Irene Quintana

Influence of homocysteine on fibrin network lysis.

To elucidate some of the links between homocysteine and vascular disease, we have evaluated the effect of the amino acid on the formation (by kinetics studies), structure (by electron microscopy) and lysis of the fibrin network, using tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). We have studied whether homocysteine could alter the activity of the components involved in fibrinolysis (by amidolytic and thrombolytic methods). The results showed that homocysteine-associated networks were more compact and branched than controls (52 ± 6 vs 44 ± 5 fibers/field, P = 0.008), and were formed by shorter and thicker fibers. This clot proved to be more resistant to fibrinolysis with u-PA than control [lysis time 50%: 257 ± 16 (homocysteine) vs 187 ± 6 min (control); P < 0.004], but there were no differences with t-PA. Homocysteine did not affect the biological activities of plasmin, or plasminogen activation by t-PA and u-PA. Defective fibrinolysis with u-PA was therefore associated with homocysteine–fibrin structural alterations rather than the homocysteine effect on the biological activities of the fibrinolytic components evaluated. Results suggest that hyperhomocysteinemic patients could produce tight clots, were more resistant to lysis, and generated a procoagulant environment in situ. We believe that our findings may contribute to understanding the mechanisms involved in the homocysteine harmful effect.

Anticoagulant activity of a unique sulfated pyranosic (1->3)-β-L-arabinan through direct interaction with thrombin.

Background: Many seaweed polysaccharides have anticoagulant activity, but the mechanism of action was elucidated in a few cases. Results: A highly sulfated pyranosic β-arabinan exerts its activity through direct and indirect inhibition of thrombin. Conclusion: The structure and mechanism of action of the arabinan are different from those found for other polysaccharides. Significance: This arabinan could be an alternative anticoagulant in certain specific cases. A highly sulfated 3-linked β-arabinan (Ab1) with arabinose in the pyranose form was obtained from green seaweed Codium vermilara (Bryopsidales). It comprised major amounts of units sulfated on C-2 and C-4 and constitutes the first polysaccharide of this type isolated in the pure form and fully characterized. Ab1 showed anticoagulant activity by global coagulation tests. Less sulfated arabinans obtained from the same seaweed have less or no activity. Ab1 exerts its activity through direct and indirect (antithrombin- and heparin cofactor II-mediated) inhibition of thrombin. Direct thrombin inhibition was studied in detail. By native PAGE, it was possible to detect formation of a complex between Ab1 and human thrombin (HT). Ab1 binding to HT was measured by fluorescence spectroscopy. CD spectra of the Ab1 complex suggested that ligand binding induced a small conformational change on HT. Ab1-thrombin interactions were studied by molecular dynamic simulations using the persulfated octasaccharide as model compound. Most carbohydrate-protein contacts would occur by interaction of sulfate groups with basic amino acid residues on the surface of the enzyme, more than 60% of them being performed by the exosite 2-composing residues. In these interactions, the sulfate groups on C-2 were shown to interact more intensely with the thrombin structure. In contrast, the disulfated oligosaccharide does not promote major conformational modifications at the catalytic site when complexed to exosite 1. These results show that this novel pyranosic sulfated arabinan Ab1 exerts its anticoagulant activity by a mechanism different from those found previously for other sulfated polysaccharides and glycosaminoglycans.

Plasma homocysteine cutoff values for venous thrombosis

Abstract Background: Hyperhomocysteinemia is considered an independent risk factor for vascular occlusive diseases. To date, there is no general agreement on hyperhomocysteinemia cutoff values. Methods: To establish a homocysteine cutoff value, we performed a case-control study in 118 patients suffering from venous thrombosis and in 115 healthy subjects. We calculated odds ratios at different cutoff points and considered hyperhomocysteinemia as homocysteine levels above which the risk of venous thrombosis was increased. Results: Initially we used the 97.5th percentiles for fasting homocysteine levels in the control group to calculate odds ratios (95% CI) of 9.5 (2.6–35.3), 3.7 (0.8–17.9) and 4.5 (1.7–123.8) for the total population, women and men, respectively. When individuals with well-known thrombotic risk factors were excluded (selected population), odds ratios were 10.5 (2.7– 41.1), 6.5 (1.3–32.1) and 11.2 (1.2–103.1), respectively, confirming hyperhomocysteinemia as an independent risk factor for venous thrombosis. We did not find any association of venous thrombosis with the homozygous methylenetetrahydrofolate reductase C677T mutation. When the hyperhomocysteinemia cutoff was set at other arbitrary points, odds ratios for the selected population were statistically significant only at >12 μmol/L. Conclusions: Based on our results, we propose 12 μmol/L as the hyperhomocysteinemia cutoff value. Clin Chem Lab Med 2007;45:232–6.

Prevalence of hyperhomocysteinemia in an elderly population

BACKGROUND Currently, total hyperhomocysteinemia (tHHcy) is a well-known condition linked to a higher risk of vascular disease. Prevalence of HHcy increases in elderly persons as the risk associated with it persists. Because factors can be potentially reduced in the elderly, it is important to carry out epidemiologic studies of HHcy. PROCEDURE Previously we described the prevalence of hypertension control in an elder population; now, in an observational cross-sectional simple blind study, total homocysteine (tHcy) concentration was determined in 196 of 400 patients from the original cohort. RESULTS Mean Hcy concentration was 13.2 ,amol/L (95% confidence interval 12.4-14.0; range, 5.0 to 48.9); 15.0 ,imol/L for men and 12.3 pAmol/L for women. Mean serum folic acid levels were 4.9 + 3.1 ng/mL (range, 2.0 to 20.0 ng/mL), and vitamin B12 levels were 384.8 314.1 pg/mL (range, 48.0 to 1500.0 pg/mL). Taking into account the reference values established by the Third National Health and Nutrition Examination Survey III study, HHcy was detected in 69.8% of all the subjects evaluated. The study showed that 76.2% of the men and 66.4% of the women had high Hcy levels. CONCLUSIONS The very high prevalence of tHHcy in the elderly population, and the consequent risks associated with it suggest that although there are no trials that effectively prove the benefit of tHcy decrease, nutritional intervention is still justified.

Heparin cofactor II in diabetic patients.

Levels of heparin cofactor II (HCII) activity and antigen and electrophoretic pattern were studied both in normal subjects and in type I diabetic patients with high and normal levels of glycosylated haemoglobin. There was a significant reduction in HCII activity (83 ± 7%) in patients with high levels of glycosylated haemoglobin compared with controls (95 ± 17%; P < 0.001). However, plasma HCII antigen levels were not decreased in these patients.

Unsuspected hyperhomocysteinemia in chronically anticoagulated patients.

Hyperhomocysteinemia is a risk factor for arterial and venous thrombosis. The aim of this study was to evaluate plasmatic homocysteine levels in patients under chronic anticoagulant treatment with dietary restriction of green vegetables. This kind of food is a very important source not only of vitamin K but also of folates, which are involved in Hcy metabolism. It is known that the lower the folate levels, the higher the Hcy concentration, so we suspected that these patients could show hyperhomocysteinemia. A group of patients receiving oral anticoagulant treatment and a restricted diet (Group I, n = 20) was compared with a group of untreated subjects of a similar age that were not on a restricted diet (Group II, n = 35). Group I showed significantly higher levels of plasmatic Hcy and significantly lower levels of serum folate than Group II. Therefore, a diet restricted in vitamin K applied to oral anticoagulated patients could induce an unwanted increase of homocysteine levels.

Plasmatic homocysteine response to vitamin supplementation in elderly people

Homocysteine (Hcy) increase is now widely accepted as a risk factor for vascular disease. The effects of folic acid (FA) and vitamins B12 and B6 in lowering Hcy have been extensively studied, but there is still little data on the response to FA dietary administration. Our purpose was to evaluate the impact of the diet and the degree of response to different doses of pharmacological FA supplementation. In a prospective, randomized, and simple blind study, 50 elderly subjects were given a 400-microg/day FA diet and were randomly assigned to one of the following treatments: Group I = placebo tablet; Group II = tablet containing 1-mg folic acid, 1-mg B12, and 25-mg B6; and Group III = tablet containing 2.5-mg folic acid and same B6 and B12 doses as Group II. Forty-four subjects completed the study, and their plasmas were evaluated. Hcy concentration significantly decreased even in patients with normal basal values, and there were no differences in the response between individuals receiving diet plus placebo and those receiving diet plus pharmacological supplementation. After the treatment, the mean decrease of plasmatic Hcy levels was 10.8 (9.4, 12.5) micromol/l, geometric mean [95% confidence interval (95% CI)], and particularly, the values for Group I were 10.6 (7.4, 14.8) micromol/l. In 31% of the subjects, the post-treatment Hcy levels were less than or = 5 micromol/l. These results show that a special diet, with or without pharmacological FA and B12 and B6 supplementation, significantly decreases the Hcy levels in elderly people. Therefore, a diet with high contents of FA might have an enormous impact on the morbidity and mortality of atherothrombosis.

Novel sulfated xylogalactoarabinans from green seaweed Cladophora falklandica: Chemical structure and action on the fibrin network

The water-soluble sulfated xylogalactoarabinans from green seaweed Cladophora falklandica are constituted by a backbone of 4-linked β-l-arabinopyranose units partially sulfated mainly on C3 and also on C2. Besides, partial glycosylation mostly on C2 with single stubs of β-d-xylopyranose, or single stubs of β-d-galactofuranose or short chains comprising (1→5)- and/or (1→6)-linkages, was also found. These compounds showed anticoagulant activity, although much lower than that of heparin. The effect of a purified fraction (F1) on the fibrin network was studied in detail. It modifies the kinetics of fibrin formation, suggesting an impaired polymerization process. Scanning electron microscopy showed a laxer conformation, with larger interstitial pores than the control. Accordingly, this network was lysed more easily. These fibrin properties would reduce the time of permanence of the clot in the blood vessel, inducing a lesser thrombogenic state. One of the possible mechanisms of its anticoagulant effect is direct thrombin inhibition.

Alterations of fibrin networks mediated by sulfated polysaccharides from green seaweeds

Fil: Arata, Paula Ximena. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Biologia Aplicada y Alimentos. Catedra de Quimica de Biomoleculas; Argentina