MAC-2, red color, fourth row) and neutrophils (Ly-6G, green color, bottom row). Arrows show examples of positive cells. Macrophage and neutrophil infiltration were expressed as the absolute number of Mac-2⁺ and Ly-6G⁺ cells per plaque. (B) Administration of either Dabigatran etexilate or rAPC rescued the phenotype and pronouncedly reduced atherosclerotic plaque burden (Placebo: 154.3±35.5*10³ µm²; Dabigatran Etexilate: 3.3±4.4*10³ µm², p<0.001; rAPC: 7.9±5.5*10³ µm², p<0.01; n = 10 per group). (C, F, G) These findings were further consolidated by a significant decrease in the degree of stenosis (with ∼80%), intima/media ratio and outward remodeling within the treatment arms of the study (n = 10 per group). (D, E) Except for a significant reduction of the necrotic core area in the Dabigatran etexilate-treated mice as compared to placebo group (n = 10 per group, p<0.05), no other effects were observed with regard to necrotic core formation or fibrous cap thickness. Of note, only mice having advanced lesions were included in these analyses (Dabigatran etexilate: n = 3; rAPC: n = 5). (H, I) In addition, TM^Pro/Pro:ApoE^−/− mice treated with direct thrombin inhibitor or natural anticoagulant rAPC developed an anti-inflammatory stable plaque phenotype, associated with substantially reduced levels of macrophage and neutrophil recruitment. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. Error bars represent mean ± SD. Arrows indicate examples of positive staining/fibrous cap thickness. DNA was counterstained with Hoechst-33342 (blue). Abbreviations: HFD – high-fat diet; AL – advanced atherosclerotic lesion; MФ- macrophage; rAPC – recombinant murine activated protein C.</p

Morphometrical analysis of periadventitial cuff-induced atherosclerosis in mice with genetically imposed alterations in blood coagulation potential.

<p>(A) Representative hematoxylin and eosin (H&E)-stained sections of carotid arteries of FII^−/+:ApoE^−/−, TM^Pro/Pro:ApoE^−/− and control ApoE^−/− 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 (26.5±12.6*10³ in FII^−/+:ApoE^−/− vs. 69.2±18.4*10³ µm² in ApoE^−/− control mice, n = 10 per group, p<0.0001), pro-thrombotic mice developed severe and occlusive atherosclerotic burden (146.4±52.7*10³ in TM^Pro/Pro:ApoE^−/− vs. 53.9±27.0*10³ µm² in ApoE^−/− control mice, n = 10 per group, p = 0.0001). The degree of stenosis in TM^Pro/Pro:ApoE^−/− reached an average of 88.6±8.1% (vs. 62.2±16.1% in ApoE^−/− mice, n = 10 per group, p = 0.0002), whereas it was substantially lower in FII^−/+:ApoE^−/− mice (36.8±11.9% vs. 64.9±9.6% in ApoE^−/− mice, n = 10 per group, p<0.0001). (A, D) Pearson's chi-squared test (<i>χ</i>²) detected a significant difference in the number of advanced atherosclerotic lesions (presence of fibrous cap atheromata <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055784#pone.0055784-Virmani1" target="_blank">[54]</a>) formed between FII^−/+:ApoE^−/− (4 out of 10) and TM^Pro/Pro:ApoE^−/− 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: 56.2±10.8% of the total plaque area, as compared to 29.0±17.7% in the control ApoE^−/− 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^−/− mice (n = 10 per group, p = 0.0081). (F) Intima/media ratio was significantly increased in TM^Pro/Pro:ApoE^−/− mice, whereas profoundly decreased in FII^−/+:ApoE^−/− mice. Of note, the average outer diameter of the common carotid artery is 0.36 mm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055784#pone.0055784-vonderThusen1" target="_blank">[21]</a>, thus suggesting that TM^Pro/Pro:ApoE^−/− atherosclerotic plaques undergo a dramatic outward remodeling as indicated in panel (G). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. Error bars represent mean ± SD. Arrows indicate examples of positive staining/fibrous cap thickness. Abbreviations: H&E – hematoxylin and eosin; AL – advanced atherosclerotic lesion.</p

Hypercoagulability induces oxidative stress in granulocytes within the bone marrow compartment.

<p>Granulocytes and monocytes cell fractions in the bone marrow were significantly increased in TM^Pro/Pro:ApoE^−/− as compared to ApoE^−/− control mice after 8 weeks on a regular chow diet (Granulocytes: 26.3±3.6% vs. 22.9±3.4%; n = 12 per group, p = 0.0292)(Monocytes: 12.3±0.6% vs. 8.8±0.7%; n = 12 per group, p<0.0001) (A, B). The significant increase in monocytes can be explained by the higher relative numbers of Ly6C^HIGH monocyte cells in TM^Pro/Pro:ApoE^−/− mice bone marrow (Ly6C^HIGH cells: 9.4±1.3% vs. 6.3±0.8%; n = 12 per group, p = 0.0002) (C). Using DHR123 FACS analysis, we analyzed the amount of oxidative burst activity in granulocytes and monocytes in the bone marrow after PMA stimulation. The monocytes did not show any differences in DHR signal and thus ROS activity, whereas in the granulocytes of the TM^Pro/Pro:ApoE^−/− mice, a significant increase was observed in the DHR signal when compared to ApoE^−/− mice, indicating enhanced oxidative stress upon PMA stimulation in the TM^Pro/Pro:ApoE^−/− granulocytes present in the bone marrow (D, E). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. Error bars represent mean ± SD. Abbreviations: DHR123– Dihydrorhodamine 123; ROS – Reactive Oxygen Species; PMA - Phorbol 12-Myristate 13-Acetate.</p

Hypercoagulable TM^Pro/Pro:ApoE^−/− mice – a new mouse model of atherosclerotic plaque vulnerability.

<p>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^−/− 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^−/− 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^−/− mice plaques <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055784#pone.0055784-Jackson1" target="_blank">[55]</a>, 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).</p

The role of hypo- and hypercoagulability in plaque fibrosis.

<p>Picrosirius red-stained sections assessed by light (<b>A, top row</b>) and polarized light (<b>A, second row</b>), indicate a significant decrease in the levels of collagen in TM^Pro/Pro:ApoE^−/− carotid atherosclerotic plaques (6.7±4.3% vs. 14.3±7.8% of total plaque area in ApoE^−/− mice, n = 10 per group, p = 0.0193) (<b>B</b>). Hypocoagulable FII^−/+:ApoE^−/− mice lesions showed a pro-fibrotic appearance, testified by increased collagen deposition (24.4±14.1% vs. 12.0±6.1% of total plaque area in ApoE^−/− mice, n = 10 per group, p = 0.0435) and α-smooth muscle actin content (25.5±13.6% vs. 6.9±3.2% of total plaque area in ApoE^−/− mice, n = 10 per group, p = 0.0003) (<b>B, C</b>). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. Error bars represent mean ± SD. Arrows indicate examples of positive staining. Abbreviations: SR – (Picro)sirius red; α-SMA - α-smooth muscle actin.</p