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Abnormal plasma fibrin clot characteristics are associated with worse clinical outcome in patients with peripheral arterial disease and thromboangiitis obliterans
Atherosclerosis 215 (2011) 481–486
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Abnormal plasma fibrin clot characteristics are associated with worse clinical outcome in patients with peripheral arterial disease and thromboangiitis obliterans夽 Anetta Undas a,b,∗ , Tomasz Nowakowski c , Mariola Cie´sla-Dul b , Jerzy Sadowski a,b a
Institute of Cardiology, Jagiellonian University School of Medicine, Krakow, Poland John Paul II Hospital, Krakow, Poland c Department of Medicine, Jagiellonian University School of Medicine, Krakow, Poland b
a r t i c l e
i n f o
Article history: Received 19 October 2010 Received in revised form 19 December 2010 Accepted 24 December 2010 Available online 19 January 2011 Keywords: Fibrin clot Fibrinolysis Inflammation PAD Thromboangiitis obliterans
a b s t r a c t Background: A role of blood coagulation in the pathogenesis of peripheral arterial disease (PAD) and Buerger’s disease, or thromboangiitis obliterans (TAO), remains unclear. Objective: To test the hypothesis that PAD and TAO are associated with prothrombotic phenotype of a fibrin clot. Patients and methods: Ex vivo plasma fibrin clot permeability, turbidimetry and efficiency of fibrinolysis were investigated in 106 patients with PAD and 20 patients with TAO and compared with the respective control groups matched for age, sex, and cardiovascular risk factors. The progression of PAD and TAO were evaluated during follow-up of 3–7.5 years. PAD patients were characterized by lower clot permeability (−18.8%, p = 0.005), shorter lag phase (−35.3%, p < 0.001), higher maximum clot absorbancy (+22.4%, p < 0.001), prolonged clot lysis time (+30.6%, p = 0.003), and lower rate of D-dimer release from clots in the presence of recombinant tissue plasminogen activator (−16.5%, p = 0.009), but twofold lower maximum D-dimer levels released from clots during lysis (p < 0.001) than the controls. Similar, but more pronounced abnormalities were observed in TAO patients versus controls (all p < 0.01). Seventeen PAD (16%) and 3 (15%) TAO patients were lost to follow-up. The progression observed in 47 (52.8%) PAD patients and 10 (59%) TAO patients was associated with lower clot permeability (−14.6%, p = 0.009, and −17.5%, p = 0.02) and prolonged clot lysis (+11.3%, p = 0.004, and +12.4%, p = 0.03, respectively). Conclusions: Unfavorably altered fibrin clot properties are observed in both PAD and TAO. Denser fibrin clots with reduced susceptibility to lysis might characterize the progression of both diseases during long-term follow-up. © 2011 Elsevier Ireland Ltd. All rights reserved.
Peripheral arterial disease (PAD), resulting from progressive narrowing or occlusion of the major arteries in the lower limbs caused by atherosclerosis, is a common disease with a prevalence between 3% and 10% [1]. PAD affects 20–30% of elderly patients in general practice and approximately two-thirds of patients with the disease are asymptomatic [2]. Symptoms of PAD vary from intermittent claudication to critical limb ischemia leading to rest pain, ulceration, and necrosis. The ankle-brachial index (ABI) is regarded as a good measure of leg functioning in PAD [3]. It has been reported that PAD is associated with other manifestations of cardiovascular
夽 Funding sources: A grant of Jagiellonian University School of Medicine no. K/ZDS/000565 (to A.U.). ∗ Corresponding author at: Institute of Cardiology, Jagiellonian University School of Medicine, 80 Pradnicka St., 31-202 Krakow, Poland. Tel.: +48 12 6143004; fax: +48 12 4233900. E-mail address: [email protected] (A. Undas). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.12.040
disease. The 5-year incidence of PAD in adult elderly men is 29.4% for subjects following cerebrovascular events and 25% for patients with coronary artery disease (CAD) [4]. CAD is present in at least 50% of the PAD patients [5]. Another inflammatory vascular disease that occurs in smokers commonly before the age of 45 years is Buerger’s disease, or thromboangiitis obliterans (TAO). This disease typically affects small- and medium-sized arteries of the lower extremities and rarely also superficial veins [6]. It has been suggested that anti-endothelial cell, anti-collagen and anti-elastin antibodies, endothelin, inflammatory cytokines are involved in the pathogenesis of TAO [6]. Recently, it has been demonstrated that during the exacerbation phase endothelial-dependent vasodilatation is not altered, while aortic stiffness is observed [7]. There is evidence that enhanced inflammation and blood coagulation have a key role in the pathogenesis of PAD. It has been shown that fibrinogen, C-reactive protein (CRP), serum amyloid A and Ddimer are higher in PAD patients than those without PAD, and CRP is
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associated with the severity of PAD [8]. CRP, fibrinogen and D-dimer have been associated with progressive PAD with the predominance of inflammatory markers over coagulation factors [9]. However, patients with PAD display elevated platelet activation markers and circulating tissue factor procoagulant activity compared to healthy controls [10,11]. To date, no specific coagulation markers in PAD patients have been identified [12]. Less is known about coagulation markers in TAO [6]. The thrombin-mediated conversion of plasma fibrinogen into fibrin and the formation of clots relatively resistant to lysis are the final step of the blood coagulation cascade. Fibrin has been demonstrated to be a consistent component of atherosclerotic plaques, which can promote their growth [13]. The structure and function of a fibrin clot is influenced by environmental and genetic factors [14]. It has been demonstrated that fibrin clots composed of tightly packed thin fibers with small pores are relatively resistant to lysis and individuals with this type of fibrin network architecture are at a higher risk for CAD [14]. Compact fibrin fiber network less susceptible to lysis has been shown in patients with acute or previous myocardial infarction (MI) [15–18], ischemic stroke [19] as well as those following venous thromboembolism (VTE) [20]. Bhasin et al. reported 34 relatively young patients with mild to moderate PAD in whom plasma fibrin clots were poorly permeable, rigid, and resistant to lysis [21]. We hypothesized that PAD and TAO are associated with prothrombotic phenotype of a fibrin clot, including low clot permeability, increased fiber thickness and clot mass, together with impaired efficiency of lysis. Therefore, the aim of the study was to evaluate plasma fibrin clot properties in patients with PAD and TAO. 1. Patients and methods Between 2003 and 2007, we recruited two groups: (1) 106 patients with PAD aged 70 years or less and (2) 20 patients with TAO diagnosed based on an Adar score of 4 or more [22] and evidence for distal arterial disease on arteriography. The PAD was defined as an ABI ≤ 0.9 measured using established methods [2]. All TAO patients were studied during remission. The exclusion criteria were a history of VTE, recent (<6 months) arterial thrombotic event, active cancer, liver cirrhosis, renal failure (serum creatinine > 177 mol/L), any acute illness, pregnancy and anticoagulant therapy. None of the patients took clopidogrel at the time of enrolment. Age-, sex- and cardiovascular (CV) risk factor (smoking, diabetes, hypertension)-matched volunteers from outpatients and hospital staff served as “disease” controls for the PAD and TAO groups (n = 106 and n = 20, respectively). They had no signs or symptoms of PAD or TAO; all of them had ABI values above 0.9. CAD was defined as a history of MI, coronary revascularization, or hospitalization for angina symptoms. Diabetes and stroke were diagnosed according to the WHO criteria. Left ventricular ejection fraction (LVEF) was measured by the modified Simpson method on transthoracic echocardiography. All the patients were followed for at least 3 years and visits were scheduled every 6 months. The progression of PAD was defined as ABI progression (decrease in ABI of >0.15) with shorter claudication distance, MI, stroke, amputation, or CV death. The progression of TAO was defined as recurring ulcerations or amputation. Cardiovascular events were also recorded in TAO patients. The Jagiellonian University Ethical Committee approved the study, and patients provided written, informed consent. 1.1. Laboratory investigations Blood was drawn from an antecubital vein with minimal stasis after an overnight fast between 8 and 10 AM. Platelet count, glu-
cose, lipid profile and creatinine were assayed by routine laboratory techniques. Fibrinogen was determined using the Clauss method and hs-CRP by the latex nephelometry (Dade Behring, Marburg, Germany). Commercially available immunoenzymatic assays were used to determine plasma fibrinopeptide A (FPA), D-dimer, tissuetype plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) (all, American Diagnostica, Greenwich, CT). Serum interleukin-6 (IL-6) was measured by an ELISA (R@D Systems, Abingdon, United Kingdom). Total homocysteine (tHcy) in plasma was assayed by the HPLC method as described [23]. All intra-assay and inter-assay coefficients of variation were below 7%. 1.2. Plasma fibrin clot analysis 1.2.1. Clot permeability Permeation properties of fibrin clots were investigated as described [15,20]. Briefly, 20 mmol/L calcium chloride and 1 U/mL human thrombin (Sigma, St. Louis, MO) were added to citrated plasma. After incubation, tubes containing the clots were connected to a reservoir of a buffer (0.01 M Tris, 0.1 M NaCl, pH 7.5) and its volume flowing through the gels was measured within 60 min. A permeation coefficient (Ks ), which indicates the pore size, was calculated from the equation: Ks = Q × L × /t × A × p, where Q is the flow rate in time t, L is the length of a fibrin gel, is the viscosity of liquid (in poise), A is the cross-sectional area (in cm2 ), and p is a differential pressure (in dyne/cm2 ). A lower limit of detection is 0.5 × 10−9 cm2 . 1.2.2. Turbidity measurements Plasma samples were diluted 1:1 with a buffer (0.05 mol/L Tris–HCl, 0.15 mol/L NaCl, pH 7.4) and addition of 1 U/mL human thrombin (Sigma, St. Louis, MO) and 15 mmol/L calcium chloride to plasma-initiated polymerization [14]. Absorbance was read at 405 nm for 15 min with a Perkin-Elmer Lambda 4B spectrophotometer (Molecular Devices Corp., Menlo Park, CA). The lag phase of the turbidity curve, which reflects the time required for clot formation, and maximum absorbance at plateau reached by all individuals (Absmax ), which reflects the number of protofibrils per fiber and indirectly fiber thickness, were recorded. Lower limits of detection are 10 s and 0.1, respectively. 1.2.3. Turbidometric clot lysis assay Fibrinolysis in the presence of recombinant tissue plasminogen activator (rt-PA, Boerhinger Ingelheim, Ingelheim, Germany) was evaluated as previously described [15]. Briefly, 100-L citrated plasma was diluted with 100 L of the same buffer containing 20 mmol/L calcium chloride, 1 U/mL human thrombin (Sigma) and 14 mol/L rtPA. Assembly kinetics was monitored by absorbance at 405 nm for 30 min in duplicates. The time required for a 50% decrease in clot turbidity (t50% ) from a peak value was chosen as a marker of the clot susceptibility to fibrinolysis. A lower limit of detection is 1 min. 1.2.4. Perfusion clot lysis assay Fibrin clots formed as described above were perfused with the same buffer containing 0.2 mol/L rtPA as described [15]. The lysis rate was determined by measuring levels of D-dimer, a marker of plasmin-mediated fibrin degradation, every 20 min in the effluent. Maximum rates of increase in D-dimer levels (D-Drate , mg/(L min)), and maximum concentrations (D-Dmax ) detected at 80 or 100 min were analyzed in each subject. Lower limits of detection are 0.012 mg/(L min) and 0.5 mg/L, respectively. The experiment was stopped, usually after 80–120 min, while the fibrin gel collapsed under the pressure.
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Table 1 Characteristics of patients with peripheral arterial disease (PAD) and thromboangiitis obliterans (TAO) vs the respective control subjects.
Age, years Male gender, n (%) BMI, kg/m2 Current smoking, n (%) Hypertension, n (%) Diabetes, n (%) MI, n (%) CAD, n (%) LVEF, % Medications Aspirin, n (%) Statins, n (%) -Blockers, n (%) ACEIs, n (%) Laboratory parameters TC, mmol/L LDL-C, mmol/L HDL-C, mmol/L TG, mmol/L Glucose, mmol/L Creatinine, mol/L Fibrinogen, g/L CRP, mg/L IL-6, pg/mL D-dimer, mg/dL tPA, ng/mL PAI-1, ng/mL FPA, ng/mL tHcy, mol/L
PAD patients (n = 106)
PAD controls (n = 106)
p-Value
TAO patients (n = 20)
TAO controls (n = 20)
p-Value
57.1 ± 6.9 82 (77.4) 26.5 (24.2–28.4) 53 (50) 78 (73.6) 24 (22.6) 19 (17.9) 47 (44.3) 52 ± 11
56.4 ± 6.8 79 (74.5) 26.6 (24.6–28.5) 47 (44.3) 69 (65.1) 20 (18.9) 16 (15.1) 41 (38.7) 55 ± 8
0.63 0.83 0.84 0.41 0.18 0.5 0.56 0.4 0.26
45.4 ± 4.9 11 (55.0) 26.1 ± 5.2 8 (40.0) 3 (15.0) 0 (0) 0 (0) 2 (10.0) 61 ± 6
46.4 ± 6.8 12 (60.0) 26.2 ± 4.8 7 (35.0) 4 (20.0) 0 (0) 0 (0) 1 (5.0) 63 ± 7
0.81 0.75 0.83 0.7 0.68 – – 0.54 0.62
96 (90.6) 82 (77.4) 22 (20.8) 70 (66.0)
98 (92.5) 79 (74.5) 24 (22.6) 68 (74.2)
0.62 0.63 0.74 0.77
8 (40.0) 2 (10.0) 2 (10.0) 3 (15.0)
9 (45.0) 1 (5.0) 1 (5.0) 4 (20.0)
0.75 0.54 0.54 0.68
5.74 ± 1.48 3.48 ± 1.34 1.42 ± 0.47 1.69 ± 0.81 5.34 ± 0.67 78 (59–103) 3.38 ± 0.98 3.29 (1.2–6.94) 3.02 (1.14–4.4) 295.2 ± 85.0 13.87 ± 3.62 18.54 ± 4.83 4.98 ± 0.94 14.6 ± 3.9
5.81 ± 1.36 3.31 ± 0.98 1.39 ± 0.42 1.48 ± 0.59 5.22 ± 0.69 69 (55–94) 3.11 ± 0.96 1.37 (0.68–2.9) 1.22 (0.52–2.16) 167.4 ± 56.7 12.46 ± 3.98 11.41 ± 3.38 3.24 ± 0.75 11.3 ± 2.9
0.52 0.34 0.68 0.09 0.63 0.18 0.24 0.003 0.01 0.03 0.7 0.001 0.005 0.03
5.19 ± 1.15 2.98 ± 0.94 1.34 ± 0.48 1.74 ± 0.89 4.61 ± 0.83 67 (57–98) 3.48 ± 1.19 3.48 (0.8–8.82) 3.22 (1.04–6.81) 364.2 ± 74.7 11.98 ± 2.52 19.33 ± 5.48 4.26 ± 1.22 12.1 ± 2.1
5.17 ± 1.06 2.86 ± 0.87 1.38 ± 0.39 1.75 ± 0.79 4.56 ± 0.75 68 (55–96) 2.45 ± 0.76 1.27 (0.56–2.44) 1.24 (0.78–1.94) 136.7 ± 45.3 7.22 ± 1.08 8.95 ± 1.18 2.64 ± 0.48 11.4 ± 2.5
0.92 0.53 0.69 0.85 0.68 0.88 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.24
Values are given as mean ± SD, median (interquartile range), or number (percentage). BMI denotes body mass index; MI, myocardial infarction; LVEF, left ventricular ejection fraction; ACEIs, angiotensin-converting enzyme inhibitors; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglycerides; CRP, C-reactive protein; IL-6, interleukin-6; tPA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; FPA, fibrinopeptide A; tHcy, total homocysteine.
The intraassay and interassay coefficients of variation for all fibrin tests were 5.6–8.1%. The tests were performed by technicians blinded to the origin of plasma samples. 1.3. Statistical analysis Continuous variables are expressed as mean ± SD or otherwise stated. Continuous variables were checked for normal distribution by the Shapiro–Wilk statistic and compared by Student’s t-test when normally distributed or by the Mann–Whitney or Wilcoxon test for non-normally distributed variables. ANOVA was used to compare the differences in fibrin clot properties. Categorical variables were compared by 2 -test or Fisher’s exact test as appropriate. The Pearson or Spearman rank correlation coefficients were calculated to test the association between two variables with a normal or non-normal distribution, respectively. A p-value < 0.05 was considered statistically significant. The study was powered to have an 80% chance of detecting a 20% difference in clot permeability between the two groups using a p-value of 0.05, based on mean values published previously [20]. In order to demonstrate such a change or greater, 12 patients was required in each group. Corresponding number of subjects for t50% was calculated to be 9. 2. Results 2.1. PAD A total of 106 PAD patients with a mean ABI of 0.69 ± 0.1 were recruited and compared with the controls with a mean ABI of 1.06 ± 0.11. The PAD patients and control subjects did not differ with regard to demographics, clinical variables and routine laboratory parameters (Table 1). PAD patients had higher CRP and IL-6,
but not fibrinogen. Moreover, PAD was associated with elevated PAI-1, D-dimer, FPA, and tHcy (Table 1). Analysis of functional fibrin clot tests (Table 2) revealed that PAD patients had reduced clot permeability by 18.8% and prolonged t50% by 30.6%. Interestingly, there were lower maximum D-dimer levels and maximum rate of the increase in D-dimer levels released from clots in the PAD group. In a turbidimetric assay, Absmax was elevated by 22.4% in PAD patients, while the lag phase was shorter by 35.3%. The presence of clinically overt CAD in PAD patients (n = 47) was associated with slightly lower clot permeability (7.2 ± 1.0 × 10−9 cm2 vs 8.0 ± 1.1 × 10−9 cm2 , p = 0.04), higher maximum absorbancy (1.07 ± 0.12 vs 0.98 ± 0.13, p = 0.04) and prolonged lysis time (10.7 ± 1.1 min vs 9.2 ± 1.1 min, p = 0.02) compared with the 59 PAD patients without CAD. Diabetes and smoking affected only lysis time among the fibrin clot measures determined in the PAD group (10.9 ± 1.4 min vs 9.2 ± 1.2 min, p = 0.02 and 10.8 ± 1.2 min vs 9.1 ± 1.2 min, p = 0.01, respectively). Medications did not affect fibrin variables in the PAD group (data not shown). In the PAD group, associations of fibrin clot characteristics have been shown in Table 3. Permeability and lysis time were associated with fibrinogen and CRP. Parameters describing efficiency of lysis showed correlations with PAI-1. None of the fibrin clot measures showed significant correlations with age or ABI (Table 3). Seventeen PAD patients (16%) were lost to follow-up. During a mean follow-up of 57.9 months (range, 34–90 months) PAD progression as a composite endpoint was observed in 47 (52.8%) of the remaining 89 patients, including 5 MIs, 4 strokes and 2 CV deaths. Two other patients (2.3%) died of lung cancer. PAD progression was associated with the presence of diabetes (p = 0.02), higher tHcy (15.9 ± 1.5 mol/L vs 12.8 ± 2.2 mol/L, p = 0.01) and
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Table 2 Plasma fibrin clot characteristics.
−9
2
Ks , 10 cm Lag phase, s Absmax (405 nm) t50% , min D-Dmax , mg/L D-Drate , mg/(L min)
PAD patients (n = 106)
PAD controls (n = 106)
p-Value
TAO patients (n = 20)
TAO controls (n = 20)
p-Value
7.8 ± 1.0 66 (43–82) 1.04 (0.95–1.09) 9.9 ± 1.1 1.94 ± 0.41 0.071 ± 0.006
9.7 ± 0.9 102 (95–109) 0.85 (0.76–0.92) 6.9 ± 1.0 3.88 ± 0.45 0.085 ± 0.007
0.005 <0.001 <0.001 0.003 <0.001 0.009
5.4 ± 0.9 62 (43–79) 1.21 (0.98–1.26) 10.9 ± 1.3 0.64 ± 0.09 0.029 ± 0.009
10.9 ± 1.2 119 (98–128) 0.79 (0.70–0.91) 6.5 ± 1.1 3.53 ± 0.41 0.071 ± 0.011
<0.001 <0.001 <0.001 0.008 <0.001 <0.001
Values are given as mean ± SD or median (IQR). Ks indicates permeability coefficient; (Absmax (405 nm), maximum absorbance of a fibrin gel at 405 nm determined by using turbidimetry; t50% , half-lysis time; D-Dmax , maximum D-dimer levels measured in the percolating buffer (see Section 1); D-Drate , maximum rate of increase in D-dimer levels in the buffer. Table 3 Correlation coefficients (r) of fibrin clot characteristics in the PAD group.
Fibrinogen CRP PAI-1:Ag tPA:Ag tHcy D-dimer ABI Age
Ks
t50%
D-Dmax
D-Drate
Abs
Lag phase
−0.63* −0.71* −0.12 0.13 −0.56* 0.08 −0.09 −0.14
0.67* 0.44* 0.47* −0.04 0.07 0.38* 0.14 0.11
0.65* 0.15 0.62* −0.05 0.17 0.12 0.07 0.12
0.11 0.13 −0.39* −0.08 0.17 0.14 0.08 −0.15
0.45* 0.43* 0.14 −0.17 0.08 0.18 0.14 0.19
−0.13 −0.16 −0.42* −0.44* −0.11 −0.12 −0.2 −0.13
Abbreviations: See Tables 1 and 2. * p < 0.05 for all r-values. Table 4 Plasma fibrin clot characteristics in patients with progression of PAD or TAO compared with the remaining patients.
Ks , 10−9 cm2 Lag phase, s Absmax (405 nm) t50% , min D-Dmax , mg/L D-Drate , mg/(L min)
PAD progression (n = 47)
No PAD progression (n = 41)
p-Value
TAO progression (n = 10)
No TAO progression (n = 7)
p-Value
7.12 ± 0.93 98 (93–103) 1.09 (1.03–1.12) 10.9 ± 1.2 2.03 ± 0.49 0.082 ± 0.006
8.34 ± 0.87 107 (99–111) 0.98 (0.92–1.02) 9.6 ± 1.0 1.83 ± 0.48 0.086 ± 0.008
0.009 0.1 0.001 0.004 0.1 0.4
4.7 (3.9–4.9) 114 (97–121) 1.23 (1.18–1.32) 11.3 (11.9–10.9) 0.58 (0.53–0.64) 0.022 (0.018–0.03)
5.7 (4.7–6.1) 122 (118–131) 1.15 (0.96–1.22) 9.9 (9.2–10.8) 0.62 (0.56–0.67) 0.024 (0.019–0.028)
0.02 0.2 0.1 0.03 0.2 0.6
Values are given as mean ± SD or median (IQR). Abbreviations see Table 2.
CRP (4.49 ± 1.92 mg/L vs 1.85 ± 2.14 mg/L, p = 0.003). All clinical variables, including LVEF, were similar in a subgroup with PAD progression and the remainder (data not shown). Patients who reached the combined endpoint were characterized by 14.6% lower clot permeability, 10.2% higher maximum clot absorbancy, and 11.3% prolonged clot lysis time than the remainder; other fibrin measures were similar (Table 3). 2.2. TAO Twenty TAO patients and controls matched for demographics, body weight and smoking habit were recruited (Table 1). The only significant intergroup differences were found for fibrinogen, CRP, IL-6, tPA, PAI-1, and FPA; all the 6 variables were higher in the TAO patients than in controls. As shown in Table 2, marked differences in fibrin clot measures were noted between the TAO patients and the controls. Clot permeability was twofold lower in TAO patients compared with controls. This variable was inversely correlated with fibrinogen (r = −0.56; p < 0.01), CRP (r = −0.61; p < 0.01) and tHcy (r = −0.5; p = 0.03). Compared with controls, TAO patients had 47.9% shorter lag phase and 34.7% greater maximum clot absorbancy. In the TAO group, t50% was longer by 40.9% compared with controls and showed positive associations with fibrinogen (r = 0.48; p = 0.02), PAI-1 (r = 0.49; p = 0.02) and CRP (r = 0.55; p < 0.01). Interestingly, markedly twofold slower D-dimer release from clots and very low maximum D-dimer levels were also observed in TAO. Three TAO patients (15%) were lost to follow-up. During a median follow-up of 83 months (range, 61–89 months) the pro-
gression of TAO as a composite endpoint was observed in 10 (58.8%) of the remaining 17 patients, including 4 amputations. We noted 1 MI and 1 stroke; no CV deaths were recorded. Three patients declared they quitted smoking during follow-up. The TAO patients with the disease progression were similar to the remainder in terms of demographics or clinical variables (data not shown). Lower clot permeability and prolonged clot lysis time at study entry were observed in the TAO patients who achieved the endpoint compared with the remainder, while other fibrin parameters were similar (Table 4). 3. Discussion This study demonstrates that in patients with both PAD and TAO, plasma fibrin clot characteristics are unfavorably altered compared with the well matched control subjects. We found that plasma fibrin clots in both patient groups are less permeable, are formed more rapidly, and are lysed at a reduced rate, compared to those made from plasma obtained from controls. In contrast to MI patients [15], maximum D-dimer levels released from fibrin clots in the permeation lysis assay are lower than in controls, suggesting impaired release of the D-dimer molecules from the tight fibrin networks compared to the controls. Overall, this study provided evidence that patients with PAD share common clot features with those with CAD [15–18]. This indicates that abnormal fibrin clot phenotype may characterize all patients with atherosclerotic vascular disease. It should also be highlighted that despite a large atherosclerotic burden in PAD, even in these patients, a subgroup with diagnosed CAD displayed lower clot permeability, longer clot lysis, and
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faster polymerization compared to individuals free of CAD symptoms. Little is known about whether blood coagulation is involved in the pathogenesis of a rare smoking-related disease such as TAO. The current study expands our knowledge by showing several procoagulant alterations in TAO, including increased thrombin formation, impaired fibrinolysis and unfavorable clot phenotype despite a relatively young age and lack of evidence for CAD or diabetes. TAO with enhanced inflammatory state, reflected by elevated fibrinogen, IL-6 and CRP, has been for the first time shown to be characterized by more pronounced unfavorable alterations in fibrin clot characteristics compared to older PAD patients with several comorbidities, including CAD. This observation, along with the association of clot phenotype with clinical progression, suggests that rapid formation of poorly lysable compact clots in small vessels might contribute to leg ischemia in TAO. A larger study is needed to validate this observation. Of note, the permeation-based lysis assay performed in the TAO group showed very low maximum D-dimer levels and their extremely slow increase over time in the percolating buffer compared to both controls and PAD patients (Table 2). This suggests that a release of D-dimer induced by rtPA is markedly impaired in TAO and despite a dense clot structure reflected by low clot permeability a peak D-dimer concentration in this assay remains low even prior to the clot collapse. It might be speculated that the TAO patients tend to form extremely compact and irregular plasma fibrin clots. An important finding is a significant difference in fibrin clot properties between PAD patients with progression of the disease compared to the remainder, suggesting that unfavorable fibrin clot phenotype, reflected by the tendency to form compact and poorly lysable clots, might predispose to occlusive vascular complications and herald worse prognosis. Similar findings for TAO might indicate that coagulation factors and hypercoagulability leading to altered fibrin(ogen) characteristics contribute to narrowing and/or occlusion of both small-sized and large arteries. If so, it might be hypothesized that anticoagulant therapy could be beneficial in a certain subgroup of PAD and TAO patients with a particularly prothrombotic clot phenotype, however, combination of oral anticoagulation with aspirin may lead to excessive bleeding risk and oral anticoagulation alone have not been investigated in these diseases [24]. Mechanisms underlying alterations in fibrin clot structure/function in PAD or TAO patients are unclear. The Leeds family study showed that genetic factors contribute modestly to variance in fibrin clot measures, while the contribution of environmental factors is much larger [25]. Acquired factors, particularly those associated with enhanced inflammation, appear to be of major importance in PAD and TAO. It is known that increased levels of fibrinogen, being a major predictor of fibrin clot properties, are associated with a faster formation of denser fibrin networks of reduced lysability [14,26]. Elevated CRP has been shown to be associated with lower clot permeability and impaired lysis in patients at cardiovascular risk and those with acute MI [15,27]. Similar correlations were observed in PAD and TAO patients. It has been reported that hyperhomocysteinemia can reduce clot permeability and susceptibility to lysis [28], and the current study corroborates that in PAD patients tHcy correlates with some fibrin clot measures despite the presence of many potential confounders. Although elevated thrombin concentrations alter fibrin structure in experiments on purified fibrinogen [29], we did not observe any associations between plasma FPA levels and any fibrin variables in both patient groups, like in some previous studies [20]. We failed to show differences in fibrin clot properties associated with the administration of statins, which may increase fibrin clot permeability and enhance clot lysis [15,27]. Lack of drug-induced differences might be related to the patients’ characteristics and a low percentage of subjects who did not receive statins.
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An original observation is a markedly increased absorbancy in the PAD group despite similar fibrinogen concentrations in the patients and controls. Bhasin et al. [21] also observed a large difference between PAD patients with intermittent claudication and controls, which might corroborate our findings given a positive association between absorbancy in turbidimetry and fiber thickness. Elevated CRP in PAD patients (2.4-fold higher concentration compared with the controls) is likely to largely contribute to this intergroup difference in clot absorbancy. Some impact could be also attributed to increased tHcy in the PAD group. The current study has several limitations. First, the size of the study populations, particularly the TAO group, is limited, which may have introduced type II errors, especially in calculations of the correlation coefficients. Given the small number of subjects for the outcome study, with 16% lost to follow-up, the results regarding associations between fibrin clot characteristics and disease progression should be regarded as exploratory and need confirmation in a larger study. Secondly, our analysis was based on a determination of each variable at a single time point, at the time of enrolment, and significant changes in clot variables with time cannot be excluded. Third, scanning electron microscopy of fibrin clots has not been performed. However, we believe that functional plasma-based assays without clot dehydration provide more relevant information on a role of fibrin clot alterations in human pathology. Our experimental approach did not allow to analyze the effect of blood cells and platelets on fibrin clot structure/function, which can further impair for example fibrinolysis [30]. Additional fibrin clot modifiers that have not been investigated in the current study include oxidative stress, typically enhanced in atherosclerotic vascular disease [31]. Finally, genetic factors reported to alter fibrin clot structure/function [32] have not been studied. The concept of an influence of other as yet unidentified genetic factors has been supported by the study by Bhasin et al. [33] showing that first-degree relatives of PAD patients showed similarly altered clot variables with increased fiber thickness and factor XIII-mediated cross-linking. Further studies are needed to elucidate the genetics of fibrin clot architecture. In conclusion, our findings demonstrate that unfavorably altered fibrin clot properties associated with reduced efficiency of clot lysis occur in PAD and to a larger extent in a rare TAO. This study adds new information on the links between atherosclerosis, inflammation and coagulation, suggesting a major role of fibrin formation and degradation characteristics in the progression and vascular complications of vascular inflammatory diseases involving the lower limbs. Conflict of interest All authors have no conflict of interest to disclose. References [1] Aslam F, Haque A, Foody J, Lee LV. Peripheral arterial disease: current perspectives and new trends in management. South Med J 2009;102:1141–9. [2] McDermott MM, Kerwin DR, Liu K, et al. Prevalence and significance of unrecognized lower extremity peripheral arterial disease in general practice. J Gen Intern Med 2001;16:384–90. [3] McDermott MM, Greenland P, Liu K, et al. The ankle brachial index as a measure of leg functioning and physical activity in peripheral arterial disease: the Walking And Leg Claudication Study. Ann Intern Med 2002;136:873–83. [4] Merino J, Planos A, Elosua R, et al. Incidence and risk factors of peripheral arterial occlusive disease in a prospective cohort of 700 adult elderly men followed for 5 years. World J Surg 2010;34:1975–9. [5] Diehm C, Kareem S, Lawall H. Epidemiology of peripheral arterial disease. Vasa 2004;33:183–9. [6] Olin JW. Thromboangiitis obliterans (Buerger’s disease). N Engl J Med 2000;343:864–9. [7] Aziz M, Boutouyrie P, Bura-Riviere A, et al. Thromboangiitis obliterans and endothelial function. Eur J Clin Invest 2010;40:518–26.
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[8] Unlu Y, Karapolat S, Karaca Y, Kiziltunc A. Comparison of levels of inflammatory markers and hemostatic factors in the patients with and without peripheral arterial disease. Thromb Res 2006;117:357–64. [9] Tzoulaki I, Murray GD, Price JF, et al. Hemostatic factors, inflammatory markers, and progressive peripheral atherosclerosis: the Edinburgh Artery Study. Am J Epidemiol 2006;63:334–41. [10] Bennett PC, Silverman SH, Gill PS, Lip GY. Peripheral arterial disease and Virchow’s triad. Thromb Haemost 2009;101:1032–40. [11] Rao AK, Vaidyula VR, Bagga S, et al. Effect of antiplatelet agents clopidogrel, aspirin, and cilostazol on circulating tissue factor procoagulant activity in patients with peripheral arterial disease. Thromb Haemost 2006;96:738–43. [12] Cooke JP, Wilson AM. Biomarkers of peripheral arterial disease. J Am Coll Cardiol 2010;55:2017–33. [13] Bini A, Fenoglio J, Mesa-Tejada E, Kudryk B, Kaplan K. Identification and distribution of fibrinogen, fibrin, and fibrin(ogen) degradation products in atherosclerosis. Atherosclerosis 1989;69:1038–45. [14] Scott EM, Ariëns RAS, Grant PJ. Genetic and environmental determinants of fibrin structure and function. Relevance to clinical disease. Arterioscler Thromb Vasc Biol 2004;24:1558–66. [15] Undas A, Szuldrzynski K, Stepien E, et al. Reduced clot permeability and susceptibility to lysis in patients with acute coronary syndrome: effects of inflammation and oxidative stress. Atherosclerosis 2007;196:551–8. [16] Fatah K, Hamsten A, Blombäck B, Blombäck M. Fibrin gel network characteristics and coronary heart disease: relations to plasma fibrinogen concentration, acute phase protein, serum lipoproteins and coronary atherosclerosis. Thromb Haemost 1992;68:130–5. [17] Collet JP, Allali Y, Lesty C, et al. Altered fibrin architecture is associated with hypofibrinolysis and premature coronary atherothrombosis. Arterioscler Thromb Vasc Biol 2006;26:2567–73. [18] Ajjan RA, Standeven KF, Khanbhai M, et al. Effects of aspirin on clot structure and fibrinolysis using a novel in vitro cellular system. Arterioscler Thromb Vasc Biol 2009;29:712–7. [19] Undas A, Podolec P, Zawilska K, et al. Altered fibrin clot structure/function as a novel risk factor for cryptogenic ischemic stroke. Stroke 2009;40: 1499–501. [20] Undas A, Zawilska K, Ciesla-Dul M, et al. Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives. Blood 2009;114:4272–8.
[21] Bhasin N, Parry DJ, Scott DJ, et al. Regarding “Altered fibrin clot structure and function in individuals with intermittent claudication”. J Vasc Surg 2009;49:1088–9. [22] Papa MZ, Rabi I, Adar R. A point scoring system for the clinical diagnosis of Buerger’s disease. Eur J Vasc Endovasc Surg 1996;11:335–9. [23] Glowacki R, Bald E. Fully automated method for simultaneous determination of total cysteine, cysteinylglycine, glutathione and homocysteine in plasma by HPLC with UV absorbance detection. J Chromatogr B: Anal Technol Biomed Life Sci 2009;877:3400–4. [24] Tangelder MJD, van Hattum ES. Antithrombotic therapy in peripheral artery disease—antiplatelet therapy, anticoagulants, both or none. Thromb Haemost 2010;104:196–9. [25] Carter AM, Cymbalista CM, Spector TD, Grant PJ. Heritability of clot formation, morphology, and lysis. The EuroCLOT Study. Arterioscler Thromb Vasc Biol 2007;27:2783–9. [26] Blombäck B, Carlsson K, Hessel B, et al. Native fibrin gel networks observed by 3D microscopy, permeation and turbidity. Biochim Biophys Acta 1989;997:96–110. [27] Undas A, Topor-Madry R, Tracz W. Simvastatin increases clot permeability and susceptibility to lysis in patients with LDL cholesterol below 3.4 mmol/l. Pol Arch Med Wewn 2009;119:354–9. [28] Undas A, Brozek J, Jankowski M, et al. Plasma homocysteine affects fibrin clot permeability and resistance to lysis in human subjects. Arterioscler Thromb Vasc Biol 2006;26:1397–404. [29] Wolberg AS. Thrombin generation and fibrin clot structure. Blood Rev 2007;21:131–42. [30] Weisel JW, Litvinov RI. The biochemical and physical process of fibrinolysis and effects of clot structure and stability on the lysis rate. Cardiovasc Hematol Agents Med Chem 2008;6:161–80. [31] Hoffman M. Alterations of fibrinogen structure in human disease. Cardiovasc Hematol Agents Med Chem 2008;6:161–80. [32] Standeven KF, Uitte de Willige S, Carter AM, Grant PJ. Heritability of clot formation. Semin Thromb Haemost 2009;35:458–67. [33] Bhasin N, Ariëns RA, West RM, et al. Altered fibrin clot architecture and function in the healthy first-degree relatives of subjects with intermittent claudication. J Vasc Surg 2008;48:1497–503.
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Abnormal plasma fibrin clot characteristics are associated with worse clinical outcome in patients with peripheral arterial disease and thromboangiitis obliterans夽 Anetta Undas a,b,∗ , Tomasz Nowakowski c , Mariola Cie´sla-Dul b , Jerzy Sadowski a,b a
Institute of Cardiology, Jagiellonian University School of Medicine, Krakow, Poland John Paul II Hospital, Krakow, Poland c Department of Medicine, Jagiellonian University School of Medicine, Krakow, Poland b
a r t i c l e
i n f o
Article history: Received 19 October 2010 Received in revised form 19 December 2010 Accepted 24 December 2010 Available online 19 January 2011 Keywords: Fibrin clot Fibrinolysis Inflammation PAD Thromboangiitis obliterans
a b s t r a c t Background: A role of blood coagulation in the pathogenesis of peripheral arterial disease (PAD) and Buerger’s disease, or thromboangiitis obliterans (TAO), remains unclear. Objective: To test the hypothesis that PAD and TAO are associated with prothrombotic phenotype of a fibrin clot. Patients and methods: Ex vivo plasma fibrin clot permeability, turbidimetry and efficiency of fibrinolysis were investigated in 106 patients with PAD and 20 patients with TAO and compared with the respective control groups matched for age, sex, and cardiovascular risk factors. The progression of PAD and TAO were evaluated during follow-up of 3–7.5 years. PAD patients were characterized by lower clot permeability (−18.8%, p = 0.005), shorter lag phase (−35.3%, p < 0.001), higher maximum clot absorbancy (+22.4%, p < 0.001), prolonged clot lysis time (+30.6%, p = 0.003), and lower rate of D-dimer release from clots in the presence of recombinant tissue plasminogen activator (−16.5%, p = 0.009), but twofold lower maximum D-dimer levels released from clots during lysis (p < 0.001) than the controls. Similar, but more pronounced abnormalities were observed in TAO patients versus controls (all p < 0.01). Seventeen PAD (16%) and 3 (15%) TAO patients were lost to follow-up. The progression observed in 47 (52.8%) PAD patients and 10 (59%) TAO patients was associated with lower clot permeability (−14.6%, p = 0.009, and −17.5%, p = 0.02) and prolonged clot lysis (+11.3%, p = 0.004, and +12.4%, p = 0.03, respectively). Conclusions: Unfavorably altered fibrin clot properties are observed in both PAD and TAO. Denser fibrin clots with reduced susceptibility to lysis might characterize the progression of both diseases during long-term follow-up. © 2011 Elsevier Ireland Ltd. All rights reserved.
Peripheral arterial disease (PAD), resulting from progressive narrowing or occlusion of the major arteries in the lower limbs caused by atherosclerosis, is a common disease with a prevalence between 3% and 10% [1]. PAD affects 20–30% of elderly patients in general practice and approximately two-thirds of patients with the disease are asymptomatic [2]. Symptoms of PAD vary from intermittent claudication to critical limb ischemia leading to rest pain, ulceration, and necrosis. The ankle-brachial index (ABI) is regarded as a good measure of leg functioning in PAD [3]. It has been reported that PAD is associated with other manifestations of cardiovascular
夽 Funding sources: A grant of Jagiellonian University School of Medicine no. K/ZDS/000565 (to A.U.). ∗ Corresponding author at: Institute of Cardiology, Jagiellonian University School of Medicine, 80 Pradnicka St., 31-202 Krakow, Poland. Tel.: +48 12 6143004; fax: +48 12 4233900. E-mail address: [email protected] (A. Undas). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.12.040
disease. The 5-year incidence of PAD in adult elderly men is 29.4% for subjects following cerebrovascular events and 25% for patients with coronary artery disease (CAD) [4]. CAD is present in at least 50% of the PAD patients [5]. Another inflammatory vascular disease that occurs in smokers commonly before the age of 45 years is Buerger’s disease, or thromboangiitis obliterans (TAO). This disease typically affects small- and medium-sized arteries of the lower extremities and rarely also superficial veins [6]. It has been suggested that anti-endothelial cell, anti-collagen and anti-elastin antibodies, endothelin, inflammatory cytokines are involved in the pathogenesis of TAO [6]. Recently, it has been demonstrated that during the exacerbation phase endothelial-dependent vasodilatation is not altered, while aortic stiffness is observed [7]. There is evidence that enhanced inflammation and blood coagulation have a key role in the pathogenesis of PAD. It has been shown that fibrinogen, C-reactive protein (CRP), serum amyloid A and Ddimer are higher in PAD patients than those without PAD, and CRP is
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associated with the severity of PAD [8]. CRP, fibrinogen and D-dimer have been associated with progressive PAD with the predominance of inflammatory markers over coagulation factors [9]. However, patients with PAD display elevated platelet activation markers and circulating tissue factor procoagulant activity compared to healthy controls [10,11]. To date, no specific coagulation markers in PAD patients have been identified [12]. Less is known about coagulation markers in TAO [6]. The thrombin-mediated conversion of plasma fibrinogen into fibrin and the formation of clots relatively resistant to lysis are the final step of the blood coagulation cascade. Fibrin has been demonstrated to be a consistent component of atherosclerotic plaques, which can promote their growth [13]. The structure and function of a fibrin clot is influenced by environmental and genetic factors [14]. It has been demonstrated that fibrin clots composed of tightly packed thin fibers with small pores are relatively resistant to lysis and individuals with this type of fibrin network architecture are at a higher risk for CAD [14]. Compact fibrin fiber network less susceptible to lysis has been shown in patients with acute or previous myocardial infarction (MI) [15–18], ischemic stroke [19] as well as those following venous thromboembolism (VTE) [20]. Bhasin et al. reported 34 relatively young patients with mild to moderate PAD in whom plasma fibrin clots were poorly permeable, rigid, and resistant to lysis [21]. We hypothesized that PAD and TAO are associated with prothrombotic phenotype of a fibrin clot, including low clot permeability, increased fiber thickness and clot mass, together with impaired efficiency of lysis. Therefore, the aim of the study was to evaluate plasma fibrin clot properties in patients with PAD and TAO. 1. Patients and methods Between 2003 and 2007, we recruited two groups: (1) 106 patients with PAD aged 70 years or less and (2) 20 patients with TAO diagnosed based on an Adar score of 4 or more [22] and evidence for distal arterial disease on arteriography. The PAD was defined as an ABI ≤ 0.9 measured using established methods [2]. All TAO patients were studied during remission. The exclusion criteria were a history of VTE, recent (<6 months) arterial thrombotic event, active cancer, liver cirrhosis, renal failure (serum creatinine > 177 mol/L), any acute illness, pregnancy and anticoagulant therapy. None of the patients took clopidogrel at the time of enrolment. Age-, sex- and cardiovascular (CV) risk factor (smoking, diabetes, hypertension)-matched volunteers from outpatients and hospital staff served as “disease” controls for the PAD and TAO groups (n = 106 and n = 20, respectively). They had no signs or symptoms of PAD or TAO; all of them had ABI values above 0.9. CAD was defined as a history of MI, coronary revascularization, or hospitalization for angina symptoms. Diabetes and stroke were diagnosed according to the WHO criteria. Left ventricular ejection fraction (LVEF) was measured by the modified Simpson method on transthoracic echocardiography. All the patients were followed for at least 3 years and visits were scheduled every 6 months. The progression of PAD was defined as ABI progression (decrease in ABI of >0.15) with shorter claudication distance, MI, stroke, amputation, or CV death. The progression of TAO was defined as recurring ulcerations or amputation. Cardiovascular events were also recorded in TAO patients. The Jagiellonian University Ethical Committee approved the study, and patients provided written, informed consent. 1.1. Laboratory investigations Blood was drawn from an antecubital vein with minimal stasis after an overnight fast between 8 and 10 AM. Platelet count, glu-
cose, lipid profile and creatinine were assayed by routine laboratory techniques. Fibrinogen was determined using the Clauss method and hs-CRP by the latex nephelometry (Dade Behring, Marburg, Germany). Commercially available immunoenzymatic assays were used to determine plasma fibrinopeptide A (FPA), D-dimer, tissuetype plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) (all, American Diagnostica, Greenwich, CT). Serum interleukin-6 (IL-6) was measured by an ELISA (R@D Systems, Abingdon, United Kingdom). Total homocysteine (tHcy) in plasma was assayed by the HPLC method as described [23]. All intra-assay and inter-assay coefficients of variation were below 7%. 1.2. Plasma fibrin clot analysis 1.2.1. Clot permeability Permeation properties of fibrin clots were investigated as described [15,20]. Briefly, 20 mmol/L calcium chloride and 1 U/mL human thrombin (Sigma, St. Louis, MO) were added to citrated plasma. After incubation, tubes containing the clots were connected to a reservoir of a buffer (0.01 M Tris, 0.1 M NaCl, pH 7.5) and its volume flowing through the gels was measured within 60 min. A permeation coefficient (Ks ), which indicates the pore size, was calculated from the equation: Ks = Q × L × /t × A × p, where Q is the flow rate in time t, L is the length of a fibrin gel, is the viscosity of liquid (in poise), A is the cross-sectional area (in cm2 ), and p is a differential pressure (in dyne/cm2 ). A lower limit of detection is 0.5 × 10−9 cm2 . 1.2.2. Turbidity measurements Plasma samples were diluted 1:1 with a buffer (0.05 mol/L Tris–HCl, 0.15 mol/L NaCl, pH 7.4) and addition of 1 U/mL human thrombin (Sigma, St. Louis, MO) and 15 mmol/L calcium chloride to plasma-initiated polymerization [14]. Absorbance was read at 405 nm for 15 min with a Perkin-Elmer Lambda 4B spectrophotometer (Molecular Devices Corp., Menlo Park, CA). The lag phase of the turbidity curve, which reflects the time required for clot formation, and maximum absorbance at plateau reached by all individuals (Absmax ), which reflects the number of protofibrils per fiber and indirectly fiber thickness, were recorded. Lower limits of detection are 10 s and 0.1, respectively. 1.2.3. Turbidometric clot lysis assay Fibrinolysis in the presence of recombinant tissue plasminogen activator (rt-PA, Boerhinger Ingelheim, Ingelheim, Germany) was evaluated as previously described [15]. Briefly, 100-L citrated plasma was diluted with 100 L of the same buffer containing 20 mmol/L calcium chloride, 1 U/mL human thrombin (Sigma) and 14 mol/L rtPA. Assembly kinetics was monitored by absorbance at 405 nm for 30 min in duplicates. The time required for a 50% decrease in clot turbidity (t50% ) from a peak value was chosen as a marker of the clot susceptibility to fibrinolysis. A lower limit of detection is 1 min. 1.2.4. Perfusion clot lysis assay Fibrin clots formed as described above were perfused with the same buffer containing 0.2 mol/L rtPA as described [15]. The lysis rate was determined by measuring levels of D-dimer, a marker of plasmin-mediated fibrin degradation, every 20 min in the effluent. Maximum rates of increase in D-dimer levels (D-Drate , mg/(L min)), and maximum concentrations (D-Dmax ) detected at 80 or 100 min were analyzed in each subject. Lower limits of detection are 0.012 mg/(L min) and 0.5 mg/L, respectively. The experiment was stopped, usually after 80–120 min, while the fibrin gel collapsed under the pressure.
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Table 1 Characteristics of patients with peripheral arterial disease (PAD) and thromboangiitis obliterans (TAO) vs the respective control subjects.
Age, years Male gender, n (%) BMI, kg/m2 Current smoking, n (%) Hypertension, n (%) Diabetes, n (%) MI, n (%) CAD, n (%) LVEF, % Medications Aspirin, n (%) Statins, n (%) -Blockers, n (%) ACEIs, n (%) Laboratory parameters TC, mmol/L LDL-C, mmol/L HDL-C, mmol/L TG, mmol/L Glucose, mmol/L Creatinine, mol/L Fibrinogen, g/L CRP, mg/L IL-6, pg/mL D-dimer, mg/dL tPA, ng/mL PAI-1, ng/mL FPA, ng/mL tHcy, mol/L
PAD patients (n = 106)
PAD controls (n = 106)
p-Value
TAO patients (n = 20)
TAO controls (n = 20)
p-Value
57.1 ± 6.9 82 (77.4) 26.5 (24.2–28.4) 53 (50) 78 (73.6) 24 (22.6) 19 (17.9) 47 (44.3) 52 ± 11
56.4 ± 6.8 79 (74.5) 26.6 (24.6–28.5) 47 (44.3) 69 (65.1) 20 (18.9) 16 (15.1) 41 (38.7) 55 ± 8
0.63 0.83 0.84 0.41 0.18 0.5 0.56 0.4 0.26
45.4 ± 4.9 11 (55.0) 26.1 ± 5.2 8 (40.0) 3 (15.0) 0 (0) 0 (0) 2 (10.0) 61 ± 6
46.4 ± 6.8 12 (60.0) 26.2 ± 4.8 7 (35.0) 4 (20.0) 0 (0) 0 (0) 1 (5.0) 63 ± 7
0.81 0.75 0.83 0.7 0.68 – – 0.54 0.62
96 (90.6) 82 (77.4) 22 (20.8) 70 (66.0)
98 (92.5) 79 (74.5) 24 (22.6) 68 (74.2)
0.62 0.63 0.74 0.77
8 (40.0) 2 (10.0) 2 (10.0) 3 (15.0)
9 (45.0) 1 (5.0) 1 (5.0) 4 (20.0)
0.75 0.54 0.54 0.68
5.74 ± 1.48 3.48 ± 1.34 1.42 ± 0.47 1.69 ± 0.81 5.34 ± 0.67 78 (59–103) 3.38 ± 0.98 3.29 (1.2–6.94) 3.02 (1.14–4.4) 295.2 ± 85.0 13.87 ± 3.62 18.54 ± 4.83 4.98 ± 0.94 14.6 ± 3.9
5.81 ± 1.36 3.31 ± 0.98 1.39 ± 0.42 1.48 ± 0.59 5.22 ± 0.69 69 (55–94) 3.11 ± 0.96 1.37 (0.68–2.9) 1.22 (0.52–2.16) 167.4 ± 56.7 12.46 ± 3.98 11.41 ± 3.38 3.24 ± 0.75 11.3 ± 2.9
0.52 0.34 0.68 0.09 0.63 0.18 0.24 0.003 0.01 0.03 0.7 0.001 0.005 0.03
5.19 ± 1.15 2.98 ± 0.94 1.34 ± 0.48 1.74 ± 0.89 4.61 ± 0.83 67 (57–98) 3.48 ± 1.19 3.48 (0.8–8.82) 3.22 (1.04–6.81) 364.2 ± 74.7 11.98 ± 2.52 19.33 ± 5.48 4.26 ± 1.22 12.1 ± 2.1
5.17 ± 1.06 2.86 ± 0.87 1.38 ± 0.39 1.75 ± 0.79 4.56 ± 0.75 68 (55–96) 2.45 ± 0.76 1.27 (0.56–2.44) 1.24 (0.78–1.94) 136.7 ± 45.3 7.22 ± 1.08 8.95 ± 1.18 2.64 ± 0.48 11.4 ± 2.5
0.92 0.53 0.69 0.85 0.68 0.88 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.24
Values are given as mean ± SD, median (interquartile range), or number (percentage). BMI denotes body mass index; MI, myocardial infarction; LVEF, left ventricular ejection fraction; ACEIs, angiotensin-converting enzyme inhibitors; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglycerides; CRP, C-reactive protein; IL-6, interleukin-6; tPA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; FPA, fibrinopeptide A; tHcy, total homocysteine.
The intraassay and interassay coefficients of variation for all fibrin tests were 5.6–8.1%. The tests were performed by technicians blinded to the origin of plasma samples. 1.3. Statistical analysis Continuous variables are expressed as mean ± SD or otherwise stated. Continuous variables were checked for normal distribution by the Shapiro–Wilk statistic and compared by Student’s t-test when normally distributed or by the Mann–Whitney or Wilcoxon test for non-normally distributed variables. ANOVA was used to compare the differences in fibrin clot properties. Categorical variables were compared by 2 -test or Fisher’s exact test as appropriate. The Pearson or Spearman rank correlation coefficients were calculated to test the association between two variables with a normal or non-normal distribution, respectively. A p-value < 0.05 was considered statistically significant. The study was powered to have an 80% chance of detecting a 20% difference in clot permeability between the two groups using a p-value of 0.05, based on mean values published previously [20]. In order to demonstrate such a change or greater, 12 patients was required in each group. Corresponding number of subjects for t50% was calculated to be 9. 2. Results 2.1. PAD A total of 106 PAD patients with a mean ABI of 0.69 ± 0.1 were recruited and compared with the controls with a mean ABI of 1.06 ± 0.11. The PAD patients and control subjects did not differ with regard to demographics, clinical variables and routine laboratory parameters (Table 1). PAD patients had higher CRP and IL-6,
but not fibrinogen. Moreover, PAD was associated with elevated PAI-1, D-dimer, FPA, and tHcy (Table 1). Analysis of functional fibrin clot tests (Table 2) revealed that PAD patients had reduced clot permeability by 18.8% and prolonged t50% by 30.6%. Interestingly, there were lower maximum D-dimer levels and maximum rate of the increase in D-dimer levels released from clots in the PAD group. In a turbidimetric assay, Absmax was elevated by 22.4% in PAD patients, while the lag phase was shorter by 35.3%. The presence of clinically overt CAD in PAD patients (n = 47) was associated with slightly lower clot permeability (7.2 ± 1.0 × 10−9 cm2 vs 8.0 ± 1.1 × 10−9 cm2 , p = 0.04), higher maximum absorbancy (1.07 ± 0.12 vs 0.98 ± 0.13, p = 0.04) and prolonged lysis time (10.7 ± 1.1 min vs 9.2 ± 1.1 min, p = 0.02) compared with the 59 PAD patients without CAD. Diabetes and smoking affected only lysis time among the fibrin clot measures determined in the PAD group (10.9 ± 1.4 min vs 9.2 ± 1.2 min, p = 0.02 and 10.8 ± 1.2 min vs 9.1 ± 1.2 min, p = 0.01, respectively). Medications did not affect fibrin variables in the PAD group (data not shown). In the PAD group, associations of fibrin clot characteristics have been shown in Table 3. Permeability and lysis time were associated with fibrinogen and CRP. Parameters describing efficiency of lysis showed correlations with PAI-1. None of the fibrin clot measures showed significant correlations with age or ABI (Table 3). Seventeen PAD patients (16%) were lost to follow-up. During a mean follow-up of 57.9 months (range, 34–90 months) PAD progression as a composite endpoint was observed in 47 (52.8%) of the remaining 89 patients, including 5 MIs, 4 strokes and 2 CV deaths. Two other patients (2.3%) died of lung cancer. PAD progression was associated with the presence of diabetes (p = 0.02), higher tHcy (15.9 ± 1.5 mol/L vs 12.8 ± 2.2 mol/L, p = 0.01) and
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Table 2 Plasma fibrin clot characteristics.
−9
2
Ks , 10 cm Lag phase, s Absmax (405 nm) t50% , min D-Dmax , mg/L D-Drate , mg/(L min)
PAD patients (n = 106)
PAD controls (n = 106)
p-Value
TAO patients (n = 20)
TAO controls (n = 20)
p-Value
7.8 ± 1.0 66 (43–82) 1.04 (0.95–1.09) 9.9 ± 1.1 1.94 ± 0.41 0.071 ± 0.006
9.7 ± 0.9 102 (95–109) 0.85 (0.76–0.92) 6.9 ± 1.0 3.88 ± 0.45 0.085 ± 0.007
0.005 <0.001 <0.001 0.003 <0.001 0.009
5.4 ± 0.9 62 (43–79) 1.21 (0.98–1.26) 10.9 ± 1.3 0.64 ± 0.09 0.029 ± 0.009
10.9 ± 1.2 119 (98–128) 0.79 (0.70–0.91) 6.5 ± 1.1 3.53 ± 0.41 0.071 ± 0.011
<0.001 <0.001 <0.001 0.008 <0.001 <0.001
Values are given as mean ± SD or median (IQR). Ks indicates permeability coefficient; (Absmax (405 nm), maximum absorbance of a fibrin gel at 405 nm determined by using turbidimetry; t50% , half-lysis time; D-Dmax , maximum D-dimer levels measured in the percolating buffer (see Section 1); D-Drate , maximum rate of increase in D-dimer levels in the buffer. Table 3 Correlation coefficients (r) of fibrin clot characteristics in the PAD group.
Fibrinogen CRP PAI-1:Ag tPA:Ag tHcy D-dimer ABI Age
Ks
t50%
D-Dmax
D-Drate
Abs
Lag phase
−0.63* −0.71* −0.12 0.13 −0.56* 0.08 −0.09 −0.14
0.67* 0.44* 0.47* −0.04 0.07 0.38* 0.14 0.11
0.65* 0.15 0.62* −0.05 0.17 0.12 0.07 0.12
0.11 0.13 −0.39* −0.08 0.17 0.14 0.08 −0.15
0.45* 0.43* 0.14 −0.17 0.08 0.18 0.14 0.19
−0.13 −0.16 −0.42* −0.44* −0.11 −0.12 −0.2 −0.13
Abbreviations: See Tables 1 and 2. * p < 0.05 for all r-values. Table 4 Plasma fibrin clot characteristics in patients with progression of PAD or TAO compared with the remaining patients.
Ks , 10−9 cm2 Lag phase, s Absmax (405 nm) t50% , min D-Dmax , mg/L D-Drate , mg/(L min)
PAD progression (n = 47)
No PAD progression (n = 41)
p-Value
TAO progression (n = 10)
No TAO progression (n = 7)
p-Value
7.12 ± 0.93 98 (93–103) 1.09 (1.03–1.12) 10.9 ± 1.2 2.03 ± 0.49 0.082 ± 0.006
8.34 ± 0.87 107 (99–111) 0.98 (0.92–1.02) 9.6 ± 1.0 1.83 ± 0.48 0.086 ± 0.008
0.009 0.1 0.001 0.004 0.1 0.4
4.7 (3.9–4.9) 114 (97–121) 1.23 (1.18–1.32) 11.3 (11.9–10.9) 0.58 (0.53–0.64) 0.022 (0.018–0.03)
5.7 (4.7–6.1) 122 (118–131) 1.15 (0.96–1.22) 9.9 (9.2–10.8) 0.62 (0.56–0.67) 0.024 (0.019–0.028)
0.02 0.2 0.1 0.03 0.2 0.6
Values are given as mean ± SD or median (IQR). Abbreviations see Table 2.
CRP (4.49 ± 1.92 mg/L vs 1.85 ± 2.14 mg/L, p = 0.003). All clinical variables, including LVEF, were similar in a subgroup with PAD progression and the remainder (data not shown). Patients who reached the combined endpoint were characterized by 14.6% lower clot permeability, 10.2% higher maximum clot absorbancy, and 11.3% prolonged clot lysis time than the remainder; other fibrin measures were similar (Table 3). 2.2. TAO Twenty TAO patients and controls matched for demographics, body weight and smoking habit were recruited (Table 1). The only significant intergroup differences were found for fibrinogen, CRP, IL-6, tPA, PAI-1, and FPA; all the 6 variables were higher in the TAO patients than in controls. As shown in Table 2, marked differences in fibrin clot measures were noted between the TAO patients and the controls. Clot permeability was twofold lower in TAO patients compared with controls. This variable was inversely correlated with fibrinogen (r = −0.56; p < 0.01), CRP (r = −0.61; p < 0.01) and tHcy (r = −0.5; p = 0.03). Compared with controls, TAO patients had 47.9% shorter lag phase and 34.7% greater maximum clot absorbancy. In the TAO group, t50% was longer by 40.9% compared with controls and showed positive associations with fibrinogen (r = 0.48; p = 0.02), PAI-1 (r = 0.49; p = 0.02) and CRP (r = 0.55; p < 0.01). Interestingly, markedly twofold slower D-dimer release from clots and very low maximum D-dimer levels were also observed in TAO. Three TAO patients (15%) were lost to follow-up. During a median follow-up of 83 months (range, 61–89 months) the pro-
gression of TAO as a composite endpoint was observed in 10 (58.8%) of the remaining 17 patients, including 4 amputations. We noted 1 MI and 1 stroke; no CV deaths were recorded. Three patients declared they quitted smoking during follow-up. The TAO patients with the disease progression were similar to the remainder in terms of demographics or clinical variables (data not shown). Lower clot permeability and prolonged clot lysis time at study entry were observed in the TAO patients who achieved the endpoint compared with the remainder, while other fibrin parameters were similar (Table 4). 3. Discussion This study demonstrates that in patients with both PAD and TAO, plasma fibrin clot characteristics are unfavorably altered compared with the well matched control subjects. We found that plasma fibrin clots in both patient groups are less permeable, are formed more rapidly, and are lysed at a reduced rate, compared to those made from plasma obtained from controls. In contrast to MI patients [15], maximum D-dimer levels released from fibrin clots in the permeation lysis assay are lower than in controls, suggesting impaired release of the D-dimer molecules from the tight fibrin networks compared to the controls. Overall, this study provided evidence that patients with PAD share common clot features with those with CAD [15–18]. This indicates that abnormal fibrin clot phenotype may characterize all patients with atherosclerotic vascular disease. It should also be highlighted that despite a large atherosclerotic burden in PAD, even in these patients, a subgroup with diagnosed CAD displayed lower clot permeability, longer clot lysis, and
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faster polymerization compared to individuals free of CAD symptoms. Little is known about whether blood coagulation is involved in the pathogenesis of a rare smoking-related disease such as TAO. The current study expands our knowledge by showing several procoagulant alterations in TAO, including increased thrombin formation, impaired fibrinolysis and unfavorable clot phenotype despite a relatively young age and lack of evidence for CAD or diabetes. TAO with enhanced inflammatory state, reflected by elevated fibrinogen, IL-6 and CRP, has been for the first time shown to be characterized by more pronounced unfavorable alterations in fibrin clot characteristics compared to older PAD patients with several comorbidities, including CAD. This observation, along with the association of clot phenotype with clinical progression, suggests that rapid formation of poorly lysable compact clots in small vessels might contribute to leg ischemia in TAO. A larger study is needed to validate this observation. Of note, the permeation-based lysis assay performed in the TAO group showed very low maximum D-dimer levels and their extremely slow increase over time in the percolating buffer compared to both controls and PAD patients (Table 2). This suggests that a release of D-dimer induced by rtPA is markedly impaired in TAO and despite a dense clot structure reflected by low clot permeability a peak D-dimer concentration in this assay remains low even prior to the clot collapse. It might be speculated that the TAO patients tend to form extremely compact and irregular plasma fibrin clots. An important finding is a significant difference in fibrin clot properties between PAD patients with progression of the disease compared to the remainder, suggesting that unfavorable fibrin clot phenotype, reflected by the tendency to form compact and poorly lysable clots, might predispose to occlusive vascular complications and herald worse prognosis. Similar findings for TAO might indicate that coagulation factors and hypercoagulability leading to altered fibrin(ogen) characteristics contribute to narrowing and/or occlusion of both small-sized and large arteries. If so, it might be hypothesized that anticoagulant therapy could be beneficial in a certain subgroup of PAD and TAO patients with a particularly prothrombotic clot phenotype, however, combination of oral anticoagulation with aspirin may lead to excessive bleeding risk and oral anticoagulation alone have not been investigated in these diseases [24]. Mechanisms underlying alterations in fibrin clot structure/function in PAD or TAO patients are unclear. The Leeds family study showed that genetic factors contribute modestly to variance in fibrin clot measures, while the contribution of environmental factors is much larger [25]. Acquired factors, particularly those associated with enhanced inflammation, appear to be of major importance in PAD and TAO. It is known that increased levels of fibrinogen, being a major predictor of fibrin clot properties, are associated with a faster formation of denser fibrin networks of reduced lysability [14,26]. Elevated CRP has been shown to be associated with lower clot permeability and impaired lysis in patients at cardiovascular risk and those with acute MI [15,27]. Similar correlations were observed in PAD and TAO patients. It has been reported that hyperhomocysteinemia can reduce clot permeability and susceptibility to lysis [28], and the current study corroborates that in PAD patients tHcy correlates with some fibrin clot measures despite the presence of many potential confounders. Although elevated thrombin concentrations alter fibrin structure in experiments on purified fibrinogen [29], we did not observe any associations between plasma FPA levels and any fibrin variables in both patient groups, like in some previous studies [20]. We failed to show differences in fibrin clot properties associated with the administration of statins, which may increase fibrin clot permeability and enhance clot lysis [15,27]. Lack of drug-induced differences might be related to the patients’ characteristics and a low percentage of subjects who did not receive statins.
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An original observation is a markedly increased absorbancy in the PAD group despite similar fibrinogen concentrations in the patients and controls. Bhasin et al. [21] also observed a large difference between PAD patients with intermittent claudication and controls, which might corroborate our findings given a positive association between absorbancy in turbidimetry and fiber thickness. Elevated CRP in PAD patients (2.4-fold higher concentration compared with the controls) is likely to largely contribute to this intergroup difference in clot absorbancy. Some impact could be also attributed to increased tHcy in the PAD group. The current study has several limitations. First, the size of the study populations, particularly the TAO group, is limited, which may have introduced type II errors, especially in calculations of the correlation coefficients. Given the small number of subjects for the outcome study, with 16% lost to follow-up, the results regarding associations between fibrin clot characteristics and disease progression should be regarded as exploratory and need confirmation in a larger study. Secondly, our analysis was based on a determination of each variable at a single time point, at the time of enrolment, and significant changes in clot variables with time cannot be excluded. Third, scanning electron microscopy of fibrin clots has not been performed. However, we believe that functional plasma-based assays without clot dehydration provide more relevant information on a role of fibrin clot alterations in human pathology. Our experimental approach did not allow to analyze the effect of blood cells and platelets on fibrin clot structure/function, which can further impair for example fibrinolysis [30]. Additional fibrin clot modifiers that have not been investigated in the current study include oxidative stress, typically enhanced in atherosclerotic vascular disease [31]. Finally, genetic factors reported to alter fibrin clot structure/function [32] have not been studied. The concept of an influence of other as yet unidentified genetic factors has been supported by the study by Bhasin et al. [33] showing that first-degree relatives of PAD patients showed similarly altered clot variables with increased fiber thickness and factor XIII-mediated cross-linking. Further studies are needed to elucidate the genetics of fibrin clot architecture. In conclusion, our findings demonstrate that unfavorably altered fibrin clot properties associated with reduced efficiency of clot lysis occur in PAD and to a larger extent in a rare TAO. This study adds new information on the links between atherosclerosis, inflammation and coagulation, suggesting a major role of fibrin formation and degradation characteristics in the progression and vascular complications of vascular inflammatory diseases involving the lower limbs. Conflict of interest All authors have no conflict of interest to disclose. References [1] Aslam F, Haque A, Foody J, Lee LV. Peripheral arterial disease: current perspectives and new trends in management. South Med J 2009;102:1141–9. [2] McDermott MM, Kerwin DR, Liu K, et al. Prevalence and significance of unrecognized lower extremity peripheral arterial disease in general practice. J Gen Intern Med 2001;16:384–90. [3] McDermott MM, Greenland P, Liu K, et al. The ankle brachial index as a measure of leg functioning and physical activity in peripheral arterial disease: the Walking And Leg Claudication Study. Ann Intern Med 2002;136:873–83. [4] Merino J, Planos A, Elosua R, et al. Incidence and risk factors of peripheral arterial occlusive disease in a prospective cohort of 700 adult elderly men followed for 5 years. World J Surg 2010;34:1975–9. [5] Diehm C, Kareem S, Lawall H. Epidemiology of peripheral arterial disease. Vasa 2004;33:183–9. [6] Olin JW. Thromboangiitis obliterans (Buerger’s disease). N Engl J Med 2000;343:864–9. [7] Aziz M, Boutouyrie P, Bura-Riviere A, et al. Thromboangiitis obliterans and endothelial function. Eur J Clin Invest 2010;40:518–26.
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