By Gosk-Bierska, I Wysokinski, W; Karnicki, K; Adamiec, R
Aim. Thrombus formation plays a critical role in pathogenesis of cardiovascular complications in atherosclerotic peripheral arterial occlusive disease (PAOD). Tissue factor (TF) initiates the clotting cascade and is considered an important regulator of hemostasis and thrombosis. TF activity is regulated by TF pathway inhibitor (TFPI). The aim of our study was to evaluate plasma levels of the TF, TFPI and their relation to coagulation system and various other risk factors of atherosclerosis in patients with chronic limbs ischemia. Methods. Plasma TF, total TFPI, truncated TFPI, full-length TFPI were assessed by ELISA using commercially available kits (IMUBIND Tissue Factor; Total TFPI; Truncated TFPI ELISA Kit; American Diagnostica Inc. Stamford) in 62 claudicant patients with PAOD and 20 healthy controls.
Results. We observed statistically higher levels of TF (94+-52 pg/ mL), total TFPI (43+-8 ng/mL), and truncated TFPI (22+-7 ng/mL) in patients with PAOD compared to healthy individuals (TF: 66+-15 pg/ mL; total TFPI: 36+-4 ng/mL; truncated TFPI: 14+-5 ng/mL). Full- length TFPI (20 +-4 ng/mL) is lower in patients with PAOD than in controls (23+-5 ng/mL). The study indicated a positive correlation between TF and truncated TFPI (r=0.34), total TFPI and full TFPI (r=0.5), total TFPI and truncated TFPI (r=0.83) in patients with PAOD, and negative correlation between full TFPI and truncated TFPI (r=-0.65) in the control.
Conclusion. Elevated levels of TF, disorders of balance between full-length TFPI and truncated TFPI as well as significantly increased truncated TFPI level in patients with PAOD can be independent risk factors of atherosclerotic complications.
[Int Angiol 2008;27:296-301]
Key words: Thromboplastin – Lipoprotein-associated coagulation inhibitor – Atherosclerosis – Arterial occlusive diseases.
Tissue factor (TF), a membrane glycoprotein found on endothelial cells, smooth muscle cells, white blood cells and in plasma, is considered an important regulator of hemostasis and thrombosis.1-3 After vascular injury, TF interacts with circulating factor VII, forming the active TF/VIIa complex which activates factor X. This process is more efficient in the presence of phospholipids and Ca- ions. P-selectins, certain cytokines, endotoxin and immune complexes stimulate monocytes and induce the TF expression on their surfaces.4 TF may contribute to the generation of coagulation proteases and the activation of protease activated receptors on vascular cells.5 Atherosclerotic plaques and monocytes can generate TF after stimulation with lipopolysaccharides or IL-1.6 TF plays an important role in the initiation of blood coagulation after plaque rupture in patients with acute coronary syndromes.79 TF activity is regulated by TF pathway inhibitor (TFPI). Alteration in TF and TFPI levels can influence thrombotic events. TFPI is secreted from platelets, endothelial cells, mesangial cells, smooth cells, monocytes, fibroblasts, cardiomyocytes.1012 TFPI is released into circulation after unfractionated or low molecular weight heparin injections in dose-dependent manner.13, 14 TFPI is a Kunitz-type protease inhibitor containing three domains. The first domain inhibits TF/ VIIa complex, the second one interacts with Xa, and the third domain contains highly positively charged carboxyl-terminus binding negatively-charged glycosaminoglycans including heparin. TFPI inhibits the proteolytic capacity of factor Xa by binding to the active site of Xa through its second Kunitz domain and then it inhibits the activity of TF/VIIa complex by forming a quaternary complex with TF/VIIa. The initial TFPI/Xa binding potentiates inhibition of the TF/VIIa complex by the first Kunitz domain. TFPI can also directly inhibit TF/VIIa/Xa complex in presence of Ca- ions.15 TFPI mediates downregulation of cell-surface TF in fibroblasts and monocytic cells via internalization and degradation by LDL receptor-related protein.16 The C-terminus region of TFPI contributes to modulation of cell proliferation. Enzyme immunoassays can distinguish between the free, lipoprotein-associated and endothelial cell-associated forms of TFPI.17 TFPI levels in plasma increase in patients with acute myocardial infarction/unstable angina.18, 19 The aim of our study was to evaluate plasma levels of the TF, TFPI and their relation to rheology, coagulation system and others risk factors in patients with atherosclerotic chronic limb ischemia.
Materials and methods
Sixty-two patients with intermittent claudication secondary to atherosclerotic peripheral arterial occlusive disease (PAOD) (stage 2 according to Fontaine) and 20 healthy individuals without risk factors of atherosclerosis were enrolled in the study. The diagnosis of PAOD was made based on positive history of claudication, positive findings on physical examination (bruits, absent or diminished pulses), and segmental blood pressure measurements (ankle-brachial index [ABI]
The study was approved by the Ethics Committee of the Wroclaw Medical University.
Blood was collected by the antecubital vein venipuncture. Blood samples for laboratory assays were obtained at approximately 8:00 a.m. after overnight fasting.
Plasma TF, total TFPI, truncated TFPI, full-length TFPI were assessed by ELISA using commercially available kits (IMUBIND Tissue Factor ELISA Kit no. 845; IMUBIND Total TFPI ELISA Kit no. 849; IMUBIND Truncated TFPI ELISA Kit no 850, American Diagnostica Inc., Stamford). Plasminalpha2-antiplasmin (PAP) complex, thrombin- antithrombin complex (TAT) were assessed by ELISA using commercially available kits (Enzygnost PAP micro-Dade Behring Marburg, Germany; Enzygnost TAT micro-Behring, Behringwerke AG Marburg, Germany). Plasma fibrinogen concentrations were measured by the coagulometric method (Fibrintimer II, Behring, Marburg, Germany using Multifiber). Platelet count, hemoglobin, hematocrit, hemoglobin A1c, plasma lipids, homocysteine, uric acid levels were elevated using routine procedures employed in our institution.
Statistical analysis
The statistical analyses were performed using STATISTICA 5,0PL. Statistical significance was accepted as P
Results
Sixty-two patients with PAOD and 20 healthy individuals were included in our study. Demographic data are presented in Table I. Both groups were gender- and age-matched. We observed statistically higher levels of TF (94+-52 pg/mL), total TFPI (43+-8 ng/mL), and truncated TFPI (22+-7 ng/mL) in patients with POAD compared to healthy individuals (TF: 66+-15 pg/mL; total TFPI: 36+-4 ng/mL; truncated TFPI: 14+-5 ng/mL). Full-length TFPI (20+-4 ng/mL) was lower in patients with PAOD than in controls (23+-5 ng/mL) (Table II). Atherosclerotic risk factors such as diabetes, hypertension, hyperlipidemia, tobacco smoking, obesity had no influence on TF and TFPI levels in patients with PAOD (Table III). We did not observe differences in TF and TFPI levels in claudicants with and without diabetes, coronary heart disease, myocardial infarction, stroke, and endovascular procedures or vascular surgery reported in past medical history (Table IV). Present study indicates positive correlation between TF and truncated TFPI (r=0.34), total TFPI and full TFPI (r=0.5), total TFPI and truncated TFPI (r=0.83) in patients with PAOD. In the control group, only negative correlation between full TFPI and truncated TFPI (r=-0.65) was observed (Table V). Fibrinogen, TAT, PAP levels were significantly higher in patient with chronic limb ischemia compared to controls (Table VI). There was no significant correlation between TF or TFPI and fibrinogen, TAT, PAP, plasma lipids, uric acid, platelets count, THT, HT, BMI, smoking (cigarettes * years).
Discussion
Our study indicates higher plasma TF levels in patients with chronic limbs ischemia in stage 2 according to Fontaine in the course of atherosclerosis compared to controls. In the present study, total TFPI and truncated TFPI levels were significantly higher in PAOD group, but full-length TFPI levels were lower in claudicants compared to controls. We did not observe any differences in TF and TFPI levels between patients in PAOD group with and without coronary heart disease and/or myocardial infarction reported in prior medical history. In claudicants group, TF and TFPI levels were also similar in patients with and without stroke, endovascular procedures or vascular surgery in medical history. TF plasma levels did not correlate with atherosclerotic risk factors such as plasma lipids, fibrinogen, and uric acid. Our study found no influence of hypertension, hyperlipidemia, diabetes, obesity, tobacco habit, cigarettes/years index on TF plasma levels. The analysis of TF and TFPI relationship indicated only positive correlation between TF and truncated TFPI in patients with chronic limbs ischemia. We did not observe any significant correlation between TF and TFPI in controls. Higher levels of fibrinogen, TAT, PAP in PAOD patients found in the present study confirm our previous observations.20, 21 However, elevated TF levels did not correlate with these parameters in claudicating patients.
There are only a few data about the role of TF and TFPI in patients with chronic limb ischemia. Blann et al. found elevated levels of TF and lower levels of total TFPI in patients with PAOD compared to healthy individuals and have suggested that reduced levels of total TFPI and raised levels of TF may contribute to the process of atherogenesis and the increased risk of thrombosis in patients with cardiovascular disease.22 We observed lower full TFPI level in claudicants compared controls, but total and truncated TFPI levels were higher in patients with PAOD. Two TFPI variants: full- length TFPI and truncated TFPI have different inhibitory potentials.23-24 The full-length TFPI contains the C-terminal domain required for direct interactions with ApoB100 as well as cell surface glycosaminoglycans. Truncated TFPI lacks the Cterminus domain essential for its anticoagulant activity and required for optimal factor Xa inhibition and interaction with lipoproteins.25 Ettelaie et al. demonstrated that only the full-length TFPI was influenced by LDL oxidation which resulted in the loss of its anticoagulant function, probably due to its ability to associate with LDL, and that C-terminal truncated TFPI was not affected significantly by oxidation. Full-length TFPI may be partially degraded in blood to the truncated TFPI.26 The change of proportion between truncated and full-length TFPI in patients with PAOD observed in our study can increase the risk of atherosclerotic damage to arterial walls and/or thrombotic tendency mainly because truncated TFPI has reduced affinity to heparin and others proteoglicans and lipids. Mast et al. postulated that physiological concentration of TFI is not sufficient to control the TF procoagulant activity.27 Increased levels of TFPI in claudicating patients in our study can be a compensative reaction due to higher TF level. High TF activity contributes to the procoagulant activity of disrupted atherosclerotic plaque and the superimposed mural thrombus.28,29 Some studies show increased levels of plasma TF in patients with acute coronary syndrome8,9 and chronic coronary artery disease.30 Suefuji et al. postulates that higher plasma TF levels in patients with acute myocardial infarction and prodromal unstable angina compared patients with chronic stable angina may reflect enhanced intravascular procoagulant activity.9 As well TFPI can be involved in thrombus formation in patients with acute coronary syndromes. The lower total and free TFPI plasma levels in coronary sinus compared to TPFI levels in aorta before and after PTCA in patients with acute myocardial infarction and lack of these differences in patients with stable angina indicated the role of TFPI in hemostasis after acute vessel wall injury.31 The lack of significant difference between TF and TFPI level in claudicants with and without coronary heart disease in our study might be due to the fact that none of our patients had unstable angina or recent myocardial infarction during the study.
Some authors suggest the influence of atherosclerotic risk factors on TF and/or TFPI levels22, 32-35 Morange et al. indicated positive correlation between free TFPI and fibrinogen, von Willebrand factor, t-PA, thrombomodulin, and presence of diabetes, hypertension, obesity and hyperlipidemia in patients with cardiovascular risk factors. The positive correlation was found for total TFPI with LDL cholesterol and negative correlation with others parameters of metabolic syndrome.32 Our study did not reveal any correlation between fibrinogen, TAT, PAP, plasma lipids and TFPI. We also did not observe any influence of diabetes, obesity or hypertension on TFPI level in patients with PAOD. Hyperlipidemia may result in more TFPI being bound which makes it unavailable to antagonize the procoagulant properties of TF. Kawaguchi et al. found that free TFPI level is inversely correlated with HDL cholesterol in patients with coronary arterial disease.33 However, in our study we did not observe statistical differences between plasma lipids in claudicant patients with PAOD in whom hyperlipidemia had been diagnosed in 85% and control patients. This was probably due to effective statin therapy. It can explain lack of the correlation between TFPI and lipid levels in patients with PAOD. A few studies have demonstrated that smoking increases TF expression in atherosclerotic plaque,34,35 but we did not detect any differences in TF levels between smoking and non smoking patients with PAOD.
We are planning to observe patients with POAD for 2 years during which all cardiovascular events (myocardial infarction, stroke, endovascular procedures, and surgery) will be registered and correlation between TF and TFPI levels and thrombotic complications analyzed.
Conclusions
Elevated levels of TF, disorders of balance between full-length TFPI and truncated TFPI, and significantly increased truncated TFPI level in patients with PAOD can be independent risk factors of atherosclerotic complications.
Received on March 1, 2007; accepted for publication on June 10, 2007.
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I. GOSK-BIERSKA 1, W. WYSOKINSKI 2, K. KARNICKI2, R. ADAMIEC 1
1 Unit of Angiology, Hypertension and Diabetology, Medical University of Wroclaw, Wroclaw, Poland
2 Mayo Clinic and Foundation for Education and Research, Rochester, MN, USA
Address reprint requests to: I. Gosk-Bierska M.D., Ph.D., Clinic of Angiology, Hypertension and Diabetology, Medical University or Wroclaw, ul. Poniatowskiego 2, 50-326 Wroclaw, Polska. E-mail: [email protected]
Copyright Edizioni Minerva Medica Aug 2008
(c) 2008 International Angiology. Provided by ProQuest LLC. All rights Reserved.
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