The effect of homocysteine reduction by B-vitamin supplementation on markers of endothelial dysfunction

Hyperhomocysteinemia is a risk factor for arterial vascular disease and venous thrombosis. The pathophysiology of this relation is unclear, but several studies suggest that hyperhomocysteinemia impairs endothelial function. We examined the effect of homocysteine lowering by B-vitamin supplementation on tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI) and von Willebrand factor (vWf)--markers of endothelial dysfunction--in hyperhomocysteinemic and normohomocysteinemic volunteers. A total of 123 healthy volunteers were randomized to placebo or B-vitamins (5 mg folic acid, 0.4 mg hydroxycobalamin and 50 mg pyridoxine) daily for 8 weeks. Before and after the intervention period, blood samples were taken for measurements of homocysteine, tPA, PAI and vWf. There was no evident association between homocysteine concentration and concentrations of markers of endothelial dysfunction at baseline. The mean reduction of homocysteine concentration was 31% (95%CI 22.7 to 39.1) in the B-vitamin group compared to 3% reduction in the placebo group. Concentrations of tPA, PAI and vWf did not change after supplementation of B-vitamins. In conclusion, the results of our study show that homocysteine reduction by B-vitamin supplementation has no effect on markers of endothelial dysfunction in healthy volunteers.     

Effects of folic acid supplementation on inflammatory and thrombogenic markers in chronic smokers. A randomised controlled trial

INTRODUCTION: Cigarette smoking may induce pro-inflammatory and pro-thrombotic changes. It is not known whether these abnormalities are caused at least partly by increased homocysteine levels. We investigated whether lowering homocysteine by folic acid supplementation might reduce the plasma concentration of inflammatory and thrombogenic markers in chronic smokers. MATERIAL AND METHODS: Twenty-four healthy cigarette smokers (age 37.8+/-2.5 years, mean+/-SEM) were randomly assigned to 4 weeks of folic acid 5 mg/day or placebo. The following parameters were measured before and after treatment: (1) markers of inflammation (C-reactive protein, CRP, and white cell count, WCC); (2) blood coagulation screen (Activated Partial Thromboplastin time Ratio, APTR, and International Normalized Ratio, INR); (3) pro-thrombotic markers (fibrinogen, factor VIII coagulant activity, VIII:C, von Willebrand factor, vWF, and D-dimer). RESULTS: Folic acid induced a significant reduction in homocysteine (10.8+/-0.6 vs. 8.2+/-0.5 micromol/l, p<0.001), plasma fibrinogen (3.15+/-0.14 vs. 2.87+/-0.14 g/l, p<0.05), and D-dimer (102+/-44 vs. 80+/-26 microg/l, p<0.05) concentrations. By contrast, no significant changes were observed in CRP (2.2+/-0.7 vs. 1.7+/-0.7 mg/l), WCC (7.2+/-0.5 vs. 6.8+/-0.5 10(9) cells/l), APTR (0.91+/-0.02 vs. 0.93+/-0.02), INR (0.92+/-0.01 vs. 0.91+/-0.01), vWF (103+/-8 vs. 102+/-9 U/dl), and VIII:C (120+/-8 vs. 107+/-8 U/dl) levels. Changes in folic acid plasma concentrations were significantly and negatively correlated with changes in fibrinogen (r=-0.48, p=0.01) but not with changes in D-dimer (r=-0.15, p=0.5) levels. Changes in plasma homocysteine concentrations did not correlate with changes in either fibrinogen or D-dimer. No significant changes in homocysteine, inflammatory and thrombogenic markers were observed in the placebo group. CONCLUSIONS: Short-term folic acid supplementation had no significant effects on inflammatory markers but induced a significant reduction in plasma fibrinogen and D-dimer concentrations in healthy chronic smokers. Thus, folic acid might have an anti-thrombotic effect in this high-risk group independent of the homocysteine lowering effect.     

Apixaban decreases coagulation activity in patients with acute deep-vein thrombosis

In patients with acute deep-vein thrombosis (DVT), apixaban, a direct oral factor Xa inhibitor, showed efficacy and safety similar to low-molecular-weight heparin followed by vitamin K antagonist (LMWH/VKA). We evaluated biomarkers of coagulation activity in relation to treatment dose, duration and clinical outcome. Patients (N = 520) with symptomatic DVT were randomised to receive apixaban (5 mg bid, 10 mg bid or 20 mg qd) or LMWH/VKA for 12 weeks. Plasma D-dimer, prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) levels were measured at baseline, and weeks 3 and 12 after treatment. Median plasma levels of D-dimer, F1+2 and TAT were elevated at baseline. At weeks 3 and 12, biomarker levels were normalised in most patients in all treatment groups, consistent with the low rate of venous thromboembolism (VTE) observed. Median reduction in D-dimer was similar in all treatment groups; percentage of patients with D-dimer above upper limit of normal decreased from 95% to 24-40% at week 12. F1+2 decline was greater with LMWH/VKA than apixaban. F1+2 in the apixaban groups changed to a similar extent (>84% of patients had F1+2 within reference range at week 12). Magnitude of TAT reduction was not quantifiable. In conclusion, levels of coagulation biomarkers decreased over 12 weeks of treatment with apixaban or LMWH/VKA in most patients with acute VTE. Baseline D-dimer and F1+2 were higher in patients with recurrent symptomatic VTE than in those without. Plasma levels of coagulation biomarkers did not appear to correlate with total bleeding events.     

Influence on coagulation activity by subcutaneous LMW heparin as an adjuvant treatment to fibrinolysis in acute myocardial infarction

In this study, which includes 101 patients with acute ST segment-elevated myocardial infarction, we investigated the influence on the increased coagulation activity after streptokinase treatment by adding low-molecular-weight (LMW) heparin or placebo and the relation between the coagulation activity and ischemic episodes, coronary patency, and mortality. The expected increase of prothrombin fragment 1+2 (F1+2), thrombin-antithrombin (TAT), and D-dimer were significantly attenuated at 2, 6, and 18 h (D-dimer only at 18 h) in the dalteparin group compared to placebo. Ischemic episodes during the first 24 h appeared significantly more often in patients with F1+2 levels above the median at 18 h. There was a tendency to a lower frequency of Thrombolysis In Myocardial Infarction Trial (TIMI) grade 3 flow in the infarct-related artery in patients with TAT and D-dimer levels above the median at 18 h. F1+2, TAT, and D-dimer were significantly higher after 18, 6, and 18 h, respectively, in the deceased compared to surviving patients. Also, the lack of reduction of the levels of F1+2 between 6 and 18 h was related to a raised mortality. In conclusion, adjuvant treatment with LMW heparin to streptokinase attenuates increased coagulation activity. This might be of importance as remaining high coagulation activity is associated with signs of early reocclusion and raised mortality.     

No effect of folic acid supplementation in the course of 1 year on haemostasis markers and C-reactive protein in older adults

Elevated homocysteine levels are associated with an increased cardiovascular disease (CVD) risk, but the underlying mechanism is still unclear. High homocysteine might affect the endothelium, and consequently lead to impaired haemostasis. In a randomized placebo controlled trial among 276 older adults with plasma total homocysteine concentrations above 13 mM at screening, we investigated the effect of homocysteine lowering by folic acid supplementation (0.8 mg/day) for 1 year on markers of endothelial function (von Willebrand factor), coagulation (tissue factor, factor VIIa, fragments 1+2), and fibrinolysis (fibrin degradation products, tissue-type plasminogen activator), and inflammation (C-reactive protein). Despite a 24% reduction in plasma homocysteine concentration and four-fold increase in serum folate concentration in the folic acid group compared to the placebo group, there was no clear change in any of the haemostasis markers, nor CRP. Although homocysteine is associated with vascular disease risk in the general population, marked lowering of slightly elevated homocysteine concentrations by one-year folic acid supplementation does not influence haemostasis markers.     

Markers of hemostatic system activation during treatment of deep vein thrombosis with subcutaneous unfractionated or low-molecular weight heparin

Prothrombin fragments (F1+2), thrombin-antithrombin complexes (TAT) and D-dimers, markers of hemostatic system activation, were measured in 59 consecutive patients with deep vein thrombosis (DVT). Patients were randomly treated either with subcutaneous unfractionated heparin (UH) administered in two to three subcutaneous doses adjusted to activated partial thromboplastin time (APTT) or with low-molecular weight heparin (LMWH) (dalteparin) administered in a fixed dose of 200 IU/kg body weight in one subcutaneous injection daily. Before treatment, F1+2, TAT and D-dimer were above the cut-off level in 27/59 (46%), 34/59 (58%) and all (100%) patients, respectively. Significant associations were observed between F1+2 and TAT (r=.66, P<.001), TAT and D-dimer (r=.36, P<.005) and F1+2 and D-dimer (r=.30, P<.050). On the third day of treatment, F1+2 and TAT significantly decreased to reference values in almost all patients (in 64/66 determinations of both F1+2 and TAT) and remained low on the seventh day of treatment. Compared to pretreatment values, a nonsignificant decrease of D-dimer was noted in both groups, but all values remained above the cut-off value. When markers of hemostatic system activation in the UH and LMWH groups were compared, no significant differences were observed. It was concluded that subcutaneous UH in an APTT-adjusted dose and subcutaneous LMWH in a once-daily weight-adjusted dose controlled these markers of hemostatic system activation in a similar manner.     

Coagulation and fibrinolysis after open infrarenal abdominal aortic aneurysm repair in a long-term perspective

In patients with abdominal aortic aneurysms (AAA) the coagulation and fibrinolytic systems have been found to be activated preoperatively. Does the increased activity of the coagulation and fibrinolytic systems persist after AAA surgery in a long-term perspective? Prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), tissue plasminogen activator (tPA), human plasminogen activator inhibitor type 1, and human cross-linked fibrin degradation product (D-dimer) were analysed in 18 patients after open AAA surgery (postop-AAA). The median time between surgery and blood sampling was 19 months (range, 5-37 months). Comparisons were made with both preoperative values of 23 patients with AAA (preop-AAA) as well as 20 age-matched healthy controls (AMC). F1+2, TAT, and D-dimer in preop-AAA were significantly higher compared to AMC (p<0.001). In post-op AAA patients these parameters were significantly lower compared to preop-AAA (p<0.05 for F1+2 and TAT, p<0.001 for D-dimer). However, TAT and D-dimer levels were still higher in postop-AAA than in AMC (p<0.01 for both). The activity of the coagulation and fibrinolytic systems seems to decrease after AAA surgery. However, the activity is still higher than in healthy AMC. A possible explanation may be that the thrombogenicity is lower in a vascular graft than in an aneurysmal sac but still higher than in a nonaneurysmal aorta.     

Effect of edoxaban on markers of coagulation in venous and shed blood compared with fondaparinux

Edoxaban, an oral direct factor Xa (FXa) inhibitor, is in phase III clinical development for stroke prevention in atrial fibrillation and treatment of venous thromboembolism. The shed blood model allows for study of activated coagulation at a site of standardised tissue injury due to local release of tissue factor. The objective of this study was to evaluate the effect of three doses of edoxaban on markers of coagulation in shed and venous blood versus placebo and a standard prophylactic dose of fondaparinux. A total of 100 healthy male subjects were randomised to receive single doses of one of five treatments: subcutaneously administered fondaparinux 2.5 mg; orally administered edoxaban 30, 60, or 120 mg; or placebo. The primary objective was measurement of blood coagulation markers prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin (TAT) complex, and platelet activation marker β-thromboglobulin (β-TG), in venous and shed blood. Secondary objectives included pharmacokinetics, shed blood volume, and safety of edoxaban. Single doses of edoxaban caused rapid and significant decreases of F1+2, TAT, and β-TG in the shed blood model, indicating inhibition of thrombin generation and platelet activation. Inhibition was significantly less for fondaparinux versus edoxaban. Baseline-corrected F1+2, TAT, and β-TG values demonstrated sustained inhibition up to 24 hours for shed blood in the edoxaban groups but no significant inhibition in venous blood. Overall, edoxaban treatments were well tolerated. In conclusion, single oral doses of edoxaban 30, 60, or 120 mg caused rapid and sustained inhibition of coagulation up to 24 hours in the shed blood model.     

Titre of anti-heparin/PF4-antibodies and extent of in vivo activation of the coagulation and fibrinolytic systems

Heparin-induced thrombocytopenia (HIT) is mediated by antibodies directed against the heparin/platelet factor 4 (PF4) complex. Our aim was to investigate whether the antibody titre is associated with the degree of in vivo thrombin generation. We measured the anti-heparin/PF4-antibody titre, prothrombin fragments F1+2, thrombin-antithrombin (TAT) complexes and D-dimers in plasma samples from 225 patients with suspected HIT. Antibody titres as detected by a particle gel immunoassay strongly correlated with optical density values measured by ELISA (r=0.84, p<0.0001). Patients with titres > or =4 (n=44) had significantly higher median levels of F1+2 (2.49 nmol/l), TAT (13.01 microg/l) and D-dimers (3340 microg/l) compared to patients with undetectable antibodies (n=148; F1+2 1.61 nmol/l, TAT 4.95 micro g/l, D-dimers 1911 micro g/l; p<0.0001 for all comparisons) or patients with titres of 1-2 (n=33; F1+2 1.44 nmol/l, p=0.0014; TAT 4.37 microg/l, p=0.0018; D-dimers 2231 microg/l, p=0.0016). Multivariate analysis indicated the anti-heparin/PF4-antibody titre as an independent predictor for F1+2 (p=0.0036), TAT (p=0.0176) and D-dimer (p=0.0003) levels. This relationship remained statistically significant after exclusion of patients with concomitant prothrombotic conditions and/or thromboembolic complications during heparin treatment. These data demonstrate that high anti-heparin/PF4-antibody titres are independently associated with an increased in vivo thrombin generation. Rapid determination of the anti-heparin/PF4-antibody titre could help guide clinical management, identifying a subset of HIT-patients who are at high risk of developing thromboembolic complications and possibly require alternative anticoagulation in therapeutic dosage even in the context of isolated HIT.     

Hyperhomocysteinemia in children treated with antiepileptic drugs is normalized by folic acid supplementation

PURPOSE: To assess the prevalence of hyperhomocysteinemia in pediatric patients treated with antiepileptic drugs (AEDs) and to evaluate the effect of folic acid supplementation on plasma total homocysteine (tHcy) concentrations in hyperhomocysteinemic patients. METHODS: 123 patients from three regional hospitals participated in the study. Patients with hyperhomocysteinemia were included in a 3-month double-blind randomized trial testing oral folic acid supplementation (1 mg/day) versus placebo. RESULTS: Hyperhomocysteinemia (tHcy >10.4 micromol/L) was present in 19 of 123 patients. Patients with hyperhomocysteinemia were older (13.7 +/- 4 vs. 11.0 +/- 3.9 years) and had significantly lower folate and cobalamin concentrations. Multidrug (two or more) AED treatment and duration of therapy correlated significantly with elevated total homocysteine (tHcy) and low folate. In contrast, polymorphisms in the methylene tetrahydrofolate reductase gene (MTHFR 677 C-->T, 1298 A-->C, 1793 G-->A) had no significant impact on tHcy. Nine of 19 patients with hyperhomocysteinemia were randomized to placebo, whereas the remaining 10 patients received folic acid supplementation. Folic acid supplementation resulted in a significant increase of folate and decrease of tHcy, whereas both parameters remained unchanged in the placebo group. CONCLUSIONS: Hyperhomocysteinemia is present in 15.5% of children receiving long-term AED treatment. Multidrug treatment and long duration of therapy enhance the risk for hyperhomocysteinemia. Folic acid supplementation significantly reduces tHcy. We recommend assessment of serum folate and plasma tHcy in children receiving AEDs.     

The effect of dalteparin on coagulation activation during pregnancy in women with thrombophilia. A randomized trial

Low-molecular-weight heparin (LMWH) is increasingly being used for prophylaxis of venous thromboembolism (VTE) and prevention of pregnancy associated morbidity in pregnant women with thrombophilia. We sought to determine if the administration of prophylactic doses of LMWH downregulates coagulation activation in high risk pregnant women with thrombophilia. This sub-study was planned as part of a randomized open label controlled trial (Thrombophilia in Pregnancy Prophylaxis Study [TIPPS]) in which patients at high risk of pregnancy complications with confirmed thrombophilia are randomized to receive either dalteparin (5,000 units/day until 20 weeks then 5,000 units q12h until 37 weeks or onset of labor) or no treatment. Blood samples were collected at baseline, day 7-9 (after starting study drug), week 20 (before increasing study drug), week 36 (prior to stopping study drug) and at the time of admission to the labor and delivery unit. Samples were not drawn at fixed times in relation to drug injection. These samples were analyzed for levels of thrombin-antithrombin complexes (TAT), prothrombin fragments 1 + 2 (F1.2), D-dimer and anti-Xa activity. Generalized linear mixed models were used for statistical analysis and model results were controlled for age, smoking status, type of thrombophilia and predisposing risk factors. The effect of dalteparin on TAT levels was defined as the primary outcome. Of 198 patients eligible, 114 were enrolled in TIPPS. Ninety-one were eligible for the TIPPS coagulation activation sub-study and randomized. Thirty-nine patients were analyzed in the treatment group (dalteparin) and 46 patients in the control group (no intervention). Levels of coagulation activation factors F1.2, TAT and D-dimer increased significantly throughout pregnancy in both groups (p < 0.0001). Dalteparin prophylaxis resulted in a significant increase in anti-Xa activity through pregnancy (p < 0.0001) compared to controls. Dalteparin had no significant effects on the levels of TAT, F1.2 and D-dimer throughout pregnancy in thrombophilic women. A post-hoc Monte Carlo power analysis revealed that our study had 100% and 88% power to detect reductions in TAT values on treatment of 50% and 25%, respectively. Prophylaxis with dalteparin at doses used in this study did not reduce coagulation activation in high risk thrombophilic women during pregnancy.     

The effect of B-vitamins on hyperhomocysteinemia in patients on antiepileptic drugs

Patients on antiepileptic drugs (AEDs) may have elevated levels of plasma total homocysteine (p-tHcy). The aim of this study was to assess the effect of B-vitamin supplementation on the levels of p-tHcy and markers of endothelial activation and lipid peroxidation. A total of 33 adult patients on AEDs were identified with either fasting (Group 1, n=23) or post methionine load (PML) (Group 2, n=10) hyperhomocysteinemia. Subjects were supplemented with B-vitamins for 30 days: folic acid 0.4 mg, pyridoxine 120 mg and riboflavin 75 mg per day. After supplementation, serum folate and pyridoxal phosphate had increased, while fasting and PML p-tHcy had decreased (P<0.0001) by 36 and 26%, respectively. Prior to supplementation, the Group 1 patients had elevated levels of P-selectin and von Willebrand factor (vWF) (P=0.05 and 0.03, respectively). After supplementation, the levels of intercellular cell adhesion molecules had decreased (P=0.01) and E-selectin decreased nonsignificantly (P=0.07). However, the levels of vascular cell adhesion molecules had increased (P<0.0001), while lipid peroxidation were unchanged. In conclusion, the combined supplementation with folic acid, pyridoxine and riboflavin reduced fasting and PML hyperhomocysteinemia in patients on AEDs. Patients with fasting hyperhomocysteinemia had elevated levels of P-selectin and vWF, which may indicate an increased risk of cardiovascular disease. Furthermore, B-vitamin supplementation influenced endothelial activation, although the clinical implication is uncertain.     

A randomised double-blind placebo-controlled trial of folic acid supplementation of cholinesterase inhibitors in Alzheimer's disease

OBJECTIVES: (1) to assess the effect of 1 mg folic acid supplementation of cholinesterase inhibitors (ChI) in a 6 month double-blind placebo-controlled study of patients with Alzheimer's Disease (AD) and (2) to assess whether outcome measures were affected by changes in homocysteine levels. METHOD: Fifty-seven consecutive outpatients with probable AD were treated concurrently with a ChI and either folic acid or placebo. None had conditions or medication known to interfere with folate metabolism. Fasting folate and homocysteine levels were measured prior to commencing ChI and 6 months later. Response was categorised using criteria of the National Institute of Clinical Excellence (NICE). RESULTS: Twelve males and 29 females completed treatment (mean age 76.27 SD 6.23 years, Mini-Mental State Examination (MMSE) 23.49 SD 3.53, baseline homocysteine 18.39 SD 4.62 micromoles per litre). 23 received folic acid and 18 placebo. There were no significant baseline differences or use of individual ChI between the two arms. After 6 months a significant difference was seen in the change from baseline in combined Instrumental Activities of Daily Living and Social Behaviour scores between arms (folate+1.50 (SD 5.32) vs placebo -2.29 (SD 6.16) (p=0.03) but not change in MMSE scores. Sixteen of 23 subjects receiving folic acid and 7/18 placebo subjects were classified as NICE responders (p=0.05). CONCLUSION: This pilot double blind study suggests that response to ChI in patients with AD may be improved by the use of folic acid. The relationship between any change in homocysteine levels and response to treatment is discussed.     

Treatment of Hyperhomocysteinemia with Folic Acid and Vitamins B12 and B6 Attenuates Thrombin Generation

The effect of homocysteine-lowering treatment on thrombin generation was investigated in 17 subjects with hyperhomocysteinemia (aged 22–60 years), 11 of whom had symptomatic atherosclerotic vascular disease. All subjects had fasting total homocysteine levels above 16 μmol/L. The formation of thrombin was assessed by measuring thrombin-antithrombin III complexes and prothrombin fragment 1+2 in peripheral venous blood and in the bleeding time blood collected at 30-second intervals from skin incisions on a forearm. All the tests were performed before and after an 8-week treatment with folic acid p.o. 5 mg/day, vitamin B₆ p.o. 300 mg/day, and vitamin B₁₂ i.m. 1000 μg given on a weekly basis. Following the 8-week therapy, the median plasma homocysteine concentration became significantly reduced from 20 to 10 μmol/L, while plasma levels of fibrinogen, prothrombin, and antithrombin III as well as activity of protein C, S, and factor VII showed no changes. Vitamin treatment was associated with a significant fall in thrombin-antithrombin III complexes and prothrombin fragment 1+2 concentrations in peripheral venous blood. Bleeding time became prolonged by about 60 seconds. At sites of hemostatic plug formation, plasma concentrations of both thrombin markers significantly decreased. Compared with pretreatment values, significantly less thrombin was produced during the first 3 minutes of bleeding after homocysteine-lowering therapy. In subjects with hyperhomocysteinemia a reduction of plasma fasting homocysteine concentration by folic acid and vitamins B₁₂ and B₆ administration is associated with attenuation of thrombin generation both in peripheral blood and at sites of hemostatic plug formation.     

High D-dimer levels predict cardiovascular events in patients with chronic atrial fibrillation during oral anticoagulant therapy

Atrial fibrillation (AF) is associated with hemostatic abnormalities and increased risk of thrombotic cardiovascular events even during oral anticoagulant therapy (OAT). The aim of our study was to evaluate the predictive value of hemostatic markers for the risk of major cardiovascular events during OAT. The study group comprised 113 patients with chronic AF (70.2 +/- 5.4 years old, 60% men), referred for OAT. Established clinical risk factors and levels of prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complexes (TAT), D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) antigen and activity, before and during OAT (after 3.9 +/- 0.7 months; INR 2.57 +/- 0.57) were determined. In all patients OAT significantly suppressed levels of F1+2 by 67%,TAT by 30% and D-dimer by 48% (all p <0.001). During an average follow-up of 44 months 22/111 (20%) patients suffered a combined cardiovascular event (stroke, myocardial infarction, peripheral vascular occlusion or vascular death). Patients with cardiovascular events were significantly older, had more frequent heart failure/systolic dysfunction and had significantly increased levels of D-dimer at entry (63 vs 39 ng/mL, p = 0.005) and during OAT (33 vs 18 ng/mL, p = 0.002), and of t-PA antigen at entry (14.3 vs 10.9 ng/mL, p = 0.02) and during OAT (15.0 vs 11.2 ng/mL, p = 0.05) (all values are medians). In multivariate Cox proportional hazard models, heart failure/systolic dysfunction (hazard ratio 2.91; 95% CI 1.17-7.26; p = 0.02), high levels of D-dimer on OAT (top vs. lower two quartiles) (hazard ratio 4.78, 95% CI 1.39-16.41; p = 0.01) and t-PA antigen levels (continuous variable) (hazard ratio 1.09; 95% CI 1.01-1.17; p = 0.02) were significantly associated with combined cardiovascular events. In conclusion, high levels of D-dimer and t-PA antigen during OAT are significant predictors of combined cardiovascular events in AF patients and, on this basis, could be useful additional markers of cardiovascular risk in such patients.     

Effects of oral and transdermal estrogen replacement therapy on markers of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in postmenopausal women

We compared the effects of oral estradiol (2 mg), transdermal estradiol (50 microg), and placebo on measures of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in 27 postmenopausal women at baseline and after 2 and 12 weeks of treatment. Oral and transdermal estradiol induced similar increases in serum free estradiol concentrations. Oral therapy increased the plasma concentrations of factor VII antigen (FVIIag) and activated factor VII (FVIIa), and the plasma concentration of the prothrombin activation marker prothrombin fragment 1+2 (F1+2). Oral but not transdermal estradiol therapy significantly lowered plasma plasminogen activator inhibitor-1 (PAI-1) antigen and tissue-type plasminogen activator (tPA) antigen concentrations and PAI-1 activity, and increased D-dimer concentrations, suggesting increased fibrinolysis. The concentration of soluble E-selectin decreased and serum C-reactive protein (CRP) increased significantly in the oral but not in the transdermal or placebo groups. In the oral but not in the transdermal or placebo estradiol groups low-density-lipoprotein (LDL) cholesterol, apolipoprotein B and lipoprotein (a) concentrations decreased while high-density-lipoprotein (HDL) cholesterol, apolipoprotein AI and apolipoprotein All concentrations increased significantly. LDL particle size remained unchanged. In summary, oral estradiol increased markers of fibrinolytic activity, decreased serum soluble E-selectin levels and induced potentially antiatherogenic changes in lipids and lipoproteins. In contrast to these beneficial effects, oral estradiol changed markers of coagulation towards hypercoagulability, and increased serum CRP concentrations. Transdermal estradiol or placebo had no effects on any of these parameters. These data demonstrate that oral estradiol does not have uniformly beneficial effects on cardiovascular risk markers and that the oral route of estradiol administration rather than the circulating free estradiol concentration is critical for any changes to be observed.     

Dose-dependent decrease of platelet activation and tissue factor by omega-3 polyunsaturated fatty acids in patients with advanced chronic heart failure

Chronic heart failure (CHF) is characterised by activation of neuroendocrine and inflammatory pathways, and both are linked to a prothrombotic state. Treatment with omega-3 polyunsaturated fatty acids (n3-PUFA) showed significant benefits including mortality reduction in CHF, but exact mechanisms of action are still unclear. We investigated the effects of n3-PUFA on markers of platelet activation and thrombogenesis in patients with severe CHF. Thirty-six patients with non-ischaemic CHF (LVEF<35%, NYHA class>2) under optimised therapy were randomised to supplementation with 1g/day or 4 g/day n3-PUFA, or placebo for 12 weeks. Using whole-blood flow cytometry, monocyte-platelet aggregates characterised by CD14+/CD42b+ co-expression and monocytic tissue factor (TF) were determined. Plasma levels of P-selectin, sCD40L, fibrinogen, prothrombin fragment F1.2, TF and pro-inflammatory markers (high sensitive[hs] interleukin-6, hsCRP, hsTNF-alpha, monocyte chemotactic protein-1) were measured by immunoassay. Supplementation with 1g/day and 4 g/day n3-PUFA but not placebo significantly reduced monocyte-platelet aggregates in a dose-dependent manner (p for trend = 0.02 across the groups). A dose of 4 g/day but not 1g/day n3-PUFA significantly decreased P-selectin (p = 0.03). Plasma TF decreased dose-dependently upon n3-PUFA supplementation (p for trend = 0.02), paralleled by a significant decrease of TF+-monocytes (p for trend = 0.01). The amount of 4 g/day n3-PUFA exhibited modest anti-inflammatory effects with a significant reduction of hs interleukin-6 (p<0.01) and a trend-wise reduction of hsTNF-alpha (p = 0.09). No changes were seen for sCD40L, fibrinogen, hsCRP and monocyte chemotactic protein-1, while F1.2 was decreased by 4 g/day n3-PUFA (P = 0.03). In patients with severe non-ischaemic CHF, treatment with n3-PUFA leads to a dose-dependent decrease of platelet activation and TF. Higher dosage exhibits also anti-inflammatory effects.     

Homocysteine but not neopterin declines in demented patients on B vitamins

Summary. Inflammation and immune system activation seem to play an important role in the development and progression of dementia. Hyperhomocysteinemia is common in various forms of dementia, and a significant relationship was found between concentrations of homocysteine and immune activation marker neopterin. B vitamin supplementation is able to slow-down homocysteine formation in patients. In an open-label study, effects of B vitamin supplementation (Beneuran compositum ^?) on concentrations of homocysteine and neopterin were investigated in 58 patients with Alzheimer’s disease (n = 30), vascular dementia (n = 12) and mild cognitive impairment (n = 16). In all groups of patients, a significant percentage of patients presented with homocysteine concentrations >15 μmol/L and with elevated concentrations of immune activation marker neopterin. Decline of homocysteine concentrations was observed after one month of B vitamin supplementation (all p < 0.01; paired Kruskal-Wallisn-test). By contrast, neopterin concentrations remained unchanged (all p > 0.05). B vitamin supplementation in patients with various forms of dementia did not influence neopterin concentrations, which indicates that the degree of immune activation and inflammation remained unchanged. The question remains, if lowering of homocysteine by folate supplementation alone could have any beneficial effect to modulate the course of dementia development and if longer period of supplementation would also ameliorate immune system activation status.     

Absence of paradoxical thrombin activation by fibrin-specific thrombolytics in acute myocardial infarction: comparison of single-bolus tenecteplase and front-loaded alteplase

OBJECTIVES: Thrombolytic therapy in patients with acute myocardial infarction is hampered by bleeding complications and procoagulant effects favoring early reocclusion. TNK-tPA was shown in vitro to have considerable fibrin specificity. We investigated the effects of tenecteplase (TNK-tPA) and alteplase (rt-PA) on the haemostasis and fibrinolytic system. METHODS AND RESULTS: We enrolled 30 patients with AMI into the study. Twenty patients received front-loaded rt-PA up to 100 mg; 10 patients were given TNK-tPA in a single bolus up to 50 mg. All patients received aspirin and intravenous heparin. During the first 2 days, the following parameters were repetitively determined: thrombin-antithrombin III complexes (TAT), antithrombin III (ATIII), prothrombin fragment F 1 + 2 (F 1 + 2), kallikrein-like activity (KK), activated factor XII (FXIIa), plasmin alpha 2-antiplasmin complexes (PAP), fibrinogen, D-dimers (DD), tissue-type plasminogen activator (t-PA). A total of 75 healthy persons served as control group. TAT increased significantly after rt-PA but not after TNK-tPA (3 h: 38.1 +/- 29.4 versus 10.5 +/- 4.2 microg/l; p < 0.01), indicating paradoxical thrombin activation. F 1 + 2 increased transiently after rt-PA but not after TNK-tPA. Fibrinogen was significantly lower after rt-PA versus TNK-tPA (3 h: 163 +/- 27 versus 380 +/- 54 mg/dl; p < 0.05). KK activities in the rt-PA group were significantly (p < 0.01) increased over 48 h versus TNK-tPA. PAP and D-dimers were lower over the time course of 48 h in the tenecteplase group versus rt-PA. CONCLUSIONS: This study indicates that tenecteplase has higher fibrin specificity not only in vitro but also in vivo versus alteplase. TNK-tPA consecutively has no paradoxical systemic procoagulant effect due to the lower extent of activation of the kallikrein-factor XII system than alteplase.     

Prophylactic antithrombin III administration during pregnancy immediately reduces the thrombin hyperactivity of congenital antithrombin III deficiency by forming thrombin-antithrombin III complexes.

We examined the changes of haemostatic molecular markers after antithrombin III (AT III) administration in a 22-year-old woman with congenital AT III deficiency in the third trimester of pregnancy who did not have thrombosis. Various markers including fibrinopeptide A (FPA), thrombin-antithrombin III complex (TAT), prothrombin fragment F1 + 2 (F1 + 2), plasmin-alpha 2antiplasmin, D-dimer, beta-thromboglobulin, and platelet factor 4 were measured before and just after 3,000 U of AT III concentrate, which was given three times per week from the 34 week of pregnancy until delivery. Just after AT III administration, F1 + 2 and FPA levels decreased on most occasions, while TAT sometimes increased. Plasma FPA levels were markedly decreased on all 8 occasions when the plasma FPA levels was above 2.0 ng/ml before AT III administration. Plasma FPA levels were always greater than or equal to 6.4 ng/ml before AT III administration on the 4 occasions when TAT increased to above 115%. The changes of plasma F1 + 2 levels were significantly correlated with the AT III level. These results suggest that prophylactic AT III administration in the third trimester immediately inactivates intravascular thrombin to form TAT and reduce the plasma FPA level. Thus, the transient TAT elevation following AT III administration may not only be due to extraction of thrombin from the fibrin clots of thrombi but also to intravascular thrombin which is not attached to thrombi. FPA is the best molecular marker for thrombin hyperactivity and it should be monitored in AT III-deficient pregnant women in the third trimester.