226 Original article
Activity of thrombin-activatable fibrinolysis inhibitor in the plasma of patients with abdominal aortic aneurysm Joanna Dubisa, Natalia Z˙uka, Ryszard Grendziakb, Norbert Zapotocznyb, Monika Pfanhauserb and Wojciech Witkiewiczb,c Patients with abdominal aortic aneurysm (AAA) experience impaired balance between fibrinolysis and coagulation, manifested by increased prothrombotic tendency and intensified inflammatory processes. The aim of this study was to evaluate the TAFI activity level (thrombin activatable fibrinolysis inhibitor) in the plasma of AAA patients. Plasma levels of PAI-1 (plasminogen activator inhibitor type 1), urokinase-type plasminogen activator and uPAR (urokinase-type plasminogen activator receptor) were measured as markers of fibrinolytic activity. The study showed that the activity of the thrombin-activatable fibrinolysis inhibitor in the plasma of AAA patients was significantly lower than in the plasma of the control individuals (64.6 W 10.1 vs. 54.2 W 10.9%, P < 0.0001). TAFI activity positively correlated with the white blood cell count (r U 0.486, P < 0.005). The uPAR concentration in the AAA patients was statistically significantly higher than in the control group and positively correlated with TAFI activity (r U 0.409, P U 0.02). The levels of PAI-1 and D-dimers (fibrin fragments) were significantly higher in patients with AAA than in the control group (44.3 W 17.5 vs. 21.7 W 8.7 ng/ml
Introduction Abdominal aortic aneurysm (AAA) involves a widening of the infrarenal aorta being the result of a progressive thinning of the vessel. It is estimated that AAA affects upto approximately 5–7% of men over 60 years [1,2]. Its course is asymptomatic and can result in an aortic rupture followed by a massive haemorrhage and death to about 1–3% of patients operated on [3]. An AAA development is a risk factor for complication of vascular comorbidities and poses an additional threat to the patient’s life [4]. Patients with AAA develop disorders in plasma haemostasis, the mechanisms of which normally help to maintain the balance between the coagulation and fibrinolysis and fluidity of blood inside the vessel. Haemostatic disorders in this group of patients primarily involve abnormalities in the mechanisms of this process regulation [5–8]. AAA patients experience intensified plasma fibrinolysis and increased concentration of prothrombotic factors. Their blood exhibits elevated levels of D-dimers (fibrin fragments), PAI-1 (plasminogen activator inhibitor type-1),thrombin-antithrombin complexes, F1þ2 (prothrombin fragments) [7,9,10]. Haemostatic abnormalities are further aggravated by inflammatory processes associated with the aneurysm. The blood concentration of inflammation markers, for 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
and 1869.6 W 1490.1 vs. 181.5 W 188.6 ng/ml, respectively). Lowered activity of the fibrinolysis inhibitor TAFI may heighten the blood fibrinolytic potential in AAA patients and contribute to the development of comorbidities. Therefore, TAFI participation in AAA pathogenesis cannot be excluded. Blood Coagul Fibrinolysis 25:226–231 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2014, 25:226–231 Keywords: abdominal aortic aneurysm, fibrinolysis, thrombin activatable fibrinolysis inhibitor a
Regional Specialist Hospital in Wrocław, Research and Development Centre, Department of Vascular Surgery, Regional Specialist Hospital in Wrocław, Research and Development Centre and cWrocław Medical University, Wrocław, Poland
b
Correspondence to Joanna Dubis, PhD, Regional Specialist Hospital in Wroclaw, ul. Kamienskiego 73a, Research and Development Center, 51-124 Wroclaw, Poland Tel: +48 71 327 05 38; e-mail: [email protected] Received 19 February 2013 Revised 10 September 2013 Accepted 13 November 2013
example, C-reactive protein and interleukin-6, tumor necrosis factor-a is markedly elevated [5,11]. This exacerbates the aortic injury and leads to an endothelial dysfunction that contributes to the loss of its antithrombotic properties. Impaired regulation of coagulation and fibrinolysis and endothelial dysfunction are the reasons for a dominating prothrombotic tendency leading to hypercoagulability [11,12]. The dilated space of large aneurysms is filled with intraluminal thrombus (ILT), the size of which correlates with the size of the aneurysm [13,14]. ILT constantly interferes with the circulating blood and undergoes continuous remodelling. Although it stabilizes the aortic wall, it also enhances inflammatory processes and proteolytic degradation of the vessel wall [6,15]. In patients with large AAA, the ILT is denser with smaller pores and is more resistant to fibrinolysis comparing to those with small AAA and those without aneurysm [14]. Apart from PAI-1 inhibitor, another important factor in fibrinolysis regulation is the thrombin-activatable fibrynolysis inhibitor (TAFI), which reduces the formation of active plasmin, thereby supporting clot lysis inhibition [16]. The fibrinolysis inhibitor TAFI is a proteolytic enzyme of carboxypeptidase activity, synthesized in the liver from where it enters the bloodstream. DOI:10.1097/MBC.0000000000000028
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Patients with abdominal aortic aneurysm Dubis et al. 227
It was also found in blood platelets [17]. TAFI is glycoprotein and circulates in the blood as an inactive enzyme precursor. As most plasma factors, TAFI is proteolytically activatable. It is activated by cleavage of the activating peptide, which obscures the active centre of the enzyme [16,18]. This results in the formation of the active form of the inhibitor, TAFIa. Inhibitor TAFI, activated by thrombin at high concentration or thrombin complexed with thrombomodulin, splits off resides of lysine at the C-terminals of fibrin, thereby preventing its binding of tPA and plasminogen. As a result, there is a decrease in catalytic efficiency of the production of plasmin from plasminogen [19,20]. TAFI is activated by thrombin, the main enzyme of the coagulation pathway, and therefore it is considered to be a molecular link between coagulation and fibrinolysis. TAFI inhibits the clot lysis, taking part in stabilization thereof. Under physiological conditions, the process of TAFI proenzyme activation to its active form occurs in the presence of thrombin and a thrombomodulin complex and is 1250 times more efficient than TAFI activation by thrombin alone [16]. The thrombin/ thrombomodulin complex activates the protein C to an active enzyme (activated protein C and APC), which complements TAFI functionality. APC suppresses the coagulation cascade by reducing the level of generated thrombin, whereas TAFI protects the developing clot against premature lysis [21]. Regardless of fibrinolysis, TAFI plays important regulatory functions in inflammatory processes. This is because the possible substrates for TAFI carboxypeptidase involve not only fibrin, but also anti-inflammatory peptides (bradykinin, anaphylatoxin C3a and C5a, osteopontin, annexin II and chemerin) [22–25]. Researchers investigating the pathological mechanisms of AAA formation and development have recently paid much attention to serine proteases of the urokinase fibrinolysis activation system urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) [26,27]. This system increases the matrix proteolytic potential and escalates aortic tissue degradation at the site of the lesion [28]. A soluble form of the uPAR receptor (suPAR) appears in pathological conditions, accompanied by enhanced inflammatory processes [29,30]. Animal studies have shown that the urokinase activator plays an important role in the process of aneurysm rupture. It has been proven that the uPA expression in ruptured AAA tissues is significantly lower [26]. However, the function of the uPA plasminogen activator in AAA pathogenesis remains unclear. Patients with AAA develop increased inflammatory processes and impaired fibrinolysis regulatory mechanisms, which lead to hypercoagulability. It is known that the PAI-1 plasma concentration in these individuals is elevated, but there is no available data on TAFIa inhibitor activity. Animal studies have shown that TAFI deficiency enhances the development of AAA in the
mouse model [31]. The aim of this study was to determine the activity level of a thrombin-activatable fibrinolysis inhibitor (TAFIa) in the plasma of AAA patients and analysis of the relationships between this factor and plasma markers of fibrinolytic activity. We also evaluated the levels of the soluble receptor of urokinase plasminogen activator uPAR, which is considered an important marker of inflammatory processes.
Methods The study included 32 patients diagnosed with AAA who were scheduled for stent graft implantation in place of the diseased vessel in the Regional Specialized Hospital in Wroclaw, Research and Development Centre. The planned study was approved by the local Bioethics Committee. The control group consisted of 45 volunteers matched for sex, age, ethnicity and smoking lifestyle, in whom an aortic widening was excluded by ultrasound. Patients receiving oral anticoagulants and those in whom a history of liver disease, autoimmune disease or venous thrombosis was ascertained in the medical interview were excluded from the study. Blood was collected from the antecubital vein of fasting patients and healthy control individuals into a tube with an anticoagulant (9 volumes of blood per 1 volume of sodium citrate). To obtain platelet poor plasma for testing, the blood was centrifuged at 2000g for 20 min. The prepared material was stored at –868C until determination of the studied parameters. The activity of the TAFI fibrinolysis inhibitor was determined with a TAFI Activity Kit (American Diagnostica, USA), developed based on a chromogenic method. Substrate of the commercial test is selective for TAFIa (active form of thrombin activatable fibrinolysis inhibitor). The concentration of clotting factors was analysed using commercially available kits, developed on the basis of the ELISA immunoassay method: IMUBIND Plasma PAI-1 ELISA Kit, IMUCLONE D-dimer ELISA kit, IMUBIND uPA ELISA and IMUBIND uPAR ELISA (American Diagnostica, USA). Other parameters – white blood cells, APTT (activated partial thromboplastin time), INR (international normalized ratio), serum creatinine and urea were determined by the standard methods employing Advia 2120i and SIEMENS BCS XP analysers in a diagnostic laboratory. All analyses were carried out using SPSS statistical software 14.0 for Windows (SPSS, Chicago, Illinois, USA). All analyses were performed using the R Statistical Package (open source, http://www.r-project.org/) and MedCalc (MedCalc Software, Belgium). The differences in findings between the study groups were assessed by chi-square tests (two-tailed without Yates correction) for categorical variables and by Mann–Whitney tests for continuous variables. The differences in findings between the study groups were evaluated by t-Student
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
228 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 3
or Welch tests (depending on homogeneity of variance). Correlation was estimated by the Pearson method. The results were considered statistically significant when the P-values were < 0.05.
Fig. 1
75 70
The study was conducted in patients diagnosed with an AAA by ultrasound and/or computed tomography (n ¼ 32). All the diagnoses were confirmed by an intraoperative assessment. The mean maximum diameter of the aneurysm was 59.3 11.5 mm. The mean thickness of the ILT was 23.6 11.8 mm. The average diameter of the aorta in the control group was 18.5 2.4 mm (14.2–24 mm). Compared with the control group, the plasma of the AAA patients showed significantly lower TAFIa fibrinolysis inhibitor activity (54.2 10.9 vs. 64.6 10.1%, P < 0.0001) and a significantly higher level of plasminogen activation inhibitor PAI-1 (44.3 17.5 vs. 21.7 8.7 ng/ml, P < 0.0001) and D-dimers (1869.6 1490.1 vs. 181.5 188.6 ng/ml, P < 0.0001). However, it was found that the concentration of its soluble receptor uPAR was significantly higher in the AAA group (3.02 1.05 vs. 2.49 1.04 ng/ml, P
Activity of thrombin-activatable fibrinolysis inhibitor in the plasma of patients with abdominal aortic aneurysm Joanna Dubisa, Natalia Z˙uka, Ryszard Grendziakb, Norbert Zapotocznyb, Monika Pfanhauserb and Wojciech Witkiewiczb,c Patients with abdominal aortic aneurysm (AAA) experience impaired balance between fibrinolysis and coagulation, manifested by increased prothrombotic tendency and intensified inflammatory processes. The aim of this study was to evaluate the TAFI activity level (thrombin activatable fibrinolysis inhibitor) in the plasma of AAA patients. Plasma levels of PAI-1 (plasminogen activator inhibitor type 1), urokinase-type plasminogen activator and uPAR (urokinase-type plasminogen activator receptor) were measured as markers of fibrinolytic activity. The study showed that the activity of the thrombin-activatable fibrinolysis inhibitor in the plasma of AAA patients was significantly lower than in the plasma of the control individuals (64.6 W 10.1 vs. 54.2 W 10.9%, P < 0.0001). TAFI activity positively correlated with the white blood cell count (r U 0.486, P < 0.005). The uPAR concentration in the AAA patients was statistically significantly higher than in the control group and positively correlated with TAFI activity (r U 0.409, P U 0.02). The levels of PAI-1 and D-dimers (fibrin fragments) were significantly higher in patients with AAA than in the control group (44.3 W 17.5 vs. 21.7 W 8.7 ng/ml
Introduction Abdominal aortic aneurysm (AAA) involves a widening of the infrarenal aorta being the result of a progressive thinning of the vessel. It is estimated that AAA affects upto approximately 5–7% of men over 60 years [1,2]. Its course is asymptomatic and can result in an aortic rupture followed by a massive haemorrhage and death to about 1–3% of patients operated on [3]. An AAA development is a risk factor for complication of vascular comorbidities and poses an additional threat to the patient’s life [4]. Patients with AAA develop disorders in plasma haemostasis, the mechanisms of which normally help to maintain the balance between the coagulation and fibrinolysis and fluidity of blood inside the vessel. Haemostatic disorders in this group of patients primarily involve abnormalities in the mechanisms of this process regulation [5–8]. AAA patients experience intensified plasma fibrinolysis and increased concentration of prothrombotic factors. Their blood exhibits elevated levels of D-dimers (fibrin fragments), PAI-1 (plasminogen activator inhibitor type-1),thrombin-antithrombin complexes, F1þ2 (prothrombin fragments) [7,9,10]. Haemostatic abnormalities are further aggravated by inflammatory processes associated with the aneurysm. The blood concentration of inflammation markers, for 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
and 1869.6 W 1490.1 vs. 181.5 W 188.6 ng/ml, respectively). Lowered activity of the fibrinolysis inhibitor TAFI may heighten the blood fibrinolytic potential in AAA patients and contribute to the development of comorbidities. Therefore, TAFI participation in AAA pathogenesis cannot be excluded. Blood Coagul Fibrinolysis 25:226–231 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2014, 25:226–231 Keywords: abdominal aortic aneurysm, fibrinolysis, thrombin activatable fibrinolysis inhibitor a
Regional Specialist Hospital in Wrocław, Research and Development Centre, Department of Vascular Surgery, Regional Specialist Hospital in Wrocław, Research and Development Centre and cWrocław Medical University, Wrocław, Poland
b
Correspondence to Joanna Dubis, PhD, Regional Specialist Hospital in Wroclaw, ul. Kamienskiego 73a, Research and Development Center, 51-124 Wroclaw, Poland Tel: +48 71 327 05 38; e-mail: [email protected] Received 19 February 2013 Revised 10 September 2013 Accepted 13 November 2013
example, C-reactive protein and interleukin-6, tumor necrosis factor-a is markedly elevated [5,11]. This exacerbates the aortic injury and leads to an endothelial dysfunction that contributes to the loss of its antithrombotic properties. Impaired regulation of coagulation and fibrinolysis and endothelial dysfunction are the reasons for a dominating prothrombotic tendency leading to hypercoagulability [11,12]. The dilated space of large aneurysms is filled with intraluminal thrombus (ILT), the size of which correlates with the size of the aneurysm [13,14]. ILT constantly interferes with the circulating blood and undergoes continuous remodelling. Although it stabilizes the aortic wall, it also enhances inflammatory processes and proteolytic degradation of the vessel wall [6,15]. In patients with large AAA, the ILT is denser with smaller pores and is more resistant to fibrinolysis comparing to those with small AAA and those without aneurysm [14]. Apart from PAI-1 inhibitor, another important factor in fibrinolysis regulation is the thrombin-activatable fibrynolysis inhibitor (TAFI), which reduces the formation of active plasmin, thereby supporting clot lysis inhibition [16]. The fibrinolysis inhibitor TAFI is a proteolytic enzyme of carboxypeptidase activity, synthesized in the liver from where it enters the bloodstream. DOI:10.1097/MBC.0000000000000028
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Patients with abdominal aortic aneurysm Dubis et al. 227
It was also found in blood platelets [17]. TAFI is glycoprotein and circulates in the blood as an inactive enzyme precursor. As most plasma factors, TAFI is proteolytically activatable. It is activated by cleavage of the activating peptide, which obscures the active centre of the enzyme [16,18]. This results in the formation of the active form of the inhibitor, TAFIa. Inhibitor TAFI, activated by thrombin at high concentration or thrombin complexed with thrombomodulin, splits off resides of lysine at the C-terminals of fibrin, thereby preventing its binding of tPA and plasminogen. As a result, there is a decrease in catalytic efficiency of the production of plasmin from plasminogen [19,20]. TAFI is activated by thrombin, the main enzyme of the coagulation pathway, and therefore it is considered to be a molecular link between coagulation and fibrinolysis. TAFI inhibits the clot lysis, taking part in stabilization thereof. Under physiological conditions, the process of TAFI proenzyme activation to its active form occurs in the presence of thrombin and a thrombomodulin complex and is 1250 times more efficient than TAFI activation by thrombin alone [16]. The thrombin/ thrombomodulin complex activates the protein C to an active enzyme (activated protein C and APC), which complements TAFI functionality. APC suppresses the coagulation cascade by reducing the level of generated thrombin, whereas TAFI protects the developing clot against premature lysis [21]. Regardless of fibrinolysis, TAFI plays important regulatory functions in inflammatory processes. This is because the possible substrates for TAFI carboxypeptidase involve not only fibrin, but also anti-inflammatory peptides (bradykinin, anaphylatoxin C3a and C5a, osteopontin, annexin II and chemerin) [22–25]. Researchers investigating the pathological mechanisms of AAA formation and development have recently paid much attention to serine proteases of the urokinase fibrinolysis activation system urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) [26,27]. This system increases the matrix proteolytic potential and escalates aortic tissue degradation at the site of the lesion [28]. A soluble form of the uPAR receptor (suPAR) appears in pathological conditions, accompanied by enhanced inflammatory processes [29,30]. Animal studies have shown that the urokinase activator plays an important role in the process of aneurysm rupture. It has been proven that the uPA expression in ruptured AAA tissues is significantly lower [26]. However, the function of the uPA plasminogen activator in AAA pathogenesis remains unclear. Patients with AAA develop increased inflammatory processes and impaired fibrinolysis regulatory mechanisms, which lead to hypercoagulability. It is known that the PAI-1 plasma concentration in these individuals is elevated, but there is no available data on TAFIa inhibitor activity. Animal studies have shown that TAFI deficiency enhances the development of AAA in the
mouse model [31]. The aim of this study was to determine the activity level of a thrombin-activatable fibrinolysis inhibitor (TAFIa) in the plasma of AAA patients and analysis of the relationships between this factor and plasma markers of fibrinolytic activity. We also evaluated the levels of the soluble receptor of urokinase plasminogen activator uPAR, which is considered an important marker of inflammatory processes.
Methods The study included 32 patients diagnosed with AAA who were scheduled for stent graft implantation in place of the diseased vessel in the Regional Specialized Hospital in Wroclaw, Research and Development Centre. The planned study was approved by the local Bioethics Committee. The control group consisted of 45 volunteers matched for sex, age, ethnicity and smoking lifestyle, in whom an aortic widening was excluded by ultrasound. Patients receiving oral anticoagulants and those in whom a history of liver disease, autoimmune disease or venous thrombosis was ascertained in the medical interview were excluded from the study. Blood was collected from the antecubital vein of fasting patients and healthy control individuals into a tube with an anticoagulant (9 volumes of blood per 1 volume of sodium citrate). To obtain platelet poor plasma for testing, the blood was centrifuged at 2000g for 20 min. The prepared material was stored at –868C until determination of the studied parameters. The activity of the TAFI fibrinolysis inhibitor was determined with a TAFI Activity Kit (American Diagnostica, USA), developed based on a chromogenic method. Substrate of the commercial test is selective for TAFIa (active form of thrombin activatable fibrinolysis inhibitor). The concentration of clotting factors was analysed using commercially available kits, developed on the basis of the ELISA immunoassay method: IMUBIND Plasma PAI-1 ELISA Kit, IMUCLONE D-dimer ELISA kit, IMUBIND uPA ELISA and IMUBIND uPAR ELISA (American Diagnostica, USA). Other parameters – white blood cells, APTT (activated partial thromboplastin time), INR (international normalized ratio), serum creatinine and urea were determined by the standard methods employing Advia 2120i and SIEMENS BCS XP analysers in a diagnostic laboratory. All analyses were carried out using SPSS statistical software 14.0 for Windows (SPSS, Chicago, Illinois, USA). All analyses were performed using the R Statistical Package (open source, http://www.r-project.org/) and MedCalc (MedCalc Software, Belgium). The differences in findings between the study groups were assessed by chi-square tests (two-tailed without Yates correction) for categorical variables and by Mann–Whitney tests for continuous variables. The differences in findings between the study groups were evaluated by t-Student
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
228 Blood Coagulation and Fibrinolysis 2014, Vol 25 No 3
or Welch tests (depending on homogeneity of variance). Correlation was estimated by the Pearson method. The results were considered statistically significant when the P-values were < 0.05.
Fig. 1
75 70
The study was conducted in patients diagnosed with an AAA by ultrasound and/or computed tomography (n ¼ 32). All the diagnoses were confirmed by an intraoperative assessment. The mean maximum diameter of the aneurysm was 59.3 11.5 mm. The mean thickness of the ILT was 23.6 11.8 mm. The average diameter of the aorta in the control group was 18.5 2.4 mm (14.2–24 mm). Compared with the control group, the plasma of the AAA patients showed significantly lower TAFIa fibrinolysis inhibitor activity (54.2 10.9 vs. 64.6 10.1%, P < 0.0001) and a significantly higher level of plasminogen activation inhibitor PAI-1 (44.3 17.5 vs. 21.7 8.7 ng/ml, P < 0.0001) and D-dimers (1869.6 1490.1 vs. 181.5 188.6 ng/ml, P < 0.0001). However, it was found that the concentration of its soluble receptor uPAR was significantly higher in the AAA group (3.02 1.05 vs. 2.49 1.04 ng/ml, P
