Early Atherosclerosis Exhibits an Enhanced Procoagulant State
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 (n=27) 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]). Tissue factor, FII, FX, and FXII activities were significantly higher in early atherosclerotic lesions than in stable advanced atherosclerotic lesions. Endogenous thrombin potential and thrombin-antithrombin complex values consolidated a procoagulant profile of early atherosclerotic lesions (endogenous thrombin potential, 1240 nmol/L · min [IQR, 1173 to 1311]; thrombin-antithrombin complex, 1045 ng/mg [IQR, 842.6 to 1376]) versus stable advanced atherosclerotic lesions (endogenous thrombin potential, 782 nmol/L · min [IQR, 0 to 1151]; thrombin-antithrombin complex, 718.4 ng/mg [IQR, 508.6 to 1151]). Tissue factor, FVII, and FX colocalized with macrophages and smooth muscle cells. In addition, multiple procoagulant and anticoagulant proteases were immunohistochemically mapped to various locations throughout the atherosclerotic vessel wall in both early and advanced atherosclerotic stages.
Conclusions— This study shows an enhanced procoagulant state of early-stage atherosclerotic plaques compared with advanced-stage plaques, which may provide novel insights into the role of coagulation during atherosclerotic plaque progression.
Received September 4, 2009; accepted June 28, 2010.
Atherosclerosis 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 atherosclerosis2,3 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-α, interleukin-110), which may downregulate local expression of anticoagulant proteins such as thrombomodulin and the endothelial protein C receptor on endothelial cells.11
Clinical Perspective on p 830
Thrombin, 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−/− mice on pharmacological inhibition of thrombin.13 Moreover, decreased expression of TF pathway inhibitor (TFPI) on an apolipoprotein E−/− background increased the atherosclerotic burden.14
Because of the reported involvement of procoagulant and anticoagulant coagulation factors during plaque progression, we hypothesized that the overall distribution and activity of coagulation proteins in the arterial vessel wall correlate with the extent and progression of atherosclerotic lesions. Moreover, we hypothesized that the amount of thrombin that can be generated from atherosclerotic tissue homogenates depends not only on the amount of TF but also on the presence and activity of other coagulation proteins that either amplify or dampen thrombin generation. Hence, we studied the localization of all coagulation proteins, in addition to the TF/FVII complex, on histologically defined early and stable advanced atherosclerotic lesions. In addition to thrombin generation, we determined the procoagulant activity of several coagulation proteins in the same lesions.
Patient Characteristics and Tissue Specimens
The tissue specimens were obtained from the Maastricht Pathology Tissue Collection. Collection, storage, and use of tissue and patient data were performed in agreement with the Code for Proper Secondary Use of Human Tissue in the Netherlands (http://www.fmwv.nl). Both early atherosclerotic lesions (EAL) and stable advanced atherosclerotic lesions (SAAL) were collected pairwise from each corresponding individual (n=27) during postmortem dissection of the abdominal aortas within 8 hours of death. Autopsy specimens were obtained from adult men and women with an age range of 45 to 84 years (mean, 55 years). Clinical characteristics of the patients are provided in Table 1. The cause of death was diverse (eg, myocardial infarction, stroke). Individuals with sepsis or cancer were excluded. All tissue specimens were histologically evaluated on hematoxylin and eosin–stained sections (4 μm). Plaque subtypes were determined in compliance with the modified American Heart Association classification, based on morphological description, proposed by Virmani et al.15 Because one of the main goals in this study was to discriminate the overall prothrombotic potential of atherosclerotic lesions between early and advanced stages of development, we classified the plaques as follows: intimal thickenings and xanthomas are uniformly termed EAL, whereas all types of stable advanced plaques are termed SAAL. Complicated lesions, including lesions with intraplaque hemorrhage, a surface defect, and/or thrombotic deposit, were not included in this study.
Preparation of Tissue Homogenates
A section of each of the collected specimens (27 EAL/27 SAAL, obtained in pairs from n=27) was snap-frozen on collection. Snap-frozen atherosclerotic tissues were freeze-dried for 3 days and pulverized, and subsequently the tissue powders were dissolved in 50 mmol/L N-octyl β-d-glucopyranoside (Sigma-Aldrich) in HN buffer (25 mmol/L HEPES, 175 mmol/L NaCl, pH 7.7), vortexed, and centrifuged twice (10 minutes, 13 000 rpm). Total protein content of the tissue homogenates was spectrophotometrically determined with the use of the Biorad DC Protein Assay system according to the manufacturer’s instructions (Bio-Rad Laboratories B.V., Veenendaal, Netherlands). All samples were further diluted into a final concentration of 5 mg/mL.
Effect of Time Delay Between Death and Postmortem Examination on Coagulation Protein Activity
See Methods in the online-only Data Supplement.
EAL and SAAL Layer Preparation and Homogenization
See Methods in the online-only Data Supplement.
Thrombin Generation, Prothrombin, FX, and FXII Activity Assays, Thrombin-Antithrombin Complex Levels, TF Activity Assay, and TFPI Antigen Assay
The calibrated automated thrombogram (Thrombinoscope, the Netherlands) was used to determine the contribution of atherosclerotic tissue homogenates to thrombin generation in human plasma (in triplicate; interassay coefficient of variation <10%). For additional information, see Methods in the online-only Data Supplement.
Effect of Phospholipid Concentration on Thrombin Generation in Normal Arterial Vessel Wall Homogenates
See Methods in the online-only Data Supplement.
RNA Isolation and Quantification, Microarray Hybridization, and Data Analysis
See Methods in the online-only Data Supplement.
Immunohistochemical and Immunofluorescence Stainings and Immunohistological Evaluation
See Methods in the online-only Data Supplement.
Data analysis was computed with SPSS, version 17.02 (SPSS Inc, Chicago, Ill) and Prism, version 5.00 (GraphPad Software Inc, San Diego, Calif). Results are expressed as median (interquartile range [IQR]). An exact-distribution Wilcoxon 2-sample test was used for all intraindividual comparisons. A 2-tailed P<0.05 was considered statistically significant. Repeated-measures ANOVA was used to assess differences in coagulation protein activity over time.
EAL Exhibit Higher Functional Activity of Key Coagulation Proteins Than SAAL Ex Vivo
We determined the dependence of the thrombin-generating potential of both EAL and SAAL on their prothrombin, FX, and FXII content. From all 54 specimens (27 pairs, EAL and corresponding SAAL) that we examined, prothrombin activity was detected in only 11 samples. From the latter 11 samples with detected activity, EAL specimens had significantly higher prothrombin activity at 0.0% (IQR, 0.0 to 7.761) compared with their paired SAAL at 0.0% (IQR, 0.0 to 0.0) (Figure 1A; Wilcoxon 2-sample test, 2-tailed exact P<0.05). The activity of FX revealed a similar trend, with a significant 3-fold upregulation in EAL at 0.276% (IQR, 0.164 to 0.536) compared with SAAL at 0.136% (IQR, 0.054 to 0.237) (Figure 1B; Wilcoxon 2-sample test, 2-tailed exact P<0.05). Furthermore, FXII also demonstrated significantly higher activity levels in EAL of 2.636% (IQR, 1.344 to 3.372) compared with levels in SAAL of 0.930% (IQR, 0.337 to 1.526) (Figure 1C; Wilcoxon 2-sample test, 2-tailed exact P<0.05).
EAL Demonstrate a 3-Fold Increase in TF Activity Versus SAAL
To better appreciate the procoagulant potential of these 2 sets of atherosclerotic plaque homogenates, we assessed the activity of TF, which is known to be a pivotal trigger of coagulation in vivo. TF activity was ≈3-fold higher (0.036 pmol/mg [IQR, 0.017 to 0.055]) in EAL compared with SAAL (0.009 pmol/mg [IQR, 0.005 to 0.022]) (Figure 1D; Wilcoxon 2-sample test, 2-tailed exact P<0.05). Twenty-six of 27 EAL homogenates indicated elevated TF activity levels compared with their corresponding SAAL specimens.
Notably, the 8-hour window between death and postmortem collection did not significantly affect the activity of TF, FII, FX, and FXII in atherosclerotic lesions harvested at various time points: 0 (baseline), 2, 4, and 8 hours. No significant differences were found between the different time points of all tested proteins and also compared with baseline values (Figure I in the online-only Data Supplement), strongly suggesting that the postmortem values reflected actual coagulation activity in vivo.
Shift of the TF/TFPI Ratio Suggests an Increased Atherothrombotic Tendency in EAL
TFPI is a potent natural inhibitor of the TF-driven pathway of the coagulation cascade and also plays an important role in regulating inflammation. Furthermore, it has been shown that TFPI modulates thrombus formation in experimental models in vivo,16 primarily by attenuating the procoagulant activity and overexpression of TF.17,18 Therefore, we tested the levels of TFPI by utilizing a homemade enzyme-linked immunosorbent assay. An ≈1.6-fold significant increase in TFPI antigen levels was found in EAL compared with SAAL. EAL demonstrated TFPI activity equal to 0.089 nmol/L per milligram (IQR, 0.072 to 0.140), whereas SAAL showed 0.056 nmol/L per milligram (IQR, 0.030 to 0.088) (Figure 1E; Wilcoxon 2-sample test, 2-tailed exact P<0.05). Despite the higher levels of TFPI antigen in the EAL homogenates, in SAAL the TF/TFPI balance in the early lesions remained in favor of TF, shown by the higher TF/TFPI ratios in EAL homogenates (Figure 1F; Wilcoxon 2-sample test, 2-tailed exact P<0.05).
Enhanced Thrombin Generation in EAL
In the absence of TF and entirely dependent on the procoagulant molecular content in the tissue homogenate, all 27 EAL induced thrombin formation in normal pooled plasma, showing significantly higher values (1240 nmol/L · min [IQR, 1173 to 1311]) compared with SAAL (782 nmol/L · min [IQR, 0 to 1151]) (Figure 2B; Wilcoxon 2-sample test, 2-tailed exact P<0.05). Twenty-six EAL induced higher endogenous thrombin potential than their corresponding advanced atheromas. For the SAAL, 10 lesions did not trigger any thrombin generation.
Furthermore, EAL showed a significantly increased thrombin generation potential compared with areas of normal aorta obtained from the same individuals (263.3 nmol/L · min [IQR, 117.8 to 350.3]; Wilcoxon 2-sample test, 2-tailed exact P<0.0001) (Figure 2B). In addition, SAAL also demonstrated significantly higher endogenous thrombin potential than their paired normal vessel homogenates (Wilcoxon 2-sample test, 2-tailed exact P=0.0053), thus consolidating the procoagulant tendency in early atherosclerosis.
Thrombin-Antithrombin Complex Levels Additionally Point to Higher Thrombin Generation in EAL Homogenates
Once generated, thrombin is inhibited on binding to antithrombin, thus forming a stable thrombin-antithrombin (TAT) complex. TAT complexes are considered a marker of in vivo intravascular thrombin generation; therefore, the main goal of this experiment was to assess whether there was an excess of activated FII generation in EAL in situ compared with their matched SAAL. The concentration of TAT complexes in EAL was significantly higher (1045 ng/mg [IQR, 842.6 to 1376]) compared with their paired SAAL homogenates (718.4 ng/mg [IQR, 508.6 to 1151]) (Figure 2C; Wilcoxon 2-sample test, 2-tailed exact P<0.05), confirming a more procoagulant state in EAL.
Layer-Selective Analysis of Coagulation Factor Activities Consolidated a More Procoagulant State of EAL Versus SAAL
To provide better insight into the procoagulant properties of the atherosclerotic lesions, we undertook a more selective, layer-specific analysis in which the potential procoagulant effects of the different vessel wall layers were studied. The activity of coagulation factors was analyzed in tissue homogenates prepared from tunica intima, media, and adventitia (histologically controlled anatomic separation; Figure 3A). All 3 layers were harvested in 42 specimens (21 pairs of EAL and SAAL from the original tissue collection). Endogenous thrombin potential values in all layers of EAL were found to be significantly higher (intima: 1489 nmol/L · min [IQR 1353 to 1680]; media: 1734 nmol/L · min [IQR, 1256 to 1983]; adventitia: 1872 nmol/L · min [IQR, 1655 to 2171]) compared with the corresponding SAAL layers (intima: 437.9 nmol/L · min [IQR, 290.3 to 549.9]; media: 392.1 nmol/L · min [IQR, 219.7 to 680.9]; adventitia: 524.1 nmol/L · min [IQR, 394.1 to 787.7]) (Figure 3B; Wilcoxon 2-sample test, 2-tailed exact P<0.05, all). This strongly pronounced procoagulant state of the EAL layers was additionally confirmed by significantly elevated prothrombin, FX, and FXII levels (Figure 3C to 3E). Intimal layers of both EAL and SAAL showed comparable TF activity, whereas TF was significantly increased in media and adventitia of EAL versus SAAL (Figure 3F). Although they demonstrated comparable activities in terms of TF, EAL intimal layers contained significantly higher TFPI levels (Figure 3G), yielding a significantly lower TF/TFPI ratio in EAL compared with SAAL. EAL and SAAL media layers did not significantly differ in TFPI levels, whereas TFPI in EAL adventitia was significantly higher compared with SAAL (Figure 3G). Tunica adventitia exhibited the most procoagulant phenotype of all vessel wall layers in terms of thrombin generation. Its values in both EAL and SAAL were significantly higher than those measured in tunica intima and media.
Gene Expression of Coagulation Genes in EAL Versus SAAL
To better explore to what extent and which coagulation proteins are expressed on the genome level within the arterial vessel wall, gene expression profiles of EAL and SAAL were obtained with the use of microarray analysis. In a separate set of patients, early and advanced carotid lesions were collected from the same patient (at autopsy), and fold changes in gene expression were assessed by comparing the advanced lesions with the early lesions. The results indicated that several coagulation factor genes were expressed in both types of atherosclerotic lesions. After correction for multiple testing, 14 coagulation genes showed significant differential transcript levels between EAL and SAAL. Figure 4 demonstrates the relative mRNA levels, described as the SAAL/EAL ratio. Of these 14 differentially regulated genes, 6 were upregulated in EAL (expressed as fold change <−1.0), whereas 8 were upregulated in SAAL (expressed as fold change >+1.0) (Table I in the online-only Data Supplement). Fold changes ranged from −1.13 to −2.96 for the upregulated genes in EAL and from 1.08 to 1.29 for the upregulated genes in SAAL. Additional information is provided in Table I in the online-only Data Supplement.
Immunohistochemical Staining: EAL
In EAL, moderate (fibrinogen/fibrin, FIX, TFPI) to strong positivity for von Willebrand factor, FX, prothrombin/thrombin, protein S, and activated protein C (APC) was observed in the endothelial luminal cells, indicated by a sharp demarcation of the endothelial lining (Figure 5 and Table 2). In addition, a positive focal endothelial distribution for TF, FVII, FXII, FXI, kallikrein, and thrombomodulin was shown. Macrophages and foam cells stained intensely positive for TF, FVII, FX, prothrombin/thrombin, kallikrein, and FXI. Despite the fact that other coagulation proteins such as FXII, FIX, protein S, protein C, and APC were also expressed by macrophages and foam cells, their expression or immunoreactivity was either scarce or focal. Furthermore, EAL were characterized by TF, FVII, and FX expression throughout the SMC-rich intima. Medial SMC-associated FVII was located in the cytoplasm and not on the membrane. FXII and FII showed enhanced expression in medial SMC. TF, FVII, FX, fibrin, kallikrein, thrombomodulin, and TFPI were also associated with medial SMC, whereas FIX demonstrated a more patchy expression. Within the adventitia, the vasa vasorum externa showed positive staining for most of the studied factors, whereas the fibroblasts were positively associated with FX, prothrombin/thrombin, kallikrein, von Willebrand factor, and FXII.
Immunohistochemical Staining: SAAL
In atherosclerotic tissues classified as SAAL, the endothelial luminal lining was moderately positive for FIX, thrombomodulin, APC, and von Willebrand factor, whereas FXI stained weakly positive (Figure 5). Some endothelial segments demonstrated a focal expression of the anticoagulant protein protein S. Moreover, all anticoagulant proteins (protein S, thrombomodulin, APC, and TFPI) were found to be associated with macrophages and foam cells. Furthermore, thrombomodulin, APC, and TFPI were also localized in the endothelial cells of the vasa vasorum and in endothelial cells of vessels sprouting into the lesions. Besides thrombomodulin, intimal and medial SMC contained most of the procoagulant proteins, as well as thrombin and fibrinogen/fibrin (Figure 5 and Figure II in the online-only Data Supplement). A slight focal association between FIX and XI with intimal SMC was observed. In contrast, SMC of the media stained moderately for FXII. Some of the medial SMC stained positive for FXI but also showed double positive staining for both CD68 and anti–smooth muscle actin, suggesting either transdifferentiation of SMC into foam-like cells or SMC outgrowth from mononuclear cells; the latter was reported recently to be a thrombin-promoted action.19 TF and FVII, FX, FXI, and FXII were either weakly or focally present on macrophages, and FXII was also found on some foam cells. FIX showed a pronounced focal distribution on both macrophages and foam cells. Some fibroblasts contained FX, FXII, and fibrin.
None of the SAAL from the current set of lesions showed a strong or even moderately positive staining for TF; this was also the case in the necrotic core. The necrotic core revealed a focal presence for most of the procoagulant proteins, except for thrombin, fibrin, and the anticoagulant APC, which stained weakly positive.
Immunofluorescence Staining: Colocalization of TF/FVII/FX with Macrophages and Vascular SMC
Double immunofluorescence staining with CD68 for macrophages and anti–smooth muscle actin for SMC suggests that most of the macrophages and SMC were involved in the synthesis of TF, FVII, and FX. The formation of the ternary complex TF/FVII/FX is a potent trigger not only for coagulation (thrombin) but also for many proinflammatory cell-signaling pathways that are pivotal in cardiovascular disease. Therefore, we also examined the presence of these procoagulant proteins on macrophages and SMC by means of immunofluorescence staining on corresponding EAL and SAAL sections, which revealed that TF/FVII/FX colocalized with both macrophage/foam cells and SMC, suggesting a local system of thrombin generation, which may regulate pathophysiological processes such as cell migration and inflammation. When EAL and SAAL are compared, colocalization is scarcer and more diffuse in SAAL, whereas EAL sections show brighter labeling and denser character (Figure 6).
The present study shows that atherosclerotic plaques exhibit functional activity of many coagulation proteins (prothrombin, FX, FXII, and TF) and represents the first study to demonstrate the presence and distribution of all coagulation proteins in both early and advanced human atherosclerotic plaques. We provide new data pointing to local synthesis of several coagulation proteins within the atherosclerotic vessel wall. Furthermore, we indicate a colocalization of key procoagulant proteins with SMC and macrophages, suggesting an active, cell-based coagulation network within the atherosclerotic plaque. Finally, the principal finding of this study is an enhanced procoagulant profile of EAL compared with SAAL homogenates, consolidated by an elevated thrombin generation potential and significantly increased TAT complex levels in early-stage atherosclerotic tissues. Thus, we provide novel evidence that may help in widening the thrombogenic spectrum of “high-risk” plaques and suggest that local coagulation factors may play an important role not only in contributing to the onset of atherothrombosis but also in contributing to progression of the atherosclerotic process.
In contrast to our expectations, these data reject the initial hypothesis that thrombin generation would positively correlate with progression of atherosclerosis. One possible mechanism that might explain the abundant presence and functional activity of coagulation proteins in the early stage of atherosclerosis is that many of the coagulation proteins help to propagate the atheromatous plaque by inducing multiple proatherogenic actions such as cellular adhesion, migration, angiogenesis, and inflammation.7,12 In addition to their prothrombotic nature, coagulation proteases induce cell proliferation,6 and the latter are of great importance in determining the stability of an atherosclerotic lesion. The abundance of almost all (intrinsic and extrinsic) coagulation proteins suggests that the generation of thrombin is an active process during atherogenesis, supporting a major role of thrombin (and possibly fibrin) in this condition. Moreover, the enhanced procoagulant state of EAL was additionally confirmed by the layer-selective analysis, which also revealed a significantly increased prothrombotic phenotype for tunica intima, media, and adventitia in EAL versus SAAL. Previous reports have also documented that adventitial fibroblasts that surround the arterial walls contain high amounts of TF, providing a “hemostatic envelope.”20 Our study confirmed this latter finding by showing that tunica adventitia was the most procoagulant vessel wall layer of all those tested. A contributory or distinct effect of other coagulation proteases on atherogenesis, including FXII, FXI, FIX, and FX, cannot be ruled out. Published data have associated FXII in blood with cardiovascular disease, and although its action has contributed mainly to promoting arterial thrombosis, its presence in atherosclerosis and in the vicinity of macrophages and foam cells suggests cell-directed actions of this protein. Indeed, in vitro work has demonstrated the localization of contact system proteins on macrophages,21 suggesting a direct effect on inflammatory pathways. Of interest, although the expression and activity of FXII diminished on progression of atherosclerosis in our data, the staining for kallikrein was more abundant in the advanced lesions in the vicinity of macrophages and foam cells. The latter may be compatible with a switch in direction from a procoagulant to a proinflammatory action of the FXII contact system, as established in a recent study.22 Switching the action of FXII to an inflammatory direction may explain in part the diminished thrombin-generating capacity in advanced lesions, which are dominated by inflammatory characteristics (including elevated interleukin-6 and tumor necrosis factor-α levels; data not shown). The apparent loss in thrombomodulin staining within the advanced plaques may also be compatible with increased inflammatory activity, as proposed previously,11 whereas apparently the vasa vasorum maintains thrombomodulin in amounts comparable to those in the early lesion.
The abundant presence of coagulation proteins in the early lesions in particular raises other questions about causes and consequences. It is well known that the initial stage of atherosclerotic development (eg, intimal thickening) is characterized by enhanced SMC migration and proliferation.23 In contrast, an advanced stage of atherosclerotic progression results in decreased cell density, primarily around the fibrous caps and necrotic core/lipid pool.24,25 However, the decreased procoagulant potential in SAAL was found to be independent of the vessel wall structure alterations that occur on atherosclerotic progression (Figure 2A and 2B), thus suggesting that differences in protein translocation from circulation toward the vessel wall, as well as local protein expression by different cell types, may also contribute significantly to variations in protein levels. Although all coagulation proteins except TF may finally appear in the vessel wall by diffusion from the circulating blood, the localization suggests that local synthesis may be involved. Moreover, the microarray analysis clearly shows that multiple coagulation proteins are expressed on the level of mRNA synthesis in the arterial vessel wall (Figure 4 and Table I in the online-only Data Supplement). However, although some of the coagulation proteins were differentially upregulated in EAL, a similar picture was also observed for other coagulation genes in SAAL, suggesting that the differences in protein expression (as revealed by immunohistochemistry) and activity levels of coagulation proteins were not reflected completely by differences in gene expression levels. Moreover, it is known that RNA expression profiles do not always correlate with protein expression and subsequent biological activity.26 Nevertheless, our data point to local synthesis of several coagulation proteins within the atherosclerotic vessel wall, suggesting that this may be part of an active regulatory mechanism, leading subsequently to the enhanced procoagulant state in EAL.
The pleiotropic effects of proteases such as thrombin and activated FX, as well as the cell growth–promoting effects of fibrin (and its split products), may be also evoked as part of a response to injury mechanism. This response action of blood coagulation is now well established in inflammatory conditions like sepsis. As a side effect of this process, the formation of fibrin may serve to protect the early lesions from rupture and contribute to plaque stability. In addition, a recent study demonstrates that hypercoagulability in transgenic mice promotes plaque stability.27
At the same time, the activity of coagulation proteases contributes to local inflammation and angiogenesis, and therefore the latter will eventually prevail over processes such as proliferation, thus compromising plaque stability. This proinflammatory state of the evolving plaque, including increased apoptosis of SMC, gradual protein loss, and enhanced angiogenesis, will herald plaque evolution and greater vulnerability. Hence, EAL may be more stable because of more clotting activity, whereas SAAL may be more vulnerable because of instability. In the case of a plaque rupture, even relatively small amounts of TF and other proteins may still be highly thrombogenic, precipitating thrombus formation and cardiovascular events.
In conclusion, our findings provide substantial new data illustrating the close involvement of coagulation proteins in the entire process of atherogenesis. Whereas in the early lesions essentially all coagulation proteins, including those from the contact/intrinsic system, are readily detectable (possibly supporting plaque stability), on transformation to advanced lesions the amount and activity of these proteins diminish. The loss in coagulation activity, possibly due to increased inflammatory pressure, may reduce plaque stability and contribute to the risk of plaque rupture. These results point to various and specific functions of coagulation proteins in regulating progression of atherosclerosis and may provide novel insights into the genesis of atherothrombosis. These data also suggest ways to modulate atherogenesis and possibly reduce atherosclerosis that may eventually be clinically useful. The fact that new specific anticoagulant agents are being clinically tested underscores the necessity of further studies in this area.
Drs Heeneman and Daemen participate in the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Commission’s Sixth Framework Program for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254). We gratefully acknowledge Diane Fens, Patricia Pluijmen, and Mathijs Groeneweg for their skillful help in processing and measuring the specimens.
Sources of Funding
Marie Curie fellowships from the European Commission were granted to Dr Borissoff (MEST-CT-2005-020706) and Peter Kaššák (QLK5-CT-2000-6007). Evren Kilinç and Dr Winckers are sponsored by the Netherlands Heart Foundation (grants 2006-B064 and 2007-B138).
Research grants as principal investigators from the Center for Translational Molecular Medicine, the Netherlands, were granted to Drs Hackeng, Daemen, and ten Cate; from the Netherlands Heart Foundation were granted to Drs Hackeng and ten Cate; and from Fonds Economische Structuurversterking were granted to Dr Hackeng. Dr ten Cate received honoraria as a consultant to Boehringer Ingelheim GmbH. Dr Hackeng received honoraria from ACS-Biomarker B.V. The other authors report no conflicts.
Levi M, van der Poll T, Buller HR. Bidirectional relation between inflammation and coagulation. Circulation. 2004; 109: 2698–2704.
Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A. 1989; 86: 2839–2843.
Wilcox JN, Noguchi S, Casanova J. Extrahepatic synthesis of factor VII in human atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 2003; 23: 136–141.
Bini A, Fenoglio JJ Jr, Mesa-Tejada R, Kudryk B, Kaplan KL. Identification and distribution of fibrinogen, fibrin, and fibrin(ogen) degradation products in atherosclerosis: use of monoclonal antibodies. Arterioscler Thromb Vasc Biol. 1989; 9: 109–121.
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.
Bea F, Kreuzer J, Preusch M, Schaab S, Isermann B, Rosenfeld ME, Katus H, Blessing E. Melagatran reduces advanced atherosclerotic lesion size and may promote plaque stability in apolipoprotein E–deficient mice. Arterioscler Thromb Vasc Biol. 2006; 26: 2787–2792.
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: 3044–3046.
Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000; 20: 1262–1275.
Ragni M, Golino P, Cirillo P, Scognamiglio A, Piro O, Esposito N, Battaglia C, Botticella F, Ponticelli P, Ramunno L, Chiariello M. Endogenous tissue factor pathway inhibitor modulates thrombus formation in an in vivo model of rabbit carotid artery stenosis and endothelial injury. Circulation. 2000; 102: 113–117.
St Pierre J, Yang LY, Tamirisa K, Scherrer D, De Ciechi P, Eisenberg P, Tolunay E, Abendschein D. Tissue factor pathway inhibitor attenuates procoagulant activity and upregulation of tissue factor at the site of balloon-induced arterial injury in pigs. Arterioscler Thromb Vasc Biol. 1999; 19: 2263–2268.
Badimon JJ, Lettino M, Toschi V, Fuster V, Berrozpe M, Chesebro JH, Badimon L. Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques: effects of tissue factor pathway inhibitor on plaque thrombogenicity under flow conditions. Circulation. 1999; 99: 1780–1787.
Martin K, Weiss S, Metharom P, Schmeckpeper J, Hynes B, O'Sullivan J, Caplice N. Thrombin stimulates smooth muscle cell differentiation from peripheral blood mononuclear cells via protease-activated receptor-1, RhoA, and myocardin. Circ Res. 2009; 105: 214–218.
Mackman N. Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler Thromb Vasc Biol. 2004; 24: 1015–1022.
Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT, Regnstrom J, Fuster V. Human monocyte-derived macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques: potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation. 1995; 92: 1565–1569.
Seehaus S, Shahzad K, Kashif M, Vinnikov IA, Schiller M, Wang H, Madhusudhan T, Eckstein V, Bierhaus A, Bea F, Blessing E, Weiler H, Frommhold D, Nawroth PP, Isermann B. Hypercoagulability inhibits monocyte transendothelial migration through protease-activated receptor-1-, phospholipase-Cbeta-, phosphoinositide 3-kinase-, and nitric oxide-dependent signaling in monocytes and promotes plaque stability. Circulation. 2009; 120: 774–784.
Apart from their well-established role in coagulation, several hemostatic factors (eg, tissue factor/activated factor VII complex, activated factor X, thrombin) have been reported to evoke multiple proatherogenic events on a wide range of arterial wall constituents. While exploring the presence and distribution of all coagulation proteins in both early and advanced human atherosclerotic plaques, we found a colocalization of key procoagulant proteins with smooth muscle cells and macrophages, thus suggesting an active cell-based coagulation network within the atherosclerotic plaque. Furthermore, we provide new evidence pointing toward local synthesis of several coagulation factors within the atherosclerotic vessel wall. The principal finding of this study, indicating enhanced procoagulant activity of early atherosclerotic plaques versus stable advanced plaques, suggests a role for the hemostatic proteins and hypercoagulability in regulating the onset and progression of atherosclerosis. These findings may become clinically relevant in the new era of selective oral anticoagulants, in which such agents may have effects on the complex process of atherosclerosis beyond their direct antithrombotic action.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.907121/DC1.