Global Fibrinolytic Activity, PAI-1 Level, and 4G/5G Polymorphism in Thai Children with Arterial Ischemic Stroke Rungrote Natesirinilkul, MD,* Werasak Sasanakul, BSc,* Ampaiwan Chuansumrit, MD,* Praguywan Kadegasem, BSc,* Anannit Visudtibhan, MD,* Pakawan Wongwerawattanakoon, BSc,† and Nongnuch Sirachainan, MD*
Background: Prolonged euglobulin clot lysis time (ECLT) and increased level of plasminogen activator inhibitor-1 (PAI-1) were reported to be risk factors of arterial ischemic stroke (AIS) by some studies; however, these findings were not supported by other studies. The objective of this study was to determine the association of ECLT, PAI-1 level, and polymorphisms of 4G and 5G of PAI-1 gene to the development of AIS in Thai children. Methods: This study included patients aged 1-18 years old. Diagnosis of AIS was confirmed by imaging study. The control group was ageand sex-matched healthy subjects. Demographic data were recorded, and blood was tested for ECLT, PAI-1 level, lipid profiles, fasting blood sugar (FBS), and 4G and 5G polymorphisms of PAI-1 gene. Results: There were 70 subjects participating in this study, consisting of 30 patients and 40 controls. Demographic data, lipid profiles, and FBS were similar between the 2 groups. Furthermore, ECLT and PAI-1 level did not differ between patient and control groups; however, both showed significant correlation (r 5 .352, P 5.006). The 4G/5G polymorphism was the most common genotype in both patient and control groups (69.0% vs. 80.0%). However, 4G and 5G polymorphisms of PAI-1 gene did not correlate with PAI-1 level in this study (P 5 .797). Conclusions: The PAI-1 level and 4G/5G polymorphism may not be a risk factor of AIS in this population. It was also found that the 4G/5G polymorphism was the most common PAI-1 genotype in this study. Key Words: Thrombosis— children—euglobulin clot lysis time—PAI-1—4 G/5G polymorphism. Ó 2014 by National Stroke Association
From the *Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok; and †Department of Nursing, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Received March 19, 2014; revision received May 6, 2014; accepted May 29, 2014. This work was supported by the Thailand Research Fund (TRF) Senior Research Scholar Award 2006 (to A.C., RTA4980005). Nongnuch Sirachainan is a recipient of the Career Development Award from the Faculty of Medicine Ramathibodi Hospital, Mahidol University. The authors indicated no potential conflicts of interest. Address correspondence to Nongnuch Sirachainan, MD, Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 270 Rama VI Road, Ratchathewi District, Bangkok 10400, Thailand. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2014.05.025
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Arterial ischemic stroke (AIS) is an uncommon disease in children but it is more recognized nowadays.1,2 The incidence of AIS in children was reported as 1.2 per 100,000 person-years, but a higher incidence of AIS was reported in black populations, at 2.62 per 100,000 person-years, while the incidence was much lower in Asian children.2 Regarding the meta-analysis, thrombophilias were risk factors for AIS and cerebral sinovenous thrombosis in children.3 The prevalence of thrombophilias in young adults (aged ,44 years) with AIS was 1.0%-4.0%4 and up to 25.0% in children with AIS.5 The common risk factors are the deficiencies of natural anticoagulants, as well as factor V Leiden, prothrombin G20210A, hyperhomocysteinemia, increased lipoprotein(a), and sickle cell disease.6 For children who have no risk factors, further
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 10 (November-December), 2014: pp 2566-2572
PAI-1 AND 4G/5G POLYMORPHISM IN CHILDREN WITH STROKE
investigations should be performed according to International Society of Thrombosis and Haemostasis guidelines, which also include euglobulin clot lysis time (ECLT) and plasminogen activator inhibitor (PAI).6 ECLT is a global measurement of fibrinolytic activity.7 The test is usually completed within 2-6 hours.7 Vuckovic et al8 reported that prolonged ECLT was significantly higher in adult population with AIS than that in the control group. PAI-1 is a plasma protein, which inhibits plasminogen function by counteracting functions of tissue plasminogen activator and urokinase. It is synthesized by the liver, spleen, adipocytes, and endothelial cells. The synthesis of PAI-1 is controlled by plasma glucose, insulin, lipids, endotoxin, and inflammatory cytokines.9 A single guanine (G) polymorphism, called 4G and 5G alleles, which are the common PAI-1 genes, are located at 675 base pairs upstream of transcriptional region.9,10 The 4G allele increases gene transcription and creates a binding only with transcriptional activator of PAI-1, whereas the 5G allele decreases PAI-1 production by binding both transcriptional activator and repressor protein.9 The 4G/4G and 4G/5G polymorphisms have been reported as risk factors of AIS in many studies in adults from different ethnic groups,11-18 which contrasted with other studies also performed in various ethnicities.19-28 In 3 meta-analyses between 4G allele of PAI-1 genes and the risk factor of AIS, there was no evidence that 4G/4G and 4G/5G polymorphisms were risk factors of AIS,29,30 whereas the study by Attia et al25 showed associations between 4G/5G polymorphism of PAI-1 and AIS with odds ratio (OR) .89 (95% confidence interval [CI], .66-1.20). Moreover, the 4G/4G polymorphism was claimed to have a protective effect for AIS proposed by Hoekstra et al27 that elevated PAI-1 level could stabilize the atherosclerotic plaque. Although there were few studies of child populations regarding the 4G/5G polymorphism of PAI-1 and AIS, all studies showed no association between these polymorphisms and the risk of AIS.31-34 However, there has been no report concerning the association the PAI-1 polymorphism and AIS in children from East Asian countries,11-13 and the correlation of the 4G allele and PAI-1 level remains controversial in patients with AIS.15,18,23,27 Therefore, this study aimed to determine the association of ECLT, PAI-1 level, and 4G/5G polymorphism of PAI-1 to the development of AIS and the polymorphism associated with increasing PAI-1 level in Thai children with AIS.
Materials and Methods Study Population The study enrolled patients diagnosed with AIS between the ages of 1-18 years old. Patients were treated in the 2008-2009 period at the Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol Uni-
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versity, Bangkok, Thailand. Demographic data, namely age at diagnosis, gender, weight, height, and body mass index (BMI), were recorded, in addition to a medical history of blood pressure and a family history of thromboembolic event (TE). Thrombophilic risk factors including protein C, protein S and antithrombin activities, homocysteine level, lupus anticoagulant and anticardiolipin, and anti beta 2-glycoprotein I (b2-GPI) antibodies were studied in all patients. Factor V Leiden and prothrombin 20210 were not performed because of the very low incidence rates in Thais.35 The control group consisted of age- and sex-matched consenting healthy Thai children. This study was approved by the Institutional Review Board of the institution.
Inclusion Criteria Following informed parental consent, patients with AIS confirmed by computed tomography, magnetic resonance imaging, or angiography were enrolled. There was at least a 1 month interval between the diagnosis of AIS and patient enrollment on the study. All patients had discontinued their use of anticoagulants, such as heparin, before enrollment for the study.
Exclusion Criteria Subjects who received blood component within 3 months before enrollment were excluded.
Laboratory Investigation After patient fasting, blood was collected into citrated, glass, sodium fluoride, and ethylene diamine tetraacetic acid (EDTA) tubes in the morning (between 6-8 a.m.). Blood in sodium fluoride and glass tubes was sent for fasting blood sugar (FBS) and lipid profiles. Citrated blood was centrifuged twice at 3500 revolution per minute for 10 minutes, and then plasma was separated and kept at 280 C before the measurement of PAI-1 level and ECLT. Buffy coat was collected from EDTA tube for DNA extraction by standard technique.
Euglobulin Clot Lysis Time ECLT was done by a modified computerized spectrophotometric microtiter plate method, as previously described.7
Plasminogen Activator Inhibitor-1 PAI-1 level was measured by sandwich technique enzyme-linked immunoabsorbent assay (Asserachrom PAI; Diagnostica Stago, Asnieres, France).
4G/5G Polymorphism DNA of all subjects was tested for 4G and 5G polymorphisms of PAI-1 gene by allele specific polymerase chain
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reaction. The specific primers for 4G, 5 -AGA GTC TGG ACA CGT GGG GA-30 , and 5G, 50 -AGA GTC TGG ACA CGT GGG GG-30 , were added to separate tubes containing the downstream primer, 50 -TGC AGC CAG CCA CGT GAT TGT CTA-30 , and upstream primer, 50 -AAG CTT TTA CCA TGG TAA CCC CTG GT-30 , to determine the 4G and 5G polymorphisms, as done in a previous research.36
Hypercholesterolemia and hypertriglyceridemia were found in 26.7% and 20.0%, respectively, but the prevalence of hypercholesterolemia and hypertriglyceridemia, diagnosed by the level over upper normal range for particular ages, were not different between patient and control groups (P 5 .343 and P 5 .227, respectively). In addition, subjects with high low-density lipoprotein (LDL) and low high-density lipoprotein (HDL) did not differ between patient and control groups (Table 2).
Statistical Analysis The PASW statistics 17.0 (SPSS Inc, Chicago, IL) was used for the analysis. ECLT and PAI-1 level were reported as mean (standard deviation) for numerical data with normal distribution and median (minimum-maximum) for numerical data, which did not have normal distribution. The student t test and Mann–Whitney U test were used for the analysis of numerical data. The chi-square and Fisher exact tests were used for analyzing categorical data. The OR was used to determine the risk of AIS with a 95% CI. Pearson correlation was used to determine the correlation of PAI-1 level to ECLT, lipid profiles, FBS, age, weight, and BMI. Statistical significance is accepted at P value less than .05.
Results Study Population There were 70 subjects in the study; 30 patients were diagnosed with AIS and 40 controls. The demographic data was not different between the 2 groups (Table 1). The underlying medical illnesses, which may signify a predisposition to AIS were found in 7 (23.3%) patients. Two of these had congenital heart diseases and autoimmune diseases, one of each had malignancy, thalassemia disease (hemoglobin H disease), and smoking. Thrombophilic risk factors were identified in 11 (36.7%) patients. Five patients had antiphospholipid antibodies, 4 patients had hyperhomocysteinemia (.15 mmol/L), and 2 patients had protein S deficiencies.
PAI-1 Level, ECLT, and Risk of AIS ECLT and PAI-1 level did not differ between patient and control groups (Table 2). Patients with known thrombophilic risk factors, including hyperhomocysteinemia, protein S deficiency, and antiphospholipid antibody, had similar levels of PAI-1 (P 5 .075) and ECLT (P 5 .234) compared with those without known thrombophilic risk factors. There was a correlation between PAI-1 level and ECLT (r 5 .352, P 5 .006). PAI-1 level correlated with age (r 5 .255, P 5 .033), BMI (r 5 .726, P , .001), triglyceride (r 5 .269, P 5 .025), and very low density lipoprotein (VLDL; r 5 .267, P 5 .026). However, there was no correlation between PAI-1 level and weight (r 5 .134, P 5 .269), cholesterol (r 5 .082, P 5 .502), LDL (r 5 .030, P 5 .803), HDL (r 5 2.049, P 5 .688), or FBS (r 5 2.100, P 5 .412; Fig 1).
PAI-1 Level and 4G/5G Polymorphism Allele frequencies of 4G and 5G were .42 and .58, respectively. The frequencies of 4G and 5G allele of the patient group were .41 and .59, respectively, whereas the frequencies of 4G and 5G alleles were .42 and .58, respectively, in the control group (P 5 .405 and .198, respectively). The 4G/5G polymorphism was the most common genotype in both patient and control groups (69.0% vs. 80.0%; Table 3). However, there were no differences in PAI-1 levels among 4G/4G, 4G/5G, and 5G/5G polymorphisms (P 5 .797).
Table 1. Demographic data of the patient and control groups Parameters
Patients, N 5 30
Controls, N 5 40
P value
Male, n (%) Age (y)* Weight (kg)* Height (m)* BMI (kg/m2)* Obesity, n (%) Hypertension, n (%) Family history of thrombosis, n (%)
12 (40.0) 9.8 (4.4) 39.7 (21.3) 1.3 (.3) 18.6 (5.2) 4 (13.3) 4 (13.3) 4 (13.3)
17 (42.5) 9.9 (5.0) 35.1 (17.9) 1.3 (.3) 18.4 (3.8) 4 (10.0) 4 (10.0) 4 (10.0)
.834 .914 .341 .854 .840 .717 .717 .717
Abbreviation: BMI, body mass index. *The values are in mean (standard deviation).
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Table 2. Lipid profiles, FBS, PAI level, and ECLT of patient and control groups Parameters
Patients, N 5 30
Controls, N 5 40
P value
Triglyceride (mg/dL)* Hypertriglyceridemia, n (%) Cholesterol (mg/dL)* Hypercholesterolemia, n (%) LDL (mg/dL)* High LDL, n (%) HDL (mg/dL)* Low HDL, n (%) VLDL (mg/dL)* FBS (mg/dL)* Hyperglycemia, n (%) PAI-1 (ng/mL)* ECLT (h)*
97.2 (68.8) 8 (26.7) 175.1 (34.7) 6 (20.0) 109.2 (27.9) 7 (23.3) 46.2 (16.5) 7 (23.3) 19.7 (12.6) 84.2 (10.4) 2 (6.7) 18.8 (17.0) 5.8 (1.5)
78.8 (41.6) 6 (15.0) 183.6 (38.8) 12 (30.0) 120.4 (36.4) 14 (35.0) 48.5 (19.5) 8 (20.0) 15.9 (8.2) 82.3 (7.3) 0 (0) 20.6 (19.0) 6.1 (1.9)
.192 .227 .339 .343 .150 .292 .597 .737 .162 .405 .180 .680 .495
Abbreviations: ELCT, euglobulin clot lysis time; FBS, fasting blood sugar; HDL, high-density lipoprotein; LDL, low-density lipoprotein; PAI, plasminogen activator inhibitor; VLDL, very low density lipoprotein. *The values are in mean (standard deviation).
Discussion Although AIS is an uncommon disease, it is more recognized nowadays.1 Besides clinical manifestation and neuroimaging studies, thrombophilic risk factors were also important to identify the etiologies of AIS in children, according to the meta-analysis by Kenet et al.3 Although the International Society of Thrombosis and Haemostasis investigation guidelines (2002) recommended 3 levels of investigation for children with TE, the types of thrombophilic risk factors are different among ethnicities.35 Patients with combined thrombophilic risk factors have an OR of developing AIS as high as 18.8 (95% CI, 6.5-54.1).37 Thus, patients who were identified as being in this category were also included in this study. As blood glucose,9 cholesterol,38 triglyceride,39 and VLDL40 have been associated with the level of PAI-1, all subjects were fully tested for all. However, the results revealed no difference in PAI-1 level, FBS, or lipid profiles in patients and controls. In the present study, VLDL and triglyceride levels showed a correlation with PAI-1 level. VLDL plays a role in stimulating PAI-1 production. This may be explained by the VLDL response site and the 4G/5G polymorphisms of PAI-1 at 2675 being located next to each other.40 Because VLDL has a major role in triglyceride transportation,41 the level of triglyceride correlates to VLDL and also PAI-1 levels. In addition, BMI associated with the level of PAI-1, which was similar to previous studies in adults9 and children.42 Adipose tissue is one of the tissues able to secrete PAI-1.9 This result might explain the higher chance of hypercoagulable states found in obese patients.42 Moreover, PAI-1 level increased according to age, a finding similar to the study by Smith et al.7 The mean level of PAI-1 in patient and control groups was not significantly different. This result supported a
study of children with AIS in Greece, which showed no difference in PAI-1 level between patient and control groups.32 In contrast, 2 studies of PAI-1 level and AIS in adults23,27 showed that high PAI-1 level was associated with an increased risk of AIS and mortality.25 Many studies from Europe reported that increased PAI-1 level was a preventive factor for AIS.25-28 In addition, the meta-analysis by Bentley et al29 showed that elevated PAI-1 level had a protective effect on AIS (OR, .95; 95% CI, .91-.99). ECLT is the result of interaction of multiple proteins in fibrinolytic pathway. A significantly longer clot lysis time was reported in adult patients with AIS.8 It was reported that ECLT had a correlation with the level of PAI antigen,7 which was similar to the present study. It is important to note that the various methods used to measure levels of PAI-1 may have an influence on the findings. In the present study, PAI-1 antigen was measured by sandwich technique, using mouse monoclonal antihuman antibody. Therefore, the result may be different to other methods using polyclonal antibody.43 The 4G/5G polymorphism was the most common genetic polymorphism of PAI-1 in the present study. This result was the same as previous studies from pediatric European populations.31-34 However, the 4G allele was less frequent in the present study than in previous studies of children and adults with AIS.13,14,19 For example, the reported frequency of 4G in Koreans was .42 in patients and .21 in controls13; in Taiwanese, it was .63 in patients and .61 in controls14; and in South Asians, it was .51 in patients and .61 in controls.19 This may be because of differences in the 4G allele of various ethnicities as mentioned by Naran et al,44 which reported a lower frequency of the 4G allele in Africans (.13) compared with Indians (.54) and whites (.58).
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Figure 1. Correlation between plasma plasminogen activator inhibitor–1 (PAI-1) level in 30 cases and 40 controls with euglobulin clot lysis time (A), age (B), body mass index (C), very low density lipoprotein (VLDL) (D), triglyceride (E), weight (F), cholesterol (G), low-density lipoprotein (LDL) (H), high-density lipoprotein (HDL) (I), and fasting blood sugar (J).
PAI-1 AND 4G/5G POLYMORPHISM IN CHILDREN WITH STROKE
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Table 3. Genetic polymorphisms of PAI-1 between patient and control groups Genotype
Patients, N 5 29
Controls, N 5 40
P value
PAI-1 level, ng/mL*
P value
4G/4G, n (%) 4G/5G, n (%) 5G/5G, n (%)
2 (6.9) 20 (69.0) 7 (24.1)
1 (2.5) 32 (80.0) 7 (17.5)
.501
17.3 (8.4-36.7) 16.1 (1.5-89.1) 15.7 (1.5-69.3)
.797
Abbreviation: PAI, plasminogen activator inhibitor. *The values are in median (minimum-maximum).
The 4G allele correlated with the increased level of PAI-1, whereas the 5G allele had a relation to decreased PAI-1 level in adult patients.8,42 Therefore, patients who have either the 4G/4G or 4G/5G polymorphism had a higher level of PAI-1, as reported by previous studies of cardiovascular disease.45 In contrast to the study by Kucukarabaci et al,15 which showed that PAI-1 activity and polymorphism may be useful in identifying patients who develop AIS, this study did not show a relation between enzyme activity and polymorphism. Moreover, the 4G/4G and 4G/5G polymorphisms did not show a difference in PAI-1 level between patient and control groups in a study in children with AIS.32 In the present study, there was no correlation between the 4G/5G polymorphism and PAI-1 level, a finding which was similar to the study reported by Matsubara el al.36
Conclusion The findings of this study were that ECLT, BMI, triglyceride, and VLDL had correlation with levels of PAI-1. 4G/ 5G polymorphism was the most common PAI-1 genotype and did not correlate with the PAI-1 level in this study. Findings also suggest that PAI-1 level and 4G/5G polymorphism may not be a risk factor of AIS in this population. Acknowledgments: The authors would like to thank all physicians and paramedic personnel who had been involved in taking care of the patients.
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2572 18. Saidi S, Slamia LB, Mahjoub T, et al. Association of PAI-1 4G/5G and -844G/A gene polymorphism and changes in PAI-1/tPA levels in stroke: a case-control study. J Stroke Cerebrovasc Dis 2007;16:153-159. 19. Chen CH, Eng HL, Chang CJ, et al. 4G/5G promoter polymorphism of plasminogen activator inhibitor-1, lipid profiles, and ischemic stroke. J Lab Clin Med 2003; 142:100-105. 20. Babu MS, Prabha TS, Kaul S, et al. Association of genetic variants of fibrinolytic system with stroke and stroke subtypes. Gene 2012;495:76-80. 21. Adamski MG, Turaj W, Slowik A, et al. A-G-4G haplotype of PAI-1 gene polymorphisms 2844 G/A, HindIII G/C, and 2675 4G/5G is associated with increased risk of ischemic stroke caused by small vessel disease. Acta Neurol Scand 2009;120:94-100. 22. van Goor ML, G omez Garcıa E, Leebeek F, et al. The plasminogen activator inhibitor (PAI-1) 4G/5G promoter polymorphism and PAI-1 levels in ischemic stroke. A case-control study. Thromb Haemost 2005;93:92-96. 23. de Paula Sabino A, Ribeiro DD, et al. Plasminogen activator inhibitor-1 4G/5G promoter polymorphism and PAI-1 plasma levels in young patients with ischemic stroke. Mol Biol Rep 2011;38:5355-5360. 24. Ding J, Nicklas BJ, Fallin MD, et al. Plasminogen activator inhibitor type 1 gene polymorphisms and haplotypes are associated with plasma plasminogen activator inhibitor type 1 levels but not with myocardial infarction or stroke. Am Heart J 2006;152:1109-1115. 25. Attia J, Thakkinstian A, Wang Y, et al. The PAI-1 4G/5G gene polymorphism and ischemic stroke: an association study and meta-analysis. J Stroke Cerebrovasc Dis 2007; 16:173-179. 26. Jood K, Ladenvall P, Tj€ arnlund-Wolf A, et al. Fibrinolytic gene polymorphism and ischemic stroke. Stroke 2005; 36:2077-2081. 27. Hoekstra T, Geleijnse JM, Kluft C, et al. 4G/4G genotype of PAI-1 gene is associated with reduced risk of stroke in elderly. Stroke 2003;34:2822-2828. 28. Boncoraglio GB, Bodini A, Brambilla C, et al. An effect of the PAI-1 4G/5G polymorphism on cholesterol levels may explain conflicting associations with myocardial infarction and stroke. Cerebrovasc Dis 2006;22:191-195. 29. Bentley P, Peck G, Smeeth L, et al. Causal relationship of susceptibility genes to ischemic stroke: comparison to ischemic heart disease and biochemical determinants. PLoS One 2010;5:e9136. 30. Tsantes AE, Nikolopoulos GK, Bagos PG, et al. Plasminogen activator inhibitor-1 4G/5G polymorphism and risk of ischemic stroke: a meta-analysis. Blood Coagul Fibrinolysis 2007;18:497-504. 31. Nowak-G€ ottl U, Str€ ater R, Kosch A, et al. The plasminogen activator inhibitor (PAI)-1 promoter 4G/4G genotype is not associated with ischemic stroke in a population of German children. Childhood Stroke Study Group. Eur J Haematol 2001;66:57-62.
R. NATESIRINILKUL ET AL. 32. Komitopoulou A, Platokouki H, Kapsimali Z, et al. Mutations and polymorphisms in genes affecting hemostasis proteins and homocysteine metabolism in children with arterial ischemic stroke. Cerebrovasc Dis 2006; 22:13-20. _ 33. Balcerzyk A, Zak I, Emich-Widera E, et al. The plasminogen activator inhibitor-1 gene polymorphism in determining the risk of pediatric ischemic stroke–case control and family-based study. Neuropediatrics 2011; 42:67-70. 34. Akar N, Akar E, Yilmaz E, et al. Plasminogen activator inhibitor-1 4G/5G polymorphism in Turkish children with cerebral infarct and effect on factor V 1691A mutation. J Child Neurol 2001;16:294-295. 35. Angchaisuksiri P, Pingsuthiwong S, Aryuchai K, et al. Prevalence of the G1691A mutation in the factor V gene (factor V Leiden) and the G20210A prothrombin gene mutation in the Thai population. Am J Hematol 2000; 65:119-122. 36. Matsubara Y, Murata M, Isshiki I, et al. Genotype frequency of plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism in healthy Japanese males and its relation to PAI-1 levels. Int J Hematol 1999; 69:43-47. 37. Mallick AA, O’Callaghan FJ. Risk factors and treatment outcomes of childhood stroke. Expert Rev Neurother 2010;14:1331-1346. 38. Mansfield MW, Stickland MH, Grant PJ. Plasminogen activator inhibitor-1 (PAI-1) polymorphism and coronary artery disease in non-insulin independent diabetes. Thromb Haemost 1995;74:1032-1034. 39. Lindgren A, Lindoff C, Norrving B, et al. Tissue plasminogen activator and plasminogen activator inhibitor-1 in stroke patients. Stroke 1996;27:1066-1071. 40. Vaughan DE. PAI-1 and atherothrombosis. J Thromb Haemost 2005;3:1879-1883. 41. Freedman DS, Bowman BA, Otvos JD, et al. Differences in the relation of obesity to serum triacylglycerol and VLDL subclass concentrations between black and white children: the Bogalusa Heart Study. Am J Clin Nutr 2002; 75:827-833. 42. Kinik ST, Ozbek N, Yuce M, et al. PAI-1 gene 4G/5G polymorphism, cytokine levels and their relations with metabolic parameters in obese children. Thromb Haemost 2008;99:352-356. 43. Ono T, Sogabe M, Ogura M, et al. Automated latex photometric immunoassay for total plasminogen activator inhibitor-1 in plasma. Clin Chem 2003;49:987-989. 44. Naran NH, Chetty N, Crowther NJ. The influence of metabolic syndrome components on plasma PAI-1 concentrations is modified by the PAI-1 4G/5G genotype and ethnicity. Atherosclerosis 2008;196:155-163. 45. Hoekstra T, Geleijnse JM, Schouten EG, et al. Plasminogen activator inhibitor-type 1: its plasma determinants and relation with cardiovascular risk. Thromb Haemost 2004;91:861-872.
Background: Prolonged euglobulin clot lysis time (ECLT) and increased level of plasminogen activator inhibitor-1 (PAI-1) were reported to be risk factors of arterial ischemic stroke (AIS) by some studies; however, these findings were not supported by other studies. The objective of this study was to determine the association of ECLT, PAI-1 level, and polymorphisms of 4G and 5G of PAI-1 gene to the development of AIS in Thai children. Methods: This study included patients aged 1-18 years old. Diagnosis of AIS was confirmed by imaging study. The control group was ageand sex-matched healthy subjects. Demographic data were recorded, and blood was tested for ECLT, PAI-1 level, lipid profiles, fasting blood sugar (FBS), and 4G and 5G polymorphisms of PAI-1 gene. Results: There were 70 subjects participating in this study, consisting of 30 patients and 40 controls. Demographic data, lipid profiles, and FBS were similar between the 2 groups. Furthermore, ECLT and PAI-1 level did not differ between patient and control groups; however, both showed significant correlation (r 5 .352, P 5.006). The 4G/5G polymorphism was the most common genotype in both patient and control groups (69.0% vs. 80.0%). However, 4G and 5G polymorphisms of PAI-1 gene did not correlate with PAI-1 level in this study (P 5 .797). Conclusions: The PAI-1 level and 4G/5G polymorphism may not be a risk factor of AIS in this population. It was also found that the 4G/5G polymorphism was the most common PAI-1 genotype in this study. Key Words: Thrombosis— children—euglobulin clot lysis time—PAI-1—4 G/5G polymorphism. Ó 2014 by National Stroke Association
From the *Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok; and †Department of Nursing, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Received March 19, 2014; revision received May 6, 2014; accepted May 29, 2014. This work was supported by the Thailand Research Fund (TRF) Senior Research Scholar Award 2006 (to A.C., RTA4980005). Nongnuch Sirachainan is a recipient of the Career Development Award from the Faculty of Medicine Ramathibodi Hospital, Mahidol University. The authors indicated no potential conflicts of interest. Address correspondence to Nongnuch Sirachainan, MD, Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 270 Rama VI Road, Ratchathewi District, Bangkok 10400, Thailand. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2014.05.025
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Arterial ischemic stroke (AIS) is an uncommon disease in children but it is more recognized nowadays.1,2 The incidence of AIS in children was reported as 1.2 per 100,000 person-years, but a higher incidence of AIS was reported in black populations, at 2.62 per 100,000 person-years, while the incidence was much lower in Asian children.2 Regarding the meta-analysis, thrombophilias were risk factors for AIS and cerebral sinovenous thrombosis in children.3 The prevalence of thrombophilias in young adults (aged ,44 years) with AIS was 1.0%-4.0%4 and up to 25.0% in children with AIS.5 The common risk factors are the deficiencies of natural anticoagulants, as well as factor V Leiden, prothrombin G20210A, hyperhomocysteinemia, increased lipoprotein(a), and sickle cell disease.6 For children who have no risk factors, further
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 10 (November-December), 2014: pp 2566-2572
PAI-1 AND 4G/5G POLYMORPHISM IN CHILDREN WITH STROKE
investigations should be performed according to International Society of Thrombosis and Haemostasis guidelines, which also include euglobulin clot lysis time (ECLT) and plasminogen activator inhibitor (PAI).6 ECLT is a global measurement of fibrinolytic activity.7 The test is usually completed within 2-6 hours.7 Vuckovic et al8 reported that prolonged ECLT was significantly higher in adult population with AIS than that in the control group. PAI-1 is a plasma protein, which inhibits plasminogen function by counteracting functions of tissue plasminogen activator and urokinase. It is synthesized by the liver, spleen, adipocytes, and endothelial cells. The synthesis of PAI-1 is controlled by plasma glucose, insulin, lipids, endotoxin, and inflammatory cytokines.9 A single guanine (G) polymorphism, called 4G and 5G alleles, which are the common PAI-1 genes, are located at 675 base pairs upstream of transcriptional region.9,10 The 4G allele increases gene transcription and creates a binding only with transcriptional activator of PAI-1, whereas the 5G allele decreases PAI-1 production by binding both transcriptional activator and repressor protein.9 The 4G/4G and 4G/5G polymorphisms have been reported as risk factors of AIS in many studies in adults from different ethnic groups,11-18 which contrasted with other studies also performed in various ethnicities.19-28 In 3 meta-analyses between 4G allele of PAI-1 genes and the risk factor of AIS, there was no evidence that 4G/4G and 4G/5G polymorphisms were risk factors of AIS,29,30 whereas the study by Attia et al25 showed associations between 4G/5G polymorphism of PAI-1 and AIS with odds ratio (OR) .89 (95% confidence interval [CI], .66-1.20). Moreover, the 4G/4G polymorphism was claimed to have a protective effect for AIS proposed by Hoekstra et al27 that elevated PAI-1 level could stabilize the atherosclerotic plaque. Although there were few studies of child populations regarding the 4G/5G polymorphism of PAI-1 and AIS, all studies showed no association between these polymorphisms and the risk of AIS.31-34 However, there has been no report concerning the association the PAI-1 polymorphism and AIS in children from East Asian countries,11-13 and the correlation of the 4G allele and PAI-1 level remains controversial in patients with AIS.15,18,23,27 Therefore, this study aimed to determine the association of ECLT, PAI-1 level, and 4G/5G polymorphism of PAI-1 to the development of AIS and the polymorphism associated with increasing PAI-1 level in Thai children with AIS.
Materials and Methods Study Population The study enrolled patients diagnosed with AIS between the ages of 1-18 years old. Patients were treated in the 2008-2009 period at the Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol Uni-
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versity, Bangkok, Thailand. Demographic data, namely age at diagnosis, gender, weight, height, and body mass index (BMI), were recorded, in addition to a medical history of blood pressure and a family history of thromboembolic event (TE). Thrombophilic risk factors including protein C, protein S and antithrombin activities, homocysteine level, lupus anticoagulant and anticardiolipin, and anti beta 2-glycoprotein I (b2-GPI) antibodies were studied in all patients. Factor V Leiden and prothrombin 20210 were not performed because of the very low incidence rates in Thais.35 The control group consisted of age- and sex-matched consenting healthy Thai children. This study was approved by the Institutional Review Board of the institution.
Inclusion Criteria Following informed parental consent, patients with AIS confirmed by computed tomography, magnetic resonance imaging, or angiography were enrolled. There was at least a 1 month interval between the diagnosis of AIS and patient enrollment on the study. All patients had discontinued their use of anticoagulants, such as heparin, before enrollment for the study.
Exclusion Criteria Subjects who received blood component within 3 months before enrollment were excluded.
Laboratory Investigation After patient fasting, blood was collected into citrated, glass, sodium fluoride, and ethylene diamine tetraacetic acid (EDTA) tubes in the morning (between 6-8 a.m.). Blood in sodium fluoride and glass tubes was sent for fasting blood sugar (FBS) and lipid profiles. Citrated blood was centrifuged twice at 3500 revolution per minute for 10 minutes, and then plasma was separated and kept at 280 C before the measurement of PAI-1 level and ECLT. Buffy coat was collected from EDTA tube for DNA extraction by standard technique.
Euglobulin Clot Lysis Time ECLT was done by a modified computerized spectrophotometric microtiter plate method, as previously described.7
Plasminogen Activator Inhibitor-1 PAI-1 level was measured by sandwich technique enzyme-linked immunoabsorbent assay (Asserachrom PAI; Diagnostica Stago, Asnieres, France).
4G/5G Polymorphism DNA of all subjects was tested for 4G and 5G polymorphisms of PAI-1 gene by allele specific polymerase chain
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reaction. The specific primers for 4G, 5 -AGA GTC TGG ACA CGT GGG GA-30 , and 5G, 50 -AGA GTC TGG ACA CGT GGG GG-30 , were added to separate tubes containing the downstream primer, 50 -TGC AGC CAG CCA CGT GAT TGT CTA-30 , and upstream primer, 50 -AAG CTT TTA CCA TGG TAA CCC CTG GT-30 , to determine the 4G and 5G polymorphisms, as done in a previous research.36
Hypercholesterolemia and hypertriglyceridemia were found in 26.7% and 20.0%, respectively, but the prevalence of hypercholesterolemia and hypertriglyceridemia, diagnosed by the level over upper normal range for particular ages, were not different between patient and control groups (P 5 .343 and P 5 .227, respectively). In addition, subjects with high low-density lipoprotein (LDL) and low high-density lipoprotein (HDL) did not differ between patient and control groups (Table 2).
Statistical Analysis The PASW statistics 17.0 (SPSS Inc, Chicago, IL) was used for the analysis. ECLT and PAI-1 level were reported as mean (standard deviation) for numerical data with normal distribution and median (minimum-maximum) for numerical data, which did not have normal distribution. The student t test and Mann–Whitney U test were used for the analysis of numerical data. The chi-square and Fisher exact tests were used for analyzing categorical data. The OR was used to determine the risk of AIS with a 95% CI. Pearson correlation was used to determine the correlation of PAI-1 level to ECLT, lipid profiles, FBS, age, weight, and BMI. Statistical significance is accepted at P value less than .05.
Results Study Population There were 70 subjects in the study; 30 patients were diagnosed with AIS and 40 controls. The demographic data was not different between the 2 groups (Table 1). The underlying medical illnesses, which may signify a predisposition to AIS were found in 7 (23.3%) patients. Two of these had congenital heart diseases and autoimmune diseases, one of each had malignancy, thalassemia disease (hemoglobin H disease), and smoking. Thrombophilic risk factors were identified in 11 (36.7%) patients. Five patients had antiphospholipid antibodies, 4 patients had hyperhomocysteinemia (.15 mmol/L), and 2 patients had protein S deficiencies.
PAI-1 Level, ECLT, and Risk of AIS ECLT and PAI-1 level did not differ between patient and control groups (Table 2). Patients with known thrombophilic risk factors, including hyperhomocysteinemia, protein S deficiency, and antiphospholipid antibody, had similar levels of PAI-1 (P 5 .075) and ECLT (P 5 .234) compared with those without known thrombophilic risk factors. There was a correlation between PAI-1 level and ECLT (r 5 .352, P 5 .006). PAI-1 level correlated with age (r 5 .255, P 5 .033), BMI (r 5 .726, P , .001), triglyceride (r 5 .269, P 5 .025), and very low density lipoprotein (VLDL; r 5 .267, P 5 .026). However, there was no correlation between PAI-1 level and weight (r 5 .134, P 5 .269), cholesterol (r 5 .082, P 5 .502), LDL (r 5 .030, P 5 .803), HDL (r 5 2.049, P 5 .688), or FBS (r 5 2.100, P 5 .412; Fig 1).
PAI-1 Level and 4G/5G Polymorphism Allele frequencies of 4G and 5G were .42 and .58, respectively. The frequencies of 4G and 5G allele of the patient group were .41 and .59, respectively, whereas the frequencies of 4G and 5G alleles were .42 and .58, respectively, in the control group (P 5 .405 and .198, respectively). The 4G/5G polymorphism was the most common genotype in both patient and control groups (69.0% vs. 80.0%; Table 3). However, there were no differences in PAI-1 levels among 4G/4G, 4G/5G, and 5G/5G polymorphisms (P 5 .797).
Table 1. Demographic data of the patient and control groups Parameters
Patients, N 5 30
Controls, N 5 40
P value
Male, n (%) Age (y)* Weight (kg)* Height (m)* BMI (kg/m2)* Obesity, n (%) Hypertension, n (%) Family history of thrombosis, n (%)
12 (40.0) 9.8 (4.4) 39.7 (21.3) 1.3 (.3) 18.6 (5.2) 4 (13.3) 4 (13.3) 4 (13.3)
17 (42.5) 9.9 (5.0) 35.1 (17.9) 1.3 (.3) 18.4 (3.8) 4 (10.0) 4 (10.0) 4 (10.0)
.834 .914 .341 .854 .840 .717 .717 .717
Abbreviation: BMI, body mass index. *The values are in mean (standard deviation).
PAI-1 AND 4G/5G POLYMORPHISM IN CHILDREN WITH STROKE
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Table 2. Lipid profiles, FBS, PAI level, and ECLT of patient and control groups Parameters
Patients, N 5 30
Controls, N 5 40
P value
Triglyceride (mg/dL)* Hypertriglyceridemia, n (%) Cholesterol (mg/dL)* Hypercholesterolemia, n (%) LDL (mg/dL)* High LDL, n (%) HDL (mg/dL)* Low HDL, n (%) VLDL (mg/dL)* FBS (mg/dL)* Hyperglycemia, n (%) PAI-1 (ng/mL)* ECLT (h)*
97.2 (68.8) 8 (26.7) 175.1 (34.7) 6 (20.0) 109.2 (27.9) 7 (23.3) 46.2 (16.5) 7 (23.3) 19.7 (12.6) 84.2 (10.4) 2 (6.7) 18.8 (17.0) 5.8 (1.5)
78.8 (41.6) 6 (15.0) 183.6 (38.8) 12 (30.0) 120.4 (36.4) 14 (35.0) 48.5 (19.5) 8 (20.0) 15.9 (8.2) 82.3 (7.3) 0 (0) 20.6 (19.0) 6.1 (1.9)
.192 .227 .339 .343 .150 .292 .597 .737 .162 .405 .180 .680 .495
Abbreviations: ELCT, euglobulin clot lysis time; FBS, fasting blood sugar; HDL, high-density lipoprotein; LDL, low-density lipoprotein; PAI, plasminogen activator inhibitor; VLDL, very low density lipoprotein. *The values are in mean (standard deviation).
Discussion Although AIS is an uncommon disease, it is more recognized nowadays.1 Besides clinical manifestation and neuroimaging studies, thrombophilic risk factors were also important to identify the etiologies of AIS in children, according to the meta-analysis by Kenet et al.3 Although the International Society of Thrombosis and Haemostasis investigation guidelines (2002) recommended 3 levels of investigation for children with TE, the types of thrombophilic risk factors are different among ethnicities.35 Patients with combined thrombophilic risk factors have an OR of developing AIS as high as 18.8 (95% CI, 6.5-54.1).37 Thus, patients who were identified as being in this category were also included in this study. As blood glucose,9 cholesterol,38 triglyceride,39 and VLDL40 have been associated with the level of PAI-1, all subjects were fully tested for all. However, the results revealed no difference in PAI-1 level, FBS, or lipid profiles in patients and controls. In the present study, VLDL and triglyceride levels showed a correlation with PAI-1 level. VLDL plays a role in stimulating PAI-1 production. This may be explained by the VLDL response site and the 4G/5G polymorphisms of PAI-1 at 2675 being located next to each other.40 Because VLDL has a major role in triglyceride transportation,41 the level of triglyceride correlates to VLDL and also PAI-1 levels. In addition, BMI associated with the level of PAI-1, which was similar to previous studies in adults9 and children.42 Adipose tissue is one of the tissues able to secrete PAI-1.9 This result might explain the higher chance of hypercoagulable states found in obese patients.42 Moreover, PAI-1 level increased according to age, a finding similar to the study by Smith et al.7 The mean level of PAI-1 in patient and control groups was not significantly different. This result supported a
study of children with AIS in Greece, which showed no difference in PAI-1 level between patient and control groups.32 In contrast, 2 studies of PAI-1 level and AIS in adults23,27 showed that high PAI-1 level was associated with an increased risk of AIS and mortality.25 Many studies from Europe reported that increased PAI-1 level was a preventive factor for AIS.25-28 In addition, the meta-analysis by Bentley et al29 showed that elevated PAI-1 level had a protective effect on AIS (OR, .95; 95% CI, .91-.99). ECLT is the result of interaction of multiple proteins in fibrinolytic pathway. A significantly longer clot lysis time was reported in adult patients with AIS.8 It was reported that ECLT had a correlation with the level of PAI antigen,7 which was similar to the present study. It is important to note that the various methods used to measure levels of PAI-1 may have an influence on the findings. In the present study, PAI-1 antigen was measured by sandwich technique, using mouse monoclonal antihuman antibody. Therefore, the result may be different to other methods using polyclonal antibody.43 The 4G/5G polymorphism was the most common genetic polymorphism of PAI-1 in the present study. This result was the same as previous studies from pediatric European populations.31-34 However, the 4G allele was less frequent in the present study than in previous studies of children and adults with AIS.13,14,19 For example, the reported frequency of 4G in Koreans was .42 in patients and .21 in controls13; in Taiwanese, it was .63 in patients and .61 in controls14; and in South Asians, it was .51 in patients and .61 in controls.19 This may be because of differences in the 4G allele of various ethnicities as mentioned by Naran et al,44 which reported a lower frequency of the 4G allele in Africans (.13) compared with Indians (.54) and whites (.58).
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Figure 1. Correlation between plasma plasminogen activator inhibitor–1 (PAI-1) level in 30 cases and 40 controls with euglobulin clot lysis time (A), age (B), body mass index (C), very low density lipoprotein (VLDL) (D), triglyceride (E), weight (F), cholesterol (G), low-density lipoprotein (LDL) (H), high-density lipoprotein (HDL) (I), and fasting blood sugar (J).
PAI-1 AND 4G/5G POLYMORPHISM IN CHILDREN WITH STROKE
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Table 3. Genetic polymorphisms of PAI-1 between patient and control groups Genotype
Patients, N 5 29
Controls, N 5 40
P value
PAI-1 level, ng/mL*
P value
4G/4G, n (%) 4G/5G, n (%) 5G/5G, n (%)
2 (6.9) 20 (69.0) 7 (24.1)
1 (2.5) 32 (80.0) 7 (17.5)
.501
17.3 (8.4-36.7) 16.1 (1.5-89.1) 15.7 (1.5-69.3)
.797
Abbreviation: PAI, plasminogen activator inhibitor. *The values are in median (minimum-maximum).
The 4G allele correlated with the increased level of PAI-1, whereas the 5G allele had a relation to decreased PAI-1 level in adult patients.8,42 Therefore, patients who have either the 4G/4G or 4G/5G polymorphism had a higher level of PAI-1, as reported by previous studies of cardiovascular disease.45 In contrast to the study by Kucukarabaci et al,15 which showed that PAI-1 activity and polymorphism may be useful in identifying patients who develop AIS, this study did not show a relation between enzyme activity and polymorphism. Moreover, the 4G/4G and 4G/5G polymorphisms did not show a difference in PAI-1 level between patient and control groups in a study in children with AIS.32 In the present study, there was no correlation between the 4G/5G polymorphism and PAI-1 level, a finding which was similar to the study reported by Matsubara el al.36
Conclusion The findings of this study were that ECLT, BMI, triglyceride, and VLDL had correlation with levels of PAI-1. 4G/ 5G polymorphism was the most common PAI-1 genotype and did not correlate with the PAI-1 level in this study. Findings also suggest that PAI-1 level and 4G/5G polymorphism may not be a risk factor of AIS in this population. Acknowledgments: The authors would like to thank all physicians and paramedic personnel who had been involved in taking care of the patients.
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