Paola Ferrazzi

Homocysteine, MTHFR C677T gene polymorphism, folic acid and vitamin B 12 in patients with retinal vein occlusion

BackgroundMany available data have suggested that hyperhomocysteinaemia, an established independent risk factor for thrombosis (arterial and venous), may be associated with an increased risk of retinal vein occlusion (RVO).Aim of the studyTo evaluate homocysteine metabolism in consecutive caucasian patients affected by RVO from Northern Italy.Patients and Methods69 consecutive patients from Northern Italy (mean age 64.1 ± 14.6 yy) with recent RVO, were tested for plasma levels of homocysteine (tHcy: fasting and after loading with methionine), cyanocobalamine and folic acid levels (CMIA-Abbot) and looking for MTHFR C677T mutation (Light Cycler-Roche) and compared to 50 volunteers, enrolled as a control group.ResultsFasting levels of tHcy were significantly higher in patients than in controls: mean value 14.7 ± 7.7 vs 10.2 ± 8 nmol/ml. Post load levels were also significantly higher: mean value 42.7 ± 23.7 vs 30.4 ± 13.3 nmol/ml; Total homocysteine increase was also evaluated (i.e. Δ-tHcy) after methionine load and was also significantly higher in patients compared to control subjects: mean Δ-tHcy 27.8 ± 21.5 vs 21.0 ± 16 nmol/ml (normal value < 25 nmol/ml). Furthermore, patients affected by RVO show low folic acid and/or vitamin B12 levels, although differences with control group did not reach statistical significance. Heterozygous and homozygous MTHFR mutation were respectively in study group 46% and 29% vs control group 56% and 4%.Conclusionour data confirm that hyperhomocysteinaemia is a risk factor for RVO, and also that TT genotype of MTHFR C677T is more frequently associated with RVO: if the mutation per se is a risk factor for RVO remains an open question to be confirmed because another study from US did not reveal this aspect.Hyperomocysteinemia is modifiable risk factor for thrombotic diseases. Therefore, a screening for tHcy plasma levels in patients with recent retinal vein occlusion could allow to identify patients who might benefit from supplementation with vitamins and normalization of homocysteine levels, in fasting and after methionine load.

Evaluation of the prevalence of severe hyperhomocysteinemia in adult patients with thrombosis who underwent screening for thrombophilia

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.

Intima-media thickness evolution after treatment with infliximab in patients with rheumatoid arthritis

Background Atherosclerosis is a well known progressive disease that recognizes risk factors such as diabetes, hypertension, smoking, dyslipidemia, and inflammation. Mechanisms underlying atherosclerotic processes during inflammation are not completely understood, but cytokines are also involved, in particular tumor necrosis factor-α (TNF-α). Chronic inflammatory diseases such as rheumatoid arthritis (RA) are commonly associated with atherosclerotic complication. Little is known about the role of treatment of chronic inflammatory disease on the evolution of atherosclerosis in this kind of disease. Usually, evolution of atherosclerosis is monitored by intima-media thickness and the presence of plaques on several arteries such as common carotid. Aim The aim of the study was to monitor atherosclerosis evolution in seven RA patients on common treatment with infliximab (an anti-TNF-α drug) compared with seven RA patients during common treatment but not treated with infliximab. Patients and methods We selected 14 patients with RA according to the American College of Rheumatology classification criteria. Seven patients were selected before and after common treatment for RA based on nonsteroidal anti-inflammatory drugs (NSAIDs), methotrexate, and steroids (12 months), and seven patients before and after treatment based on infliximab associated with NSAIDs, methotrexate, and steroids (12 months). Ultrasound vascular imaging was performed to screen intima-media thickness and the presence of atherosclerotic plaques on common carotid artery and identify evolution of atherosclerosis. Results After 12 months, patients that were treated with infliximab showed significant worsening of atherosclerosis with an increase of intima-media thickness and the presence of further atherosclerotic plaques compared to patients that were treated traditionally and showed a nonsignificant increase of the same parameters. Discussion Treatment based on anti-TNF-α such as infliximab shows a worsening evolution of atherosclerosis based on our data. If these data are associated with a poor clinical outcome such as atherothrombosis of cerebral vessels and/or coronary vessels, this should be evaluated by further studies.

Is there a relationship between factor V Leiden and type 2 diabetes

BackgroundDiabetes is well known risk factor for thrombotic events. The association between diabetes and venous thromboembolism is still matter of debate. However, during diabetes an acquired thrombophilia is present and is due to the non-enzymatic glycosilation of clotting inhibitors as antithrombin thus leading to hypercoagulable state. A possibile relationship between the presence of FVL gene variant in type 1 or type 2 diabetes has been hypothysed by several reports in the Literature with non-univocal findings.Patients and methodsRetrospectively we analysed nearly 7000 patients referred to our Thrombosis Center for venous thromboembolism (VTE) then we selected 115 patients underwent to the screening for inherited thrombophilia. All selected patients were divided in 2 groups: the first group (group A) included 64 patients with previous VTE and carriers of factor V Leiden, while the second group (group B) included 51 patients with previous VTE and evetually carriers of thrombophilic defects other than factor V Leiden. Patients of group B acted as control group. 75 g oral glucose tolerance Test (OGTT) recommended by WHO was perfomed to all subjects in the study in order to screen subjects with glucose reduced tolerance or subjects with inducible diabetes. Statistical analysis was performed with STATA 6 http://www.stata.com with Student t test for unpaired data, with χ2 test or with Fisher exact test where appropriated; differences were considered to be significant if p < 0.05.ResultsWe did not find sifferences between glycaemia at baseline and after OGTT between patients with VTE carriers of FVL compared to non-carriers of FVL. We found a relevant increase in the prevalence of IGT and diabetes between patients with VTE carriers of FVL compared to non-carriers of FVL although this increase did not raise statistical significance.Discussionour data pointed out an interesting aspect of the linking between FVL gene variant, diabetes and atherothrombosis and other vascular complications, although data on larger population are needed; this aspect may be another relevant topic of research based because also a link between the pathogenesis of venous thrombosis and atherothrombosis has been recently reported in the Literature.

The effect of parnaparin sodium on in vitro fertilization outcome: A prospective randomized controlled trial

INTRODUCTION In-vitro and in-vivo models suggest the influence of low-molecular weight heparin on conception in infertile women undergoing in vitro fertilization procedures (IVF). In this randomized controlled trial we assessed whether a low-molecular weight heparin (parnaparin) could affect IVF outcomes. MATERIALS AND METHODS 271cycles were analyzed in 247 women having a first or subsequent IVF cycle at Fertility Center of Humanitas Research Hospital. Patients, without severe thrombophilia and hormonal or active untreated autoimmune disorders, were randomly allocated (1:1) to receive for the whole cycle parnaparin, or routine hormonal therapy only. The primary endpoint was the clinical pregnancy rate and the secondary endpoints included implantation rate and live birth rate. RESULTS The clinical pregnancy and the live birth rate were similar in treated and controls (21.5% vs. 26.7%, p=0.389; 18.5% vs. 20.6%, p=0.757). The abortion rate was 10.3% vs 22.9%, p=0.319, respectively. The subgroups analysis, ≤35, 36-38, 39-40years, showed the following: comparable clinical pregnancy rate (22.5% vs 38.8%, p=0.124; 21.8% vs 17.3%, p=0.631; 19.4% vs 23.3%, p=0.762 respectively) and live birth rate (16.3% vs 32.7%, p=0.099; 20.0% vs 13.5%, p=0.443; 19.4% vs 13.3%, p=0.731 respectively) in treated vs controls. Sensitivity analyses on women with ≥3 previous attempts and first enrolment only, and subgroup analyses according to trial conclusion conditioning a small sample size with low statistical power. CONCLUSIONS Our study excludes positive effect of parnaparin, once a day for the whole cycle, on clinical pregnancy rate in infertile women undergoing in vitro fertilization techniques.

Differences in the INR evaluation of two different thromboplastins in patients with positivity to lupus anticoagulant in ongoing oral anticoagulation

Background: A possible interference between lupus anticoagulant (LAC), a well characterized clotting inhibitor, in the International Normalized Ratio (INR) determination during oral anticoagulation (OA) has been reported in the literature. Few data are available about the relationship between this kind of interference and the daily clinical management of oral anticoagulation. The aim of the study is to evaluate the role of two different thromboplastins–RecombiPlasTin 2G and HepatoComplex–in the determination of INR values of several patients’ ongoing OA for a previous thrombotic disorder with and without positivity to LAC, and to evaluate possible interferences in the daily therapeutic approach. Patients and methods: We selected 16 patients (13 females and 3 males, mean age 59 ± 16 years) with LAC positivity ongoing OA and 11 control subjects (7 females and 4 males, mean age 58 ± 14.5 years) with similar characteristics (ie, ethnic background and weight) with LAC negativity ongoing OA. 165 assays for INR determination were analyzed from both groups. Statistical analysis was performed using STATA 10 software. P values were considered significant if <0.05. Results: Mean values of INR for patients with LAC positivity were 3.79 ± 1.63 when tested with RecombiPlasTin 2G vs 3.18 ± 1.15 when tested with HepatoComplex (P < 0.001, s); while mean values of INR for patients with antiphospholipid syndrome (APS) with LAC negativity were 3.54 ± 1.39 when tested with RecombiPlasTin 2G vs 3.23 ± 1.14 when tested with HepatoComplex (P < 0.002, s). An INR value > than 4.5 was found in 31/165 samples in 9 subjects, 8 patients with LAC positivity, and 1 control group subject with LAC negativity. There was a great difference in INR values in these subjects if we use the common thromboplastin (ie, RecombiPlasTin 2G) with a INR range varying from 5.14 ± 0.35 vs 3.79 ± 0.38 if we use another thromboplastin (ie, HepatoComplex) (P < 0.001, s). A change in the therapeutic approach for OA is possible in these cases because different INR values were obtained using different thromboplastins. Discussion: Our data confirm that INR evaluation does not reveal significant changes also if tested with two different thromboplastins, for patients ongoing OA with and without LAC positivity, when the INR value is < than 4. Over this INR value there is a significant difference in patients with LAC positivity if we use a different thromboplastin for the INR determination. For this reason values obtained by RecombiPlasTin 2G need to be confirmed and matched with another thromboplastin (ie, HepatoComplex). This approach may be useful in order to have a good INR testing for the chronic long-term treatment with OA in particular in patients with LAC positivity.

Predictors of Long-Term Recurrent Vascular Events After Ischemic Stroke at Young AgeCLINICAL PERSPECTIVE

Background— Data on long-term risk and predictors of recurrent thrombotic events after ischemic stroke at a young age are limited. Methods and Results— We followed 1867 patients with first-ever ischemic stroke who were 18 to 45 years of age (mean age, 36.8±7.1 years; women, 49.0%), as part of the Italian Project on Stroke in Young Adults (IPSYS). Median follow-up was 40 months (25th to 75th percentile, 53). The primary end point was a composite of ischemic stroke, transient ischemic attack, myocardial infarction, or other arterial events. One hundred sixty-three patients had recurrent thrombotic events (average rate, 2.26 per 100 person-years at risk). At 10 years, cumulative risk was 14.7% (95% confidence interval, 12.2%–17.9%) for primary end point, 14.0% (95% confidence interval, 11.4%–17.1%) for brain ischemia, and 0.7% (95% confidence interval, 0.4%–1.3%) for myocardial infarction or other arterial events. Familial history of stroke, migraine with aura, circulating antiphospholipid antibodies, discontinuation of antiplatelet and antihypertensive medications, and any increase of 1 traditional vascular risk factor were independent predictors of the composite end point in multivariable Cox proportional hazards analysis. A point-scoring system for each variable was generated by their &bgr;-coefficients, and a predictive score (IPSYS score) was calculated as the sum of the weighted scores. The area under the receiver operating characteristic curve of the 0- to 5-year score was 0.66 (95% confidence interval, 0.61–0.71; mean, 10-fold internally cross-validated area under the receiver operating characteristic curve, 0.65). Conclusions— Among patients with ischemic stroke aged 18 to 45 years, the long-term risk of recurrent thrombotic events is associated with modifiable, age-specific risk factors. The IPSYS score may serve as a simple tool for risk estimation.

Predictors of Long-Term Recurrent Vascular Events After Ischemic Stroke at Young AgeCLINICAL PERSPECTIVE: The Italian Project on Stroke in Young Adults

Background— Data on long-term risk and predictors of recurrent thrombotic events after ischemic stroke at a young age are limited. Methods and Results— We followed 1867 patients with first-ever ischemic stroke who were 18 to 45 years of age (mean age, 36.8±7.1 years; women, 49.0%), as part of the Italian Project on Stroke in Young Adults (IPSYS). Median follow-up was 40 months (25th to 75th percentile, 53). The primary end point was a composite of ischemic stroke, transient ischemic attack, myocardial infarction, or other arterial events. One hundred sixty-three patients had recurrent thrombotic events (average rate, 2.26 per 100 person-years at risk). At 10 years, cumulative risk was 14.7% (95% confidence interval, 12.2%–17.9%) for primary end point, 14.0% (95% confidence interval, 11.4%–17.1%) for brain ischemia, and 0.7% (95% confidence interval, 0.4%–1.3%) for myocardial infarction or other arterial events. Familial history of stroke, migraine with aura, circulating antiphospholipid antibodies, discontinuation of antiplatelet and antihypertensive medications, and any increase of 1 traditional vascular risk factor were independent predictors of the composite end point in multivariable Cox proportional hazards analysis. A point-scoring system for each variable was generated by their &bgr;-coefficients, and a predictive score (IPSYS score) was calculated as the sum of the weighted scores. The area under the receiver operating characteristic curve of the 0- to 5-year score was 0.66 (95% confidence interval, 0.61–0.71; mean, 10-fold internally cross-validated area under the receiver operating characteristic curve, 0.65). Conclusions— Among patients with ischemic stroke aged 18 to 45 years, the long-term risk of recurrent thrombotic events is associated with modifiable, age-specific risk factors. The IPSYS score may serve as a simple tool for risk estimation.

SV-IV Peptide1–16 reduces coagulant power in normal Factor V and Factor V Leiden

Native Factor V is an anticoagulant, but when activated by thrombin, Factor X or platelet proteases, it becomes a procoagulant. Due to these double properties, Factor V plays a crucial role in the regulation of coagulation/anticoagulation balance.Factor V Leiden (FVL) disorder may lead to thrombophilia. Whether a reduction in the activation of Factor V or Factor V Leiden may correct the disposition to thrombophilia is unknown. Therefore we tested SV-IV Peptide 1–16 (i.e. a peptide derived by seminal protein vescicle number IV, SV-IV) to assess its capacity to inhibit the procoagulant activity of normal clotting factor V or Factor V Leiden (FVL). We found that SV-IV protein has potent anti-inflammatory and immunomodulatory properties and also exerts procoagulant activity. In the present work we show that the SV-IV Peptide 1–16, incubated with plasma containing normal Factor V or FVL plasma for 5 minutes reduces the procoagulant capacity of both substances. This is an anticoagulant effect whereas SV-IV protein is a procoagulant. This activity is effective both in terms of the coagulation tests, where coagulation times are increased, and in terms of biochemical tests conducted with purified molecules, where Factor X activation is reduced.Peptide 1–16 was, in the pure molecule system, first incubated for 5 minutes with purified Factor V then it was added to the mix of phosphatidylserine, Ca2+, Factor X and its chromogenic molecule Chromozym X. We observed a more than 50% reduction in lysis of chromogenic molecule Chromozym X by Factor Xa, compared to the sample without Peptide 1–16. Such reduction in Chromozym X lysis, is explained with the reduced activation of Factor X by partial inactivation of Factor V by Peptide 1–16. Thus our study demonstrates that Peptide 1–16 reduces the coagulation capacity of Factor V and Factor V Leiden in vitro, and, in turn, causes factor X reduced activation.