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Hyperhomocysteinemia (HHcy) affects haemostasis and shifts its balance in favour of thrombosis. In vitro and in vivo studies suggested that HHcy may impair fibrinolysis either by influencing the plasma levels of fibrinolytic factors or by altering the fibrinogen structure. We investigated the influence of mild HHcy levels on plasma fibrinolytic potential by using clot lysis time (CLT) and fibrin susceptibility to plasmin-induced lysis in 94 patients with previous pulmonary embolism and no pulmonary hypertension. CLT was measured as lysis time of tissue factor induced clots exposed to exogenous tissue plasminogen activator (t-PA). The rate of in vitro plasmin-mediated cleavage of fibrin β-chain was assessed over a 6-h period on fibrin clots, which were obtained by exposition to thrombin of purified fibrinogen. Homocysteine plasma levels were measured by Abbott Imx immunoassay and we considered as altered the values above 15 μmol/L according to the literature. In 68 patients homocysteine levels were below 15 μmol/L (NHcy) and in 26 they were above (HHcy). Significant differences were observed between the two groups regarding plasma fibrinolytic potential (p = 0.016), TAFIact (expressed as clot lysis ratio) (p = 0.02), t-PA (0.008) and PLG (0.037), but not for the other assessed components. The HHcy-patients had a threefold higher risk to have an impaired fibrinolysis. Instead, a multivariate logistic regression analysis adjusted for significances of univariate showed that HHcy (OR 5.2 95% CI 1.7-15.9; p = 0.003) and BMI (OR 5.0 95% CI 1.6-15.9; p = 0.006) resulted independently associated with impaired fibrinolytic activity. HHcy affects TAFI-mediated hypofibrinolysis but not fibrin(ogen) structure or function as documented by fibrin degradation analysis.

Introduction: Treatment with B-vitamins and betaine reduces the high risk of thrombosis in patients with homocystinuria, a metabolic syndrome that is characterized by severe hyperhomocysteinemia (HHcy). In contrast, there is no clear demonstration that B-vitamins reduce the risk of thrombosis in patients with mild HHcy: for this reason, many question the clinical utility of measuring total Hcy (tHcy) in patients with thrombosis. However, thrombosis may be the first clinical manifestation of homocystinuria in patients reaching adulthood without signs and symptoms of the syndrome., Aim: 1) to measure the prevalence of severe, previously undiagnosed, HHcy among patients with thrombosis 2) to profile these patients on the basis of their characteristics., Methods: Six Italian Thrombosis Centers completed a first questionnaire, reporting tHcy levels in patients with thrombosis who underwent thrombophilia screening, and a second questionnaire, reporting the characteristics of patients with severe HHcy (tHcy>100μmol/L)., Results: Of 19,678 cross-sectionally collected patients with thrombosis who underwent thrombophilia screening in the last 12.5years (median value, range 6-17), 38 had severe HHcy (0.2%). Their median age at diagnosis was 47years (range 19-83) and the median level of tHcy was 130μmol/L (range 101-262). Venous thromboembolism (71%) was more frequent than arterial thromboembolism (26%); recurrent thrombosis occurred in 42% of cases., Conclusions: Measurement of tHcy in adult patients with thrombosis may reveal the presence of severe HHcy. Since treatment of patients with severe HHcy decreases the risk of thrombosis, measurement of tHcy in patients with thrombosis may prove clinically useful., (© 2013.)

Aim: To assess the hypercoagulability in PBC and its relationship with homocysteine (HCY) and various components of the haemostatic system., Methods: We investigated 51 PBC patients (43F/8M; mean age: 63+/-13.9 yr) and 102 healthy subjects (86 women/16 men; 63+/-13 yr), and evaluated the haemostatic process in whole blood by the Sonoclot analysis and the platelet function by PFA-100 device. We then measured HCY (fasting and after methionine loading), tissue factor (TF), thrombin-antithrombin complexes (TAT), D-dimer (D-D), thrombomodulin (TM), folic acid, vitamin B6 and B12 plasma levels. C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphism was analyzed., Results: Sonoclot RATE values of patients were significantly (P<0.001) higher than those of controls. Sonoclot time to peak values and PFA-100 closure times were comparable in patients and controls. TAT, TF and HCY levels, both in the fasting and post-methionine loading, were significantly (P<0.001) higher in patients than in controls. Vitamin deficiencies were detected in 45/51 patients (88.2%). The prevalence of the homozygous TT677 MTHFR genotype was significantly higher in patients (31.4%) than in controls (17.5%) (P<0.05). Sonoclot RATE values correlated significantly with HCY levels and TF., Conclusion: In PBC, hyper-HCY is related to hypovitaminosis and genetic predisposing factors. Increased TF and HCY levels and signs of endothelial activation are associated with hypercoagulability and may have an important role in blood clotting activation.

Background: The mechanism by which high circulating homocysteine concentrations are a risk factor for atherothrombosis is incompletely understood. A proinflammatory state is related to atherosclerosis, and recent studies suggest that acute phase reactants correlate with circulating concentrations of homocysteine., Objective: We determined whether high concentrations of inflammatory markers are associated with hyperhomocysteinemia independently of dietary vitamin intakes, vitamin concentrations, and cardiovascular disease risk factors in a large, representative sample of the general population., Design: Five hundred eighty-six men and 734 women were randomly selected from the inhabitants of 2 small towns near Florence, Italy., Results: After adjustment for multiple potential confounders, interleukin 1 receptor antagonist (IL-1ra) and interleukin 6 (IL-6) concentrations were significantly (P < 0.001) associated with plasma homocysteine concentrations in older (>65 y) populations. Compared with participants in the lowest IL-6 tertile, those in the highest tertile had a higher risk of having homocysteine concentrations that were high (>30 micromol/L; odds ratio: 2.6; 95% CI: 1.1, 5.6; P = 0.024) or in the intermediate range 15-30 micromol/L (odds ratio: 1.6; 95% CI: 1.2, 2.2; P = 0.0014). Sedentary state, intakes of vitamin B-6 and folic acid, and serum folate, vitamin B-12, vitamin B-6, and alpha-tocopherol concentrations were significant independent correlates of homocysteine., Conclusions: High circulating concentrations of IL-1ra and IL-6 are independent correlates of hyperhomocysteinemia and may explain, at least in part, the association between homocysteine and atherosclerosis.

The aim of this study was to document, in hyperhomocysteinemic renal transplant recipients, the effect of vitamin supplementation on carotid intima-media thickness (cIMT). Fifty-six hyperhomocysteinemic stable renal transplant recipients were randomly assigned to either vitamin supplementation (group A) or placebo treatment (group B). All patients underwent high-resolution B mode ultrasound to measure IMT of common carotid arteries before and after 6 months of vitamin supplementation. In group A, cIMT significantly decreased after treatment, whereas no significant changes were observed in group B. In conclusion, our results demonstrate a beneficial effect of the treatment of hyperhomocysteinemia by vitamin supplementation on an early sign of atherosclerosis in a group of renal transplant recipients.

Background: A recent "ex-vivo" study showed that nonvalvular atrial fibrillation (NVAF) is associated with enhanced activity of metalloproteinases at the atrial level, and in animal models homocysteine (Hcy) is able to activate metalloproteinases. The aim of this case-control study was to investigate the association of total Hcy plasma levels, vitamin status (folate, vitamin B6, and vitamin B12), and methylenetetrahydrofolate reductase C677T and CBS 844ins68 polymorphisms with NVAF. Furthermore, the role of these variables in the occurrence of ischemic events was investigated., Methods: We studied 310 NVAF patients on oral anticoagulant treatment (168 patients with previous ischemic events and 142 without) and 310 controls., Results: Hyperhomocysteinemia (highest quartile) and vitamin B6 deficiency (lowest quartile) were independently associated with NVAF after multivariate analysis (Hcy: odds ratio [OR] 6.40, 95% CI 3.29-12.46; vitamin B6: OR 3.02, 95% CI 1.02-8.95). A significant correlation was found between Hcy levels and left atrial diameter (r = 0.46; P <.001). As shown by multivariate analysis, elevated Hcy levels were an independent risk factor for ischemic complications during NVAF (OR 2.66, 95% CI 1.15-6.20)., Conclusions: This study demonstrates a significant association of both elevated Hcy levels and low vitamin B6 levels with the presence of NVAF; in addition, it confirms the role of Hcy as a risk factor for ischemic events during NVAF.

Background: Nitric oxide is an endothelium-derived relaxing factor that contributes significantly to vascular tone regulation. In this study we investigated the role of endothelial nitric oxide synthase (eNOS) polymorphisms as predisposing factors to acute coronary syndromes (ACS)., Methods: In 477 consecutive patients admitted to the coronary intensive therapy unit of the University of Florence and in 537 unrelated controls, genotypes of eNOS G894T and T-786C polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism analysis and the repeat polymorphism 4a/4b was analyzed by polymerase chain reaction. The genotype distribution was in Hardy-Weinberg equilibrium for all variants., Results: The multivariate analysis showed that the homozygosity for the eNOS 4a rare variant represented an independent predisposition factor to ACS (odds ratio [OR] 2.5, 95% CI 1.1-5.4, P =.02) and in particular influenced the risk of acute myocardial infarction (OR 3.6, 95% CI 1.2-11.5, P =.03). Subjects carrying the 4a4a/-786CC haplotype showed a higher predisposition to the disease (OR 6.1, 95% CI 1.3-29.6, P =.02). The contemporary presence of hyperhomocysteinemia and homozygosity for the -786C variant influenced the predisposition to ACS (OR 9.1, 95% CI 1.7-46.7, P =.008)., Conclusions: The presence of the eNOS 4a4a genotype represents a predisposing condition to ACS and in particular to acute myocardial infarction. Moreover, our data provide the evidence that the -786CC pattern modulates the susceptibility to ACS in 4a4a homozygotes and in hyperhomocysteinemic patients.

Aims: The aim of this study was to evaluate (1) homocysteinemia and the prevalence of the C677T MTHFR polymorphism in Marfan patients and (2) whether the severity of cardiovascular manifestations is associated with homocysteinemia and/or C677T polymorphism., Methods and Results: We studied 107 patients subdivided into three subgroups based on the severity of cardiovascular manifestations: (A) no involvement (n=4); (B) mild involvement (n=45); (C) aortic dilatation or aortic dissection (n=58), and 189 controls. Total homocysteine (tHcy) was significantly higher in subgroup C than in subgroup B. In subgroup C patients with dissection tHcy was higher than in those without dissection. In subgroup C the prevalence of 677T homozygotes was higher, but not significantly, than in the subgroup B. In patients with dissection the prevalence of 677T homozygotes was significantly higher than in those without dissection and than in subgroup B. In the logistic regression analysis, severe cardiovascular manifestations and aortic dissection in Marfan patients were associated with tHcy plasma levels., Conclusions: Our data indicate an association between the severity of the cardiovascular manifestations, in particular aortic dissection, and elevated tHcy levels. This suggests an important role for tHcy in determining phenotypic variability in Marfan patients and provides further evidence for the association of homocysteinemia with the damage of the vascular system.

Background: We previously demonstrated among renal-transplant recipients (RTRs) a high prevalence of hyperhomocysteinemia, which might account for their elevated cardiovascular risk. The purpose of our study was to document, in hyperhomocysteinemic RTRs, the effect of vitamin supplementation on carotid intima-media thickness (cIMT), which is an early sign of atherosclerosis., Methods: A total of 56 stable hyperhomocysteinemic RTRs were randomly assigned to vitamin supplementation (folic acid 5 mg/day; vitamin B(6) 50 mg/day; vitamin B(12) 400 microg) (group A) or placebo treatment (group B) for 6 months. All subjects underwent cardiovascular risk-factor assessment, including fasting homocysteine (Hcy) levels assay, and high resolution B-mode ultrasound to measure the intima-media thickness of common carotid arteries, at time of enrollment and after 6 months., Results: Fasting Hcy levels markedly decreased in group A after treatment (21.8 [15.5-76.6] micromol/L vs. 9.3 [5.8-13] micromol/L; P<0.0001), whereas no significant changes were observed in group B (20.5 [17-37.6] micromol/L vs. 20.7 [15-34] micromol/L; P=not significant). In group A, cIMT significantly decreased after treatment (0.95+/-0.20 mm vs. 0.64+/-0.17 mm; P<0.0001). All except one patient showed a reduction of cIMT and the mean percentage of cIMT decrease was -32.2+/-12.9%. Patients with methylenetetrahydrofolate reductase (MTHFR) C677T +/+ genotype, with higher Hcy levels, had the major percentage of decrease of Hcy with respect to the other genotypes (mean decrease: MTHFR +/+ 74.8+/-5.7%; MTHFR +/- 58.1+/-10%; MTHFR -/- 56.3+/-8.6%). In hyperhomocysteinemic patients without vitamin supplementation (group B) we documented a significant increase in cIMT after 6 months (0.71+/-0.16 mm vs. 0.87+/-0.19 mm; P<0.05). In 19 of 28 subjects we observed an increase in cIMT, and in 9 of 28 the cIMT was unmodified. The mean percentage of cIMT increase was + 23.3+/-21.1%., Conclusions: Our results demonstrate a beneficial effect of the treatment of hyperhomocysteinemia by vitamin supplementation on cIMT in a group of RTRs.

Introduction: Moderate hyperhomocysteinemia is considered a risk factor for both venous and arterial thrombosis. A prevalence of up to 30% of fasting hyperhomocysteinemia has been recently reported in patients with retinal vein occlusion (RVO) whereas conflicting data exist on the role of C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene as a risk factor for RVO. No report has been published on cystathionine beta-synthase (CBS) 844ins68 polymorphism (another genetic determinant of blood Hcy levels) in RVO patients. Moreover, scarce information is available on the usefulness of measuring homocysteine also after methionine loading to increase the diagnostic efficacy of hyperhomocysteinemia in RVO patients., Materials and Methods: In 55 consecutive patients with diagnosis of RVO and 65 matched controls, plasma fasting total homocysteine (Hcy) levels and CBS and MTHFR polymorphisms were evaluated. In patients with normal fasting Hcy levels, post-methionine Hcy levels were determined., Results: Moderate fasting hyperhomocysteinemia was detected in 18/55 patients (32.7%). In the remaining 37 patients, Hcy was measured again post-methionine loading (PML). Only 3/37 (8.1%) patients had PML hyperhomocysteinemia. Thus, the total prevalence of moderate hyperhomocysteinemia in this cohort of RVO patients was 21/55 (38.2%). The prevalence of homozygosity for C677T MTHFR genotype, but not that of heterozygosity for CBS844ins68, was significantly higher in RVO patients than in controls., Conclusions: Differently from what has been reported for arterial and/or venous thrombosis, a single fasting Hcy measurement is able to detect most of RVO patients (85.7%) with moderate hyperhomocysteinemia. C677T MTHFR, but not CBS 844ins68, genotype may play a role as risk factor for RVO.

Hyperhomocysteinemia is a well established risk factor for atherothrombotic disease, and the request for homocysteine determinations and the number of laboratories that need to perform this assay to assess individual risk profile is increasing. Different methods to evaluate homocysteine plasma levels are at present available. In the present study three methods, an in-house high-pressure liquid chromatographic (HPLC) method (considered as reference method) and two commercial immunoassays, an enzyme-linked immunoassay (EIA) and an automated fluorescence polarization immunoassay (FPIA), were used to measure homocysteine plasma levels in 100 samples. The median of homocysteine plasma levels obtained by HPLC was 9.0 micromol/L (range 4.2-23.0); the median of values obtained by EIA and FPIA were 10.6 micromol/L (range 3.3-21.5) and 9.6 micromol/L (4.8-20.2), respectively. The FPIA method showed the lowest within-run and between-run coefficients of variation (3.6% and 4.1%, respectively). There was a significant correlation between EIA and HPLC (r=0.81; p<0.0001), and between FPIA and HPLC (r=0.85; p<0.0001). The Bland-Altman analysis showed that FPIA agreed best with HPLC; EIA displayed a relatively wide scatter of difference data points. The present results indicate that the technological characteristics of the FPIA assay make this method suitable for the determination of Hcy in clinical laboratories.

Hyperhomocysteinemia (Hcy) is an independent factor of cardiovascular disease, which is the main cause of morbidity and mortality both in uremic and kidney transplant patients. The aim of the study was to determine Hcy, plasminogen activator inhibitor (PAI-1) and lipoprotein (a) (Lp(a)) serum levels in 70 patients with a well functioning renal transplant. We also verified whether these levels were modified by a multivitamin therapy. The genetic polymorphism of the methylenetetrahydrofolate reductase (MTHFR) enzyme which plays a main role in Hcy metabolism, was studied as well. We found Hcy, PAI-1 and Lp(a) levels significantly elevated with respect to healthy control subjects. The thermolabile form of the MTHFR enzyme was linked to higher Hcy levels. After a short time on therapy with B6, B12 and folic acid vitamins, Hcy and PAI-1 decreased to normal levels. The authors conclude that high Hcy levels could be a relevant covariate for cardiovascular disease in transplant patients and they suggest that vitamin supplementation be recommended as a part of therapy.

Complications of pregnancy have been found to be related with thrombophilic polymorphisms that explain about 30% of obstetric complications. We evaluated the angiotensin converting enzyme (ACE) and the angiotensin type 1 receptor (AT1R) gene polymorphisms in the renin-angiotensin system (RAS) as possible risk factors for fetal loss. Fifty-nine women with a history of three or more first-trimester fetal losses and 70 healthy women with a history of normal pregnancies were enrolled in this study. Thrombophilic factors, ACE insertion/deletion (I/D) and AT1R A1166C polymorphisms, prothrombin G20210A and factor V Leiden mutations were analyzed. At univariate and multivariate analysis, a significant association between ACE DD and AT1R CC genotype and fetal loss was observed. The effect of the ACE DD genotype on the risk of fetal loss was higher in AT1R C allele carriers. The prevalence of hyperhomocysteinemia (Hcy) (defined as baseline plasma levels higher than the 95% percentile; cut-off, 10.5 micromol/l per l) was significantly higher in women with fetal loss, and an association between Hcy and fetal loss was detected. All patients showed normal antithrombin, protein C, protein S, and plasminogen activator inhibitor-1 (PAI-1) values. The presence of one risk factor not associated with others was found in 33 out of 59 patients (56%); ACE DD genotype was the most prevalent risk factor. Our results identify new possible predictive markers for fetal loss in RAS polymorphisms and Hcy. Large-scale studies are warranted to attribute clinical relevance to these polymorphisms as risk factors for complicated pregnancies.

Purpose: In vitro studies have recently demonstrated that homocysteine interacts with the aortic wall by inducing both elastolysis and endothelial perturbation. The aim of this study was to evaluate homocysteine plasma levels and their relationships with aortic diameter and endothelial damage in patients with abdominal aortic aneurysm., Subjects and Methods: Fifty-eight consecutive male patients (mean age, 69.5 +/- 6.6 years; age range, 49-78 years) who underwent abdominal aortic aneurysm surgery were enrolled in the study. Twenty-two of 58 patients had no clinical or instrumental evidence of atherosclerosis. Sixty control subjects were age matched and sex matched with the patients. In all of the subjects, we evaluated total homocysteine and thrombomodulin plasma levels and the distribution of the C677T methylenetetrahydrofolate reductase gene mutation., Results: Hyperhomocysteinemia was found in 26 (48%) of the 58 patients with abdominal aortic aneurysm, and homocysteine plasma levels were significantly higher in patients than in control subjects (15.7 +/- 6.5 micromol/L vs 9.6 +/- 3.9 micromol/L; P <. 0001). In addition, the subgroup of patients with abdominal aortic aneurysm who did not show evidence of atherosclerosis showed homocysteine plasma levels significantly higher than those in the controls (14.8 +/- 6.1 micromol/L vs 9.6 +/- 3.9 micromol/L; P <. 001). A larger aneurysmal size was detected in hyperhomocysteinemic patients than in those with normal homocysteine plasma levels (5.09 +/- 0.84 cm vs 5.79 +/- 1.5 cm; P <.05). The genotype distribution of the C677T methylenetetrahydrofolate reductase mutation was as follows: TT 21%, TC 55%, and CC 24% in the patients; TT 10%, TC 58%, and CC 32% in the controls. Moreover, in patients a significant correlation (P <.005) between homocysteine plasma level and 677TT methylenetetrahydrofolate reductase genotype was found. Thrombomodulin plasma levels were significantly higher (P <.00005) in patients (median, 30 ng/mL; range, 10-164 ng/mL) than in controls (median, 19 ng/mL; range, 13-44 ng/mL), and thrombomodulin levels were significantly higher (P <.005) in hyperhomocysteinemic patients (median, 39.5 ng/mL; range, 15-164 ng/mL) than in normohomocysteinemic patients (median, 27.5 ng/mL; range, 10-85 ng/mL). In addition, in patients with abdominal aortic aneurysm, a direct significant correlation (P <.005) was found between homocysteine and thrombomodulin., Conclusions: These data indicate an association between the presence of AAA in patients selected for surgical treatment of AAA and elevated homocysteine plasma levels and suggest that homocysteine may induce endothelial perturbation and stimulation in these patients.