Evaluation of the role of Nrf2/Keap1 pathway-associated novel mutations and gene expression on antioxidant status in patients with deep vein thrombosis.

Deep vein thrombosis (DVT) is a type of venous thromboembolism and a clinically complex vascular disease. Oxidative stress serves a key role in the pathogenesis of numerous cardiovascular diseases, particularly in endothelial dysfunction-associated syndromes. Nuclear factor erythroid-2-like 2(Nrf2) transcription factor is the primary regulator of antioxidant responses. The levels of reactive oxygen species (ROS) are regulated by Nrf2 and its suppressor protein Kelch-like ECH-associated protein 1 (Keap1). However, to the best of our knowledge, genetic abnormalites in the Nrf2/Keap1 pathway in DVT syndrome have not been thoroughly investigated. The aim of the present study was to investigate the association between the Nrf2/Keap1 pathway and antioxidant responses in DVT. Mutations and expression levels of genes involved in the Nrf2/Keap1 pathway were measured in 27 patients with DVT via DNA sequencing analysis and reverse transcription-quantitative PCR, respectively. The Polymorphism Phenotyping v2 program was used to identify the pathogenic mutations. Total antioxidant activity levels were determined by measuring the effect of serum samples from 27 patients with DVT on oxidation of the 2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) system. A total of 23 mutations, including seven novel mutations, were detected in the Nrf2/Keap1 pathway in 24 (89%) of the 27 patients with DVT. Keap1 mRNA expression levels were significantly higher compared with Nrf2 expression levels in patients with DVT (P=0.02). Analysis of molecular characteristics and gene expression levels demonstrated that Nrf2/Keap1-associated mutations and total antioxidant levels can be used as precursor markers in the diagnosis of DVT.

Frequency of vitamin K oxidoreductase complex subunit-1 (VKORC1) polymorphisms and warfarin dose management in patients with venous thromboembolism.

Warfarin works by inhibiting VKORC1, so polymorphisms of this gene modify the required drug dose. The aim of this study is to examine the relation between therapeutic weekly dose of warfarin and C1173T/G1639A polymorphism of VKORC1 in patients with VTE. Seventy-five patients with VTE were enrolled. Weekly warfarin doses and time (day) to reach therapeutic INR were evaluated retrospectively along with VKORC1-C1173T and G1639A alleles. The mean weekly warfarin dose was lower and time to reach therapeutic INR was shorter in homozygote alleles (AA and TT) (p < 0.05). The multivariate regression model was produced, R2 = 0.05% for age (p = 0.04), R2 = 6% for VKORC1 (p = 0.03), the model for estimating warfarin dose R2 = 17% (p > 0.05). In particular, patients who need overdose of warfarin or whose bleeding score is high, study of these polymorphisms can be considered.

An analysis of the levels of the soluble form of the endothelial protein C receptor in children with Henoch-Schönlein Purpura.

The pathogenesis of Henoch-Schönlein Purpura (HSP) has not been clearly defined. Inflammatory cytokines have been associated with HSP but there are only a few reports that have focused on coagulation. The endothelial protein C receptor (EPCR), which has anticoagulant and antiinflammatory activity, is the key component of the protein C pathway. Recent studies have implicated the soluble form of EPCR (sEPCR) in Wegener's granulomatosis, Behçet's disease, and systemic lupus erythematosus. The aim of this study was to evaluate the levels of sEPCR in HSP children. Twenty-two children with HSP and 17 healthy children were included. We found no significant differences (P > .05) between patient and control groups in the levels of von Willebrand factor and thrombomodulin. The median sEPCR values in the HSP group were lower than the control group (79 vs. 102 ng/mL, respectively) (P > .05). The mean sEPCR value in HSP patients with severe abdominal pain was lower than without (88.8 ± 54.9 vs. 108.2 ± 66.3 ng/mL, respectively) (P > .05). In addition, the mean IL-6 serum levels were significantly elevated in HSP patients during the acute stage of HSP (2.1 ± 1.7 vs. 1.5 ± 1.2 pg/mL, P = .038). We also observed a slight negative correlation between the levels of sEPCR and IL-6 (R = -.135, P > .05). To our knowledge, this was the first study to analyze sEPCR levels in HSP. Our results did not conclusively identify a direct role of sEPCR in HSP, but our findings warrant further investigations, especially in severe HSP cases characterized by gastrointestinal bleeding or renal involvement.

Evaluation of indirect microparticle activity and parameters of thrombin generation test in healthy infants.

INTRODUCTION: Circulating microparticles support thrombin generation. The aim of this study is to determine the indirect microparticle activity and the parameters of thrombin generation in healthy infants. MATERIALS AND METHODS: A total of 85 infants who were brought to follow-up visits were taken into the study. Blood samples were collected. Thrombin generation parameters and indirect microparticle activity were measured. RESULTS: The infants were divided into four groups according to the time of follow-up visits. Mean ages were 1.18 ± 0.19 months in Group 1, 6.15 ± 0.16 months in Group 2, 12.38 ± 0.46 months in Group 3 and 24.53 ± 0.39 months in Group 4, respectively. There was no statistical difference among the age-based groups with respect to the indirect microparticle activity. The lag time and the TTP levels in Group 1 were lower than that found in Group 2. The ETP and peak levels were higher in Group 1 than that of Group 2. The ETP and peak levels in Group 2 were found lower than those found in older children, but the TTP level was found relatively higher. Statistically correlations were found between indirect microparticle activity and all parameters of thrombin generation. CONCLUSIONS: The absence of a difference in terms of age-based microparticle levels may suggest that the features of microparticles in healthy children of this age group are similar. Age-dependent changes in thrombin generation parameters may suggest a regulation mechanism for the thrombin generation system over the first years of life. The results may provide mean values for indirect microparticle activity and thrombin generation in this healthy group.

Factor V Leiden and Prothrombin 20210A Mutations among Turkish Pediatric Leukemia Patients.

This study was undertaken to determine the prevalence of the Factor V 1691 G-A and PT 20210 G-A mutations in Turkish children with leukemia. We genotyped 135 pediatric leukemia patients with for these mutations. Eleven (8%) of the 135 patients were heterozygous for the FV 1691 G-A mutation. Seven (5,1%) of the patients carried the PT 20210 G-A heterozygous mutation. Of the 135 patients, only three had thrombotic event, none of which had these two mutations, which is common in Turkish population. Our findings revealed a controversial compared to the previous reports, which needs further investigation.

Prohemostatic and antithrombin activities of Ankaferd hemostat are linked to fibrinogen gamma chain and prothrombin by functional proteomic analyses.

Ankaferd blood stopper (ABS) is a novel topical hemostatic agent of plant origin registered for the management of external hemorrhages, in Turkey. The ABS-induced formation of the protein network with vital erythroid aggregation covers the whole physiological hemostatic process. The aim of this study is to assess prohemostatic and antithrombin effects of ABS on the basis of functional proteomic analyses performed in ABS-treated plasma and serum samples based on the previous hypotheses about ABS action. For this purpose, serum and plasma proteins were separated by 2-dimensional (2D) gel electrophoresis, and proteins were identified using reference plasma gel on Swiss-2DPAGE database. Our results indicated that fibrinogen gamma chain and prothrombin levels just initially decreased first and thereafter enhanced following the ABS exposure. Dual effects of ABS on those critical hemostatic molecules seem to be associated with prohemostatic and antithrombin activities of the hemostatic agent.

The Effects of Endothelial Protein C Receptor Gene Polymorphisms on the Plasma sEPCR Level in Venous Thrombosis Patients.

OBJECTIVE: The aim of this study was to investigate variations in the endothelial cell protein C receptor gene (EPCRgene) that may play a role in thrombosis and the effects of these variations on the plasma soluble endothelial cell proteinC receptor (sEPCR) level in Turkish patients with venous thrombosis. MATERIAL AND METHODS: This study included 111 thrombosis patients and 73 healthy controls. Following DNAextraction, PCR, SSCP, and DNA sequencing analysis of 4 exons of the EPCR gene was performed. Plasma sEPCR wasmeasured via enzyme-linked immunosorbent assay (ELISA). RESULTS: In all, 3 polymorphisms were detected in exons 1-4. C3998T (SNP no: rs2069952) polymorphism was detectedin intron 2 and C4678G (A1 haplotype) (SNP no: rs9574) in the 3' untranslated region (3'UTR). There weren't anysignificant differences in C3998T polymorphism between the control and patient groups. There wasn't a significantdifference in plasma sEPCR levels between both controls and patients that carried the A1 allele. A4600G substitution (A3haplotype) (SNP no: rs867186) was observed in exon 4 and was associated with a 2.04-fold higher risk of thrombosis.A3 allele carriers had higher sEPCR levels than those without the allele. Mean sEPCR level in the patients with thehomozygous A3 allele was 289 ng µL-1, versus 113.42 ng µL-1 in those with the homozygous A1 allele. CONCLUSION: The A1 haplotype might offer protection against thrombosis and the A3 haplotype might be associatedboth with elevated plasma sEPCR and elevated risk of venous thrombosis in the Turkish population. Plasma sEPCRlevels were significantly higher in those that carried the A3 allele (4600A>G) (both patients and controls). Among theparticipants that carried the A1 allele (4678C>G), plasma sEPCR did not differ significantly.

Thrombotic gene polymorphisms and postoperative outcome after coronary artery bypass graft surgery.

BACKGROUND: Emerging perioperative genomics may influence the direction of risk assessment and surgical strategies in cardiac surgery. The aim of this study was to investigate whether single nucleotide polymorphisms (SNP) affect the clinical presentation and predispose to increased risk for postoperative adverse events in patients undergoing coronary artery bypass grafting surgery (CABG). METHODS: A total of 220 patients undergoing first-time CABG between January 2005 and May 2008 were screened for factor V gene G1691A (FVL), prothrombin/factor II G20210A (PT G20210A), angiotensin I-converting enzyme insertion/deletion (ACE-ins/del) polymorphisms by PCR and Real Time PCR. End points were defined as death, myocardial infarction, stroke, postoperative bleeding, respiratory and renal insufficiency and event-free survival. Patients were compared to assess for any independent association between genotypes for thrombosis and postoperative phenotypes. RESULTS: Among 220 patients, the prevalence of the heterozygous FVL mutation was 10.9% (n = 24), and 3.6% (n = 8) were heterozygous carriers of the PT G20210A mutation. Genotype distribution of ACE-ins/del was 16.6%, 51.9%, and 31.5% in genotypes I/I, I/D, and D/D, respectively. FVL and PT G20210A mutations were associated with higher prevalence of totally occluded coronary arteries (p < 0.001). Furthermore the risk of left ventricular aneurysm formation was significantly higher in FVL heterozygote group compared to FVL G1691G (p = 0.002). ACE D/D genotype was associated with hypertension (p = 0.004), peripheral vascular disease (p = 0.006), and previous myocardial infarction (p = 0.007). CONCLUSIONS: FVL and PT G20210A genotypes had a higher prevalence of totally occluded vessels potentially as a result of atherothrombotic events. However, none of the genotypes investigated were independently associated with mortality.

Evaluation of hypercoagulability in obese children with thrombin generation test and microparticle release: effect of metabolic parameters.

Obesity is associated with a hypercoagulable state. Thrombin generation test (TGT) and microparticle levels were not studied in obese children extensively. It is aimed to determine whether any differences in the coagulation system between obese and normal weighed children exist with the use of TGT and microparticles release. A total of 120 obese and 38 healthy children were included to the study. An increase of thrombin generation and microparticles levels were found in obese children. Hyperinsulinism could not find a risk factor for hypercoagulability in our obese children. None of the parameters of TGT has been shown to be related to metabolic parameters and metabolic syndrome. Microparticles release time is found to correlate only to body mass index (BMI) Standard deviation score (SDS) in obese children. Hypercoagulability is associated with childhood obesity. Significant correlation between degree of obesity and microparticles release suggested that high adipokine levels secreted from adipose tissue can stimulate procoagulant status-independent metabolic dearrangements.

Contribution of clinical, metabolic, and genetic factors on hypertension in obese children and adolescents.

The role of ACE gene insertion (I) or deletion (D) polymorphism on blood pressure phenotype is not clear in children. The aim of this work is to examine the association between hypertension and ACE I/D polymorphism, as well as the contribution of clinical and metabolic parameters on blood pressure. The study participants were 199 obese children. Forty-four of them were hypertensive. The hypertensive subjects were older than the normotensive and most of them were pubertal. The prevalence of hypertension in obese subjects with II, ID, and DD genotype was similar. There was no difference between the hypertensive and the normotensive group according to ACE I/D genotype, BMISDS, sex, blood glucose level and total cholesterol levels. In obese children, high IR-HOMA values, puberty, presence of family history for hypertension, hypertriglyceridemia, and low HDL-cholesterol, high triglyceride/HDL-cholesterol ratio were found as increased risk factors of hypertension. In obese children and adolescents, blood pressure did not differ by ACE I/D genotype. The presence of family history, puberty, insulin resistance and hypertriglyceridemia constitute important risk factors for developing hypertension.

The relation between soluble endothelial protein C receptor and factor VIII levels and FVIII/sEPCR index in healthy infants.

OBJECTIVE: Both soluble endothelial protein C receptor (sEPCR) and factor VIII (FVIII) seem to be potential mediators in thrombotic and inflammatory states. The aim of the present study was to determine the relation between plasma sEPCR and FVIII levels in a group of healthy Turkish infants. METHODS: The study population consisted of 50 healthy infants aged 6 months (Group 1, n=23) and 12 months (Group 2, n=27) having no acute or chronic infection and/or disease. sEPCR levels and FVIII levels were measured by ELISA and one stage factor assay method, respectively. RESULTS: The sEPCR levels of the infants aged 6 months were found higher than those of the infants aged 12 months (p<0.001). There was a correlation between sEPCR and FVIII levels of the infants in Group 1 (6-month-old infants) (r=0.678, p<0.001). FVIII/sEPCR index was 0.73±0.3 and 1.0±0.5 in Group 1 and Group 2, respectively (p=0.027). A correlation between infant age and FVIII/sEPCR index was found (r=0.312, p=0.027). CONCLUSION: The FVIII/sEPCR index in healthy infants reflects the physiological condition of this population. The finding showing a positive relationship between sEPCR and FVIII levels suggests a possible interaction between these mediators in healthy infants aged six months.

The role of tumor necrosis factor-alpha -308 G/A and transforming growth factor-beta 1 -915 G/C polymorphisms in childhood idiopathic thrombocytopenic purpura.

OBJECTIVE: To increase our understanding of the etiology of idiopathic thrombocytopenic purpura (ITP) some cytokine gene polymorphisms were analyzed for susceptibility to the disease. The aim of this study was to investigate the role of tumor necrosis factor-alpha (TNF-α) -308 G/A and transforming growth factor-beta 1 (TGF-ß1) -915 G/C polymorphisms in the development and clinical progression of childhood ITP. METHODS: In all, 50 pediatric patients with ITP (25 with acute ITP and 25 with chronic ITP) and 48 healthy controls were investigated via LightCycler® PCR analysis for TNF-α -308 G/A and TGF-ß1 -915 G/C polymorphisms. RESULTS: The frequency of TNF-α -308 G/A polymorphism was 20%, 16%, and 22.9% in the acute ITP patients, chronic ITP patients, and controls, respectively (p>0.05). The frequency of TGF-ß1 -915 G/C polymorphism was 16%, 8%, and 8.3% in the acute ITP patients, chronic ITP patients, and controls, respectively (p>0.05). The risk of developing ITP and clinical progression were not associated with TNF-α -308 G/A (OR: 0.738, 95% CI: 0.275-1.981, and OR: 0.762, 95% CI: 0.179-3.249) or TGF-ß1 -915 G/C (OR: 1.5, 95% CI: 0.396-5.685, and OR: 0.457, 95% CI: 0.076-2.755) polymorphisms. CONCLUSION: The frequency of TNF-α -308 G/A and TGF-ß1 -915 G/C polymorphisms did not differ between pediatric ITP patients and healthy controls, and these polymorphisms were not associated with susceptibility to the development and clinical progression of the disease.

Factor V G1691A (Leiden) is a major etiological factor in Egyptian Budd-Chiari syndrome patients.

OBJECTIVE: Budd-Chiari syndrome is a multifactorial disease in which several prothrombotic disorders may predispose patients to the development of thrombosis at this uncommon location (hepatic veins). The aim of this study was to determine the prevalence and characteristics of inherited thrombophilia in Egyptian Budd-Chiari syndrome patients. METHODS: The study included 47 Budd-Chiari syndrome patients (20 children and 27 adults). Genotyping of Factor V G1691A (Leiden), prothrombin G20210A (PT), and methylenetetrahydrofolate reductase C677T were performed using real-time PCR and fluorescence melting curve detection analysis. RESULTS: Factor V Leiden was observed in 29 patients (61.7%). It is the only factor that caused Budd-Chiari syndrome in 18 of the patients and in 5 of the patients with inferior vena cava involvement. Myeloproliferative disease was noted in 12 (25.5%) patients, antiphospholipid syndrome in 5 (10.6%), and Behcet's disease in 3 (6.4%). Interestingly, 3 of the children with Budd-Chiari syndrome had lipid storage disease. CONCLUSION: Factor V Leiden was a major etiological factor in Egyptian Budd-Chiari syndrome patients, which may have been related to the high frequency of this mutation in the study region. Factor V Leiden was also a strong thrombophilic factor and the leading cause of inferior vena cava thrombosis in these patients. Lipid storage disease should be included as a risk factor for Budd-Chiari syndrome.

Relation of soluble endothelial protein C receptor and cytokines after allogeneic hematopoietic stem cell transplantation.

AIM: The objective of this study was to elucidate the effects of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), interleukin 2 (IL-2), interleukin 6 (IL-6), and interleukin 8 (IL-8) on the expression of soluble endothelial protein C receptor (sEPCR) in the pathogenesis of thrombotic complications after hematopoietic stem cell transplantation (HSCT). METHODS: The relationship between plasma concentrations of proinflammatory cytokines (TNF-α, IL-1ß, IL-2, IL-6, and IL-8) and sEPCR was evaluated in 32 consecutive allogeneic hematopoietic stem cell-transplanted patients prior to conditioning regimen and randomly once between +5 and +30 days after transplantation and compared these results with 20 healthy controls. RESULTS: Soluble endothelial protein C receptor levels did not indicate any significant difference between pre- and posttransplantation period, and sEPCR levels showed a significantly negative correlation between IL-6 and IL-8 (sEPCR and IL-6, r = -.43, P < .01; sEPCR and IL-8, r = -.57, P < .01). There was no correlation between sEPCR levels and TNF-α, IL-1ß, or IL-2 (sEPCR and TNF-α, r = -.13, P > .05; sEPCR and IL-1ß, r = -.1, P ≥ .05; sEPCR and IL-2, r = -.07, P > .05). CONCLUSIONS: Our results suggest that the production of sEPCR was not affected by allogeneic HSCT. Soluble endothelial protein C receptor did not show any positive correlation between these proinflammatory cytokines (TNF-α, IL-1ß, IL-2, IL-6, and IL-8), on the contrary a significantly negative correlation was determined between sEPCR and either IL-6 or IL-8. This negative correlation may be a protective mechanism in the pathway of protein C activation.

Association of methylenetetrahydrofolate reductase C677T and cystathionine ß-synthase polymorphisms in cardiovascular disease in the algerian population.

AIM: Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) and cystathionine ß-synthase (CBS) genes, involved in the intracellular metabolism of homcysteine, can result in hyperhomocysteinemia. The objective of this study was to evaluate prevalence estimates of MTHFR C677T and the CBS insertion of 68-bp (844ins68) polymorphisms among individuals with cardiovascular disease (CVD). METHODS: In total, 131 patients (61 men and 70 women) were hospitalized in the Cardiology Department in CHU of Sétif, Algeria. The control group included 147 apparently healthy adults (82 women and 65 men). The genetic analysis of the MTHFR C677T polymorphism was performed by real-time polymerase chain reaction on a Light Cycler; the CBS genotype was analyzed by polymerase chain reaction in a thermal cycler. RESULTS: The frequency of the TT genotype was 16.1% in the patient group and 14.3% in the control group. The CT genotype constituted 43.5% and 40.1% in the patient group and the control group, respectively. There was no significant difference in the occurrence of the TT genotype between the studied groups. The frequency of C677T/MTHFR in male and female patients was 16.4% and 15.7% for the TT genotype, respectively. There was no significant difference in T allele frequencies between sexes. However, the frequency of C677T homozygotes in the patients was higher in men with CVD than that in corresponding control subjects (40.2% vs. 29.2%), but the difference was not statistically significant. The coexistence of the MTHFR 677TT genotype and the common CBS 844ins68 variant was lower among patients. CONCLUSIONS: The MTHFR C677T and CBS 844ins68 variants tested in this study, individually or combined, are not associated with CVD in the Algerian population.