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Keywords:

  • aortic stenosis;
  • fibrin;
  • fibrinolysis;
  • mast cells;
  • plasminogen activator inhibitor-1

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Study limitations
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References

Background

A role of fibrinolysis in the pathogenesis of aortic valve stenosis (AS) is unknown, although fibrinolytic proteins have been detected in aortic stenotic valves.

Objective

To investigate whether impaired fibrinolysis could be associated with AS.

Methods and Results

We studied 74 patients with AS (43 male, 31 female, aged 62.7 ± 10.7 years) without documented atherosclerotic valvular disease scheduled for isolated valve replacement and 68 controls. The plasma fibrin clot lysis time (CLT) in the presence of tissue factor (TF) and tissue plasminogen activator (tPA), along with plasma plasminogen activator inhibitor-1 (PAI-1) were determined. Valvular expression of fibrin and PAI-1 together with macrophages and mast cells (MC) was evaluated by immunostaining. Patients with AS compared with controls were characterized by a prolonged CLT (median, 110 [54–153] vs. 92.5 [58–115] min, = 0.0007) and increased plasma PAI-1 (78.6 [35.5–149] vs. 38.5 [18–61] ng mL−1, < 0.0001). CLT was correlated with maximal (= 0.43, = 0.0002) and mean (= 0.38, = 0.001) transvalvular pressure gradients, and aortic valve area (= −0.59, < 0.0001). In AS patients, the CLT was positively correlated with the valve leaflet thickness (= 0.67, = 0.003), the degree of valve calcification (= 0.65, < 0.00001), valvular fibrin (= 0.54, = 0.007) and PAI-1 expression (= 0.48, = 0.007). Double-immunostaining revealed colocalization of valvular PAI-1 with MC (87 ± 17% cells) and macrophages (48 ± 11% cells) within stenotic valves.

Conclusions

Hypofibrinolysis might be a marker of severe AS and be implicated in AS progression.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Study limitations
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References

Calcific aortic valve stenosis (AS) has a prevalence of 0.2% in 55- to 64-year-old individuals [1] and 2–3% in subjects older than 65 years [2]. This disease is commonly perceived as an active inflammatory process, which shares several common characteristics with atherosclerosis, including disruption of the basement membrane, subendothelial accumulation of intracellular lipids, lipoproteins and molecular mediators of calcification, together with infiltration of the inflammatory cells and activation of local and systemic inflammation [3]. The concept of AS as an atherosclerosis-like process is supported by a number of studies showing that AS is associated with cardiovascular risk factors such as smoking, hypercholesterolemia and arterial hypertension [4]. The main biological features observed in stenotic aortic valves are calcification and remodeling of the extracellular matrix, resulting from a disturbed balance between proteases and their inhibitors involved in those processes [5, 6]. One might hypothesize that systemic and local imbalance between the plasminogen activators and their inhibitors might contribute to the progression of valve pathology to severe AS [7].

Dissolution of a fibrin clot is mediated by the interaction of tissue plasminogen activator (tPA), released by endothelial cells, and plasminogen on the fibrin surface that accelerates the conversion of plasminogen to plasmin [8].

Plasminogen and tPA bind to lysine residues exposed by plasmin-mediated proteolysis of fibrin, further enhancing plasminogen conversion to plasmin. A key inhibitor of tPA, plasminogen activator inhibitor-1 (PAI-1), is released from blood platelets and endothelial cells. The fibrin structure largely affects the rate of fibrinolysis through a number of molecular mechanisms [9] associated with differences in accessibility of the clot to fibrinolytic proteins and in the binding of tPA and plasminogen to clots with different structures [10].

Reduced activity of the fibrinolytic system has been shown to predispose to arterial and venous thromboembolic events [11, 12]. Impaired fibrinolysis has been observed in patients with advanced atherosclerosis and those who experienced acute coronary syndrome [12].

The clot lysis time (CLT) calculated from the turbidity profile of the clot formation and clot lysis is thought to represent overall plasma fibrinolytic capacity. Determination of CLT introduced by Lisman et al. [13] has been successfully used to assess the overall activity of the fibrinolytic system in circulating blood in various clinical settings, including peripheral artery disease, stroke, myocardial infarction and premature atherosclerotic vascular disease [12-15]. In the general population, the main determinants of CLT are plasma PAI-1 levels, followed by plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), prothrombin and α2-antiplasmin [16]. Moreover, Meltzer et al. [16] have shown in simple linear regression analyses that all fibrinolytic factors except plasminogen were associated with CLT, but the strongest association was found between PAI-1 and CLT.

Previously, we have demonstrated that fibrin is present in large amounts on the surface and within stenotic valves in AS patients [17]. Areas positive for fibrin detected in the AS leaflets were associated with thrombin generation and fibrin turnover in circulating blood [17]. Fibrin in large foci can be detected in advanced atherosclerotic plaques where fibrin may stimulate plaque growth [18].

To our knowledge, fibrinolysis has not been investigated in patients with AS. We tested the hypothesis that impaired fibrinolysis is associated with the severity of AS and unfavorable processes occurring in loco within stenotic aortic valves, in particular valvular accumulation of fibrin.

Patients and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Study limitations
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References

Patients

Seventy-four patients were scheduled for isolated elective aortic valve replacement for severe AS (defined as a mean transvalvular gradient ≥ 40 mmHg; mean transvalvular gradient 59.4 ± 21.5; 43 men and 31 women, mean age 62.7 ± 10.7 years) and their isolated valves were subjected to in situ analysis. Patients were recruited from September 2010 to June 2011. The exclusion criteria were: acute infection, Valsalva sinus aneurysm or rheumatic AS, angiographically documented epicardial artery stenosis (> 20% diameter on coronary angiography performed within the preceding month), carotid artery stenosis (> 30% diameter observed on ultrasound examination), peripheral artery disease (subjects with intermittent claudication or ankle brachial index < 0.9), known cancer, autoimmune disorders, endocarditis, previous cardiac surgery and, finally, a history of myocardial infarction, stroke, venous thromboembolism or major bleeding. Patients receiving hypoglycemic agents and/or having fasting glucose > 7 mm were classified as having diabetes mellitus and excluded from the study. Patients who required additional surgical intervention or had other heart defects were ineligible. Sixty-eight subjects matched for age, gender and cardiovascular risk factors, who were recruited from hospital personnel and their families, served as controls.

Arterial hypertension was diagnosed based on a history of hypertension (blood pressure > 140/90 mmHg) or preadmission antihypertensive treatment. Hyperlipidemia was diagnosed based on total cholesterol of 5.2 mm or more, or statin therapy. Patients gave their informed consent and the University Bioethical Committee approved the study.

Echocardiography

Transthoracic echocardiography was performed in each patient using a GE Vivid 7 ultrasound machine (General Electric Healthcare, Little Chalfont, UK) prior to surgery using conventional techniques in accordance with the European Society of Cardiology (ESC) guidelines. The aortic valve area (AVA) was calculated using the standard continuity equation. The transvalvular gradient was measured by Doppler echocardiography using the modified Bernoulli equation.

Laboratory tests

Fasting venous blood was drawn from patients 24 h before surgery between 07.00 and 09.00 hours. Citrated blood samples (9:1 of 0.106 m sodium citrate) were centrifuged at 2500 g at 20 °C for 10 min and stored in aliquots at −80 °C until analysis. Lipid profile, glucose and creatinine were assayed by routine laboratory techniques. High-sensitivity C-reactive protein (CRP) was determined using immunoturbidimetry (Roche Diagnostics, Mannheim, Germany). Fibrinogen was measured by the von Clauss method (Instrumentation Laboratory, Bedford, MA, USA). A commercially available ELISA was used to determine PAI-1 and tPA antigen (Hyphen BioMed, Neuville-Sur-Oise, France), α2-antiplasmin (α2-AP) and plasminogen (STA-Stachrom antiplasmin and STA-Stachrom plasminogen; Diagnostica Stago, Asniéres, France) and TAFI antigen (Diagnostica Stago) in citrated plasma. Technicians blinded to the origin of the samples performed all measurements. Intra- and inter-assay coefficients of variation were < 8%.

Plasma clot lysis

Clot lysis time was measured using a tissue factor (TF)-induced clot lysis assay as described previously with some modifications [13, 19]. Briefly, citrated plasma was mixed with calcium chloride at final concentration 15 mm, human tissue factor (Innovin; Dade Behring, Marburg, Germany) at final concentration of 0.6 pm, phospholipid vesicles (Avanti PolarLipids, Alabaster, AL, USA) at a final concentration of 12 μm, and recombinant tPA (Boerhinger Ingelheim, Ingelheim, Germany) at final concentration of 60 ng mL−1. All the dilutions were prepared in Tris-buffered saline (50 mm Tris-HCl, 0.1 m NaCl, pH 7.4). Plasma represented 50% of the mixture volume. Turbidity was measured at 405 nm at 37 °C. CLT was defined as the time from the midpoint of the baseline clear to maximum turbid transition, to the final plateau phase. Intra- and inter-assay coefficients of variation were 6.0% and 7.4%, respectively.

Analysis of aortic valves

Aortic valves were embedded in Tissue Tec-OCT compound (Sakura, Torrance, CA, USA) for tissue cryopreservation, and cryosectioned (10 μm thick) onto SuperFrost slides (Menzel-Glaser, Braunschweig, Germany) by a Leica Jung CM 3000 cryostat (Leica Microsystems, GmbH, Vienna, Austria). Sections taken transversely from the mid of the leaflet and from commissural areas were stored at −20 °C until immunostaining.

Immunostaining

Immunofluorescence and immunohistochemistry were performed on adjacent sections of aortic valves as described [17, 20]. Briefly, sections were incubated 12 h at 4 °C with primary, antibodies against human fibrin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), PAI-1 (Abcam, Cambridge, UK), tPA (Abcam), mast cell-tryptase (DAKO, Glostrup, Denmark) or macrophage-CD68 (R&D Systems, Inc., Minneapolis, MN, USA). Primary antibodies were followed by the corresponding secondary antibodies conjugated with fluorochrome (R&D Systems) or by the avidin-biotin complex immunoperoxidase. Double-immunofluorescence for tryptase-PAI-1- or tPA-PAI-1-positive cells and for macrophages- PAI-1-positive cells were also performed (= 43). A negative control (without primary antibody incubation) was included routinely. Fifteen aortic valves from age-matched apparently healthy subjects, obtained at autopsy, without morphological cardiac disorders served as negative controls.

The calcification degree was analyzed semi-quantitatively according to the following scoring system: 0, 1, 2: absence, isolated calcium deposits (10–20% of total leaflet area), abundant calcium deposits (> 20% of total leaflet area), respectively.

Morphometry

The numbers of immunopositive mast cells (MC) and macrophages were counted at × 40 magnification throughout the entire section and expressed as the cell number per square millimeter [21]. The surface areas occupied by immunostained fibrin, PAI-1 or tPA were analyzed with CellProfiler image analysis software after adjustment for non-specific staining and calculated as percentages of the total tissue area [22]. The thickness of the fibrin layer within leaflets was measured in the lesion area as described [17].

The thickness of valves was measured using image analysis system (the maximum thickness was taken under analysis). The layer was measured perpendicularly from dislocated elastica to the top of lesions in five locations per slide. Intra- and inter-observer coefficients of variations were < 6%.

Statistical analysis

Values are expressed as mean (standard deviation [SD]) or median or otherwise stated. The Kolmogorov–Smirnov test was used to assess conformity with a normal distribution. Pair-wise comparisons were made using the Tukey's test for continuous variables and the χ2 test for proportions. The Mann–Whitney U-test was used to compare non-normally distributed variables between two groups. To assess linear dependence between variables, the Pearson's correlation coefficient for normally distributed or Spearman's rank correlation coefficient for non-normally distributed variables were calculated. To identify independent predictors of the mean transvalvular gradient and valve thickness, all clinical and demographic variables that showed the association with the transvalvular gradient and valve thickness in the univariate model (P ≤ 0.2) and did not show substantial correlations ( 0.5) with another independent variable were then included in the multivariate linear regression analysis. Covariates were selected judiciously based on consistent significant correlations between the covariates and the independent or dependent variables. Models were adjusted for age, gender and/or body mass index (BMI). A two-sided P-value < 0.05 was considered statistically significant. Data were analyzed using Statsoft 7.1 PL package (StatSoft Inc., Tulsa, OK, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Study limitations
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References

Characteristics of the patients

Seventy-four consecutive patients with AS scheduled for isolated valve replacement and 68 controls were studied (Table 1). Patients with AS compared with controls were characterized by 19% prolonged CLT (= 0.0007). In both AS and control subjects, CLT correlated positively with age (= 0.4, = 0.023; = 0.47, = 0.00005, respectively). In AS patients there were positive correlations between CLT and both plasma PAI-1 (= 0.42, = 0.0003) and plasma TAFI (= 0.25, = 0.009), whereas CLT showed no correlations with plasma plasminogen (= −0.1, = 0.25) or α2-AP (= 0.018, = 0.85). The correlation between CLT and plasma PAI-1 was also observed in controls (= 0.8, < 0.000001), but no correlations of CLT with plasma fibrinogen or CRP were observed in either group (all, > 0.1). Plasma PAI-1 was also correlated with valve thickness (= 0.2, = 0.03), which was the only significant correlation of plasma PAI-1 with histological or hemodynamic parameters.

Table 1. Characteristics of aortic value stenosis (AS) and control patients
 AS (n = 74)Controls (n = 68) P
  1. Data are given as mean ± standard deviation (SD), median [min-max] and number (percentage). ACE inhibitors, angiotensin-converting enzyme inhibitors; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Male, n (%)43 (58)38 (56)0.35
Age (years)62.7 ± 10.763.5 ± 8.30.61
Body mass index (kg m−2)28.7 ± 5.127.5 ± 3.30.72
Risk factors
Arterial hypertension, n (%)24 (32)23 (34)0.35
Hypercholesterolemia, n (%)31 (42)27 (40)0.9
Current smoking, n (%)10 (13.5)11 (16.2)0.07
Treatment
Beta-blockers, n (%)25 (34)
Acetylsalicylic acid, n (%)32 (43)
ACE inhibitors, n (%)23 (31)
Statins, n (%)21 (27)
Laboratory parameters
Fibrinogen (g L−1)3.6 [1.4–7.2]3.8 [1.9–6.3]0.86
Glucose (mm)5.5 [3.8–12.4]5.42 [4.1–9.8]0.62
Creatinine (μm)87 [59–330]80.51 [55–161]0.32
Total cholesterol (mm)5.6 [2.6–8.1]4.4 [2–8.4]0.77
LDL cholesterol (mm)2.9 [0.9–4.6]2.7 [0.9–5.8]0.12
HDL cholesterol (mm)1.3 ± 0.41.2 ± 0.30.33
Triglycerides (mm)1.3 [0.4–6.7]1.3 [0.4–3.4]0.61
C-reactive protein (mg L−1)7.5 [1.7–66.8]2.25 [0.3–5.6]0.02
Plasminogen activator inhibitor-1 (ng mL−1)78.6 [35.5–149]38.53 [18–61]< 0.0001
Thrombin-activatable fibrinolysis inhibitor (%)108 [97.1–117.9]  
α2-antiplasmin (%)103 [90–115.9]  
Plasminogen (%)91.3 [81.7–104.6]  
Clot lysis time (min)110 [54–153]92.52 [58–115]0.0007

Table 2 shows the echocardiographic parameters in AS and control patients. There were positive correlations of maximal and mean transvalvular pressure gradients, as well as AVA with CLT, plasma PAI-1 and plasma TAFI levels (Table 3). Moreover, the plasma PAI-1 levels in AS patients were correlated with valve thickness (= 0.2, = 0.03), but not with the fibrin layer thickness within aortic valves. Plasma TAFI levels were correlated with the number of macrophages within leaflets (= 0.41, < 0.0003), but not with other histological parameters.

Table 2. Echocardiographic data in aortic value stenosis (AS) and control patients
 AS (n = 74)Controls (n = 68) P
  1. Data are given as mean ± standard deviation (SD), median [min-max].

Mean aortic gradient (mmHg)59.4 ± 21.57.8 ± 2.30.0007
Maximum aortic gradient (mmHg)89.8 ± 25.112.6 ± 2.90.001
Ejection fraction (%)55.7 [35–80]65.8 [55–77]< 0.0001
Aortic valve area (cm2)0.8 [0.3–1.5]
Table 3. Correlations of echocardiographic parameters with clot lysis time (CLT), plasma plasminogen activator inhibitor-1 (PAI-1) and plasma thrombin activatable fibrinolysis inhibitor (TAFI) in aortic value stenosis (AS) patients (n = 74)
 CLTPlasma PAI-1Plasma TAFI
r P r P r P
Mean aortic gradient (mmHg)0.380.0010.49< 0.00010.240.038
Maximum aortic gradient (mmHg)0.430.00020.42< 0.00010.150.194
Aortic valve area (cm2)−0.59< 0.0001−0.30.03−0.30.01

Qualitative and quantitative immunocytochemistry

In AS valves focal areas of subendothelial thickening (lesions) were seen in 74 (100%) of the valves. Total valve leaflets thickness (1.36 [0.98–2.74] mm) in AS patients showed a positive correlation with CLT (Table 4). Of note, total valve thickness was associated with the degree of valve calcification (= 0.72, = 0.002), which in turn showed a strong positive correlation with CLT (= 0.65, < 0.00001). There was also a positive association between CLT and valvular fibrin layer thickness (Table 4).

Table 4. Correlations of histologic parameters with clot lysis time (CLT) in aortic value stenosis (AS) patients (n = 74)
  r P
Total valve thickness (mm)0.670.003
Valvular fibrin layer (μm)0.540.007
Valvular fibrin (%)0.540.008
Valvular plasminogen activator inhibitor-1 (%)0.480.007
Number of macrophages (cell mm−2)0.420.04

Analysis of the stenotic valves cellular composition showed macrophages distributed dispersed all over the leaflets; however, the highest density of those cells (79 ± 21 mm−2) was observed close to calcium deposits. There was an abundance of fibrin within valve leaflets. PAI-1 antigen was detected as 11 ± 9% of the total valve area, including both the subendothelial region and the adjacent fibrosa, and colocalized with fibrin (Fig. 1A). However, areas positive for PAI-1 were also visible in fibrin-free regions, infiltrated by macrophages. There were 6.9 [2.3–18.9] MC per square millimeter of the leaflets. In calcified regions of the leaflet 97 ± 5% of MC were observed whereas in non-calcified regions MC were sparsely distributed. Double-staining revealed colocalization of MCs with PAI-1 (Fig. 1B). The majority (87 ± 17%) of the MC stained positively for PAI-1. Moreover, PAI-1 positive staining was also evident in 48 ± 11% of macrophages. In contrast to PAI-1, tPA expression was at a very low level (3.5 ± 1% of the total valve area) and was detected, mainly colocalized with macrophages. Interestingly, tryptase-positive MC did not show tPA expression.

image

Figure 1. Representative micrographs of the double-immunostaining performed on aortic valve leaflets. (A) Immunostained fibrin is green and plasminogen activator inhibitor-1 (PAI-1) is red. Co-localized areas of each factor are stained orange. Scale 500 μm. Original magnification × 40. (B) Immunostained mast cells are green. Co-localized areas of PAI-1 and mast cells are stained orange. Original magnification × 100.

Download figure to PowerPoint

Positive correlations of valvular PAI-1 with maximal (= 0.38, = 0.0045) and mean (= 0.63, = 0.00004) transvalvular gradients, valvular fibrin expression (= 0.78, = 00001) and MC (= 0.69, < 0.000001) were noted.

There were no association between demographic variables and the amounts of valvular fibrin, PAI-1or tPA expression (data not shown). Interestingly, CLT showed positive correlations with valvular fibrin and PAI-1 (Table. 4). CLT correlated positively with the number of macrophages (Table. 4) and tended to correlate with the number of MC (= 0.37, = 0.07). There were no significant differences in the amount of immunostained fibrin and PAI-1 or MC and macrophages within aortic valves between AS patients receiving β-blockers, ACE inhibitors or acetylsalicylic acid (data not shown).

Regression analysis

Multivariate regression analysis including hypertension, smoking, CRP, triglycerides and CLT identified two parameters, CLT and smoking, as the independent predictors of the mean transvalvular gradient (R2 = 0.35, < 0.006). Moreover, CLT was identified as the independent predictor of the total valve thickness (R2 = 0.44, < 0.0005). After adjustment for confounding variables, including age, gender and BMI, CLT remained the independent predictor of the mean transvalvular gradient (R2 = 0.42, = 0.003) and total valve thickness (R2 = 0.47, = 0.000006).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Study limitations
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References

To the best of our knowledge, the role of fibrinolysis in the pathophysiology of AS has not been investigated until now. Our study is the first to show that patients with AS without documented significant atherosclerotic vascular disease are characterized by prolonged plasma clot lysis time. To assess fibrinolysis, a variety of plasma-based clot lysis assays have been used, including those with the use of human thrombin added to plasma together with tPA at higher concentrations [23, 24] and those based on serial measurements of D-dimer released from a plasma fibrin clot subjected to a buffer containing tPA [25, 26]. The advantage of the CLT derives from a simultaneous effect of procoagulant and profibrinolytic effects, along with compelling evidence for its utility in patients in various clinical settings. Our concept is that subjects with a lower efficiency of lysis owing to various genetic and/or acquired factors are more likely to develop severe forms of AS. We found that in patients with AS prolonged CLT is associated with pathologic processes including an increased fibrin layer within aortic valves and larger calcium deposits resulting in a greater thickness of the stenotic valves. Of note, hypofibrinolysis showed correlations with transvalvular pressure gradients and AVA, key clinical measures of the severity of AS. Hypofibrinolysis appears to be largely determined by elevated plasma PAI-1 and TAFI in AS patients, which is in line with the findings reported in patients with cardiovascular disease and those with venous thrombosis [16, 27]. The correlation between PAI-1 and CLT was weaker in AS patients probably because of the confounding effects of concomitant disorders (e.g. arterial hypertension, systemic low-grade inflammation and smoking) known to affect blood coagulation, fibrin clot phenotype and fibrinolysis [12, 14]. As we excluded patients with documented coronary artery disease, carotid artery stenosis and peripheral artery disease, the association of CLT and echocardiographic measures of AS severity cannot be explained by concomitant advanced atherosclerosis, known to be associated with prolonged clot lysis [28]. It might be speculated that subjects with slower plasma fibrin clot lysis are predisposed to a faster progression of AS and tend to develop more severe forms of this disease. However, one might argue that the reverse is true, namely worsening of AS leads to impaired fibrinolysis. To our knowledge, there have been no reports showing impairment of fibrinolysis as the result of increased shear stress associated with significant AS. Given available data on enhanced platelet activation in AS regardless of the extent of atherosclerosis [29] and platelets as another source of PAI-1 [8], we cannot exclude that AS itself might to some extent deteriorate fibrinolytic capacity by increasing plasma PAI-1 levels. However, available data are suggestive of a minor, if any, effect of this potential modulator of clot lysis.

Since impaired fibrinolysis promotes fibrin deposition [8], not surprisingly, thicker fibrin layers on the surface of stenotic leaflets were found in patients with longer CLT. It indicates that there is a close relationship between overall efficiency of clot lysis in circulating blood and the composition of the valves in AS patients. Superficial fibrin deposition within the leaflets is most probably the consequence of increased blood clotting activation on damaged valve leaflets without efficient fibrin degradation. We have previously shown in circulating blood that AS is characterized by enhanced formation of thrombin that catalyzes conversion of fibrinogen to fibrin [29]. A subendothelial pool of fibrin could result from intravalvular fibrin formation as a result of leakage of fibrinogen from neovessels inside stenotic leaflets. The presence of prothrombin and thrombin, necessary to convert fibrinogen to fibrin, has been reported within the leaflets of sclerotic aortic valves in rabbits fed a high-fat diet [30]. Given data on proatherogenic effects of fibrin and its degradation products in atherosclerosis [31], similar consequences of this phenomenon could be observed within aortic valves. Moreover, calcium deposits and macrophage infiltrates also show positive associations with CLT, suggesting that accumulation of fibrin within the stenotic valves may promote inflammation and calcification in AS or vice versa. A vital role of fibrin and fibrin degradation products in the development of the atherosclerotic plaque was postulated [18, 32]. Fibrin not only provides a scaffold for smooth muscle cells adhesion, migration and proliferation [33], but also contributes to the inflammation by inducing production of CRP [34], interleukin-6, tumor necrosis factor-alpha and inducible nitric oxide synthase [35].

A novel finding of the present study is the observation that MC represent an additional source of PAI-1 within the stenotic aortic valves. MC are multifunctional effector cells participating in the modulation of various inflammatory and cardiovascular processes [36]. The presence of numerous MC in an activated, degranulated state has been described within human aortic valves [37]. Accumulation of MC in the fibrotic lesions of the aortic valves suggests that MC-derived mediators such as chymase, cathepsin G and transforming growth factor-beta may participate in the induction of fibrosis, neovascularization and calcification with ensuing valve stiffening [37-39]. A role of MC producing PAI-1 has been proposed in the pathogenesis of atherosclerosis [21]. As it has been determined that under chronic inflammatory conditions profibrinolytic, antithrombotic resting MC producing active t-PA, change into an antifibrinolytic, prothrombotic phenotype secreting PAI-1 [40], it is tempting to speculate that MC could be involved in locally impaired fibrinolysis, fibrin and collagen deposition, and thus AS severity. This hypothesis may be supported by the negligible amount of tPA observed within stenotic valves in the present study. Cho et al. [41] have demonstrated that stimulated MC can release PAI-1 in excess over tPA and the latter enzyme is neutralized by PAI-1.

In the previous study on 21 patients with dominant AS and 17 patients with dominant aortic insufficiency [17], we failed to observe intergroup differences in the efficiency of lysis, which disagrees with the present findings. However, to measure the efficiency of fibrinolysis, another assay was used, in which exogenous thrombin and higher amounts of tPA are added resulting in faster lysis [17], whereas in the current assay no thrombin is added and lower final tPA concentrations are used resulting in much longer lysis time. It is likely that the small size of the former study and different methodology of lysis assessment resulted in similar lysis parameters in AS patients and those with aortic insufficiency. From the methodological point of view, the current assay, CLT, appears to be the best approach to assess global efficiency of lysis in cardiovascular patients.

We have also raised the question whether drugs may affect CLT in AS patients. It has been shown that statins and aspirin could accelerate plasma fibrin clot lysis [12]. In the present study, no effects of those drugs on CLT, total valve or fibrin layer thickness were observed. This could be explained with the severity of the disease and enhanced inflammatory state, which renders the latter parameters resistant to statin-induced effects. Most probably, in AS the impact of statins on fibrinolysis is minor, if any. To our knowledge, other cardiovascular medications have not been shown to alter CLT.

Study limitations

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Study limitations
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References

First, the study group was small. However, it represents typical patients with advanced AS in clinical practice. Second, the presence of antigens expressed within aortic valves was determined using a semi-quantitative analysis system. However, a large number of the images analyzed per each valve by two independent observers can ensure reliable results. Additional analysis, such as ELISA tests performed on aortic valve homogenates to quantify antigens analyzed by immunochemistry, has not been done. To confirm our observations in humans, an animal model of AS could be developed, in which the influence of fibrinolysis inhibitors on disease progression could be corroborated. Moreover, the measurement of fibrinolytic parameters in patients after AS valve replacement would support our findings; however, long-term anticoagulation hampers such analysis. Furthermore, it may be expected that aortic valve replacement does not significantly alter the efficiency of fibrinolysis. Finally, the significant associations reported here did not necessarily mean the cause–effect relationship.

We have shown that the efficiency of fibrinolysis determined in plasma is associated with the severity of AS in patients without documented atherosclerosis. These findings provide new insights into the links between coagulation/fibrinolysis and AS. Our understanding is that subjects with a slower plasma fibrin clot lysis, their inherent genetically and environmentally determined feature, are predisposed to faster progression of AS. It might be speculated that CLT could be a novel marker of advanced AS and might be involved in the progression of this disease. A larger study may help to validate this hypothesis, contribute to identify subjects at a higher risk of developing severe AS and open novel therapeutic options in such patients. We believe that our study shows a novel link between fibrinolysis and aortic stenosis unrelated to known traditional risk factors for both atherosclerosis and this common valve defect, which might have practical implications.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Study limitations
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References

This study has been supported by a grant of Polish Ministry of Science (N N402 383338, to A.U.).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Study limitations
  8. Acknowledgements
  9. Disclosure of Conflict of Interest
  10. References
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