Isa Francalanci

D-DIMER CONCENTRATIONS DURING NORMAL PREGNANCY, AS MEASURED BY ELISA

In pregnant women a number of changes in blood clotting and fibrinolysis proteins have been reported so indicating the existence of a state of hypercoagulability. In addition to fibrinogen and antithrombin III (AT), D-dimer is frequently checked during pregnancy, in particular during at risk pregnancy, but the exact pattern of D-dimer modifications during uncomplicated pregnancy is not definitively described. The aim of this study was to establish the range values in three different periods of uncomplicated pregnancy (A: 1-20 wks; B: 21-30 wks; C: 31-40 wks). We measured plasma levels of D-dimer, clottable fibrinogen and AT in 108 consecutive normal pregnant women aged 16 to 42 years. In period A, the range of D-dimer values was 43-211 ng/mL, not different from controls, while fibrinogen levels were significantly higher (p < 0.05) than in matched non pregnant women. Mean D-dimer levels were higher in periods B (p < 0.05) and C (p < 0.05) vs period A. Similarly, mean fibrinogen levels were found more elevated in periods B and C vs period A (p < 0.05). A significant correlation was found between fibrinogen and D-dimer levels (p < 0.001). No differences in AT levels were found among the three periods of pregnancy. The results of this study indicate that levels of D-dimer up to 685 micrograms/L may be reached at the end of physiological pregnancy. This fact should be taken into account in the evaluation of hemostatic studies performed in uncomplicated and complicated pregnant women.

Effect of Medium-term Supplementation with a Moderate Dose of n-3 Polyunsaturated Fatty Acids on Blood Pressure in Mild Hypertensive Patients

Several studies have shown that n-3 polyunsaturated fatty acids (n-3 PUFA) are able to lower blood pressure (BP) in humans, but large doses of fish oils have been often used. Moreover, most of the studies available in the literature were not able to evaluate the specific effects of n-3 PUFA because they employed fish oils which contain, together with n-3 PUFA, many other different components. The aim of this preliminary study was to evaluate if medium-term supplementation with a moderate dose of highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl esters is able to reduce BP in mild hypertensive patients. Sixteen mild essential hypertensive (diastolic BP: 95-104 mm Hg), non-diabetic, normolipidemic male outpatients and 16 normotensive male controls were recruited to participate in the study. Both hypertensive and control subjects were randomly assigned to receive either EPA and DHA ethyl esters (2.04 g EPA and 1.4 g DHA) as active treatment or olive oil (4 g/day) as a placebo for a period of 4 months. These subjects were followed up with 24-hour ambulatory BP monitoring and blood chemistry analyses at 2 and 4 months of treatment and 2 months after its discontinuation. The intake of n-3 PUFA was checked by red blood cell (RBC) phosphatidylcholine (PC) fatty acid composition. The effect of n-3 PUFA on BP in the active group was maximum after 2 months. Both systolic (-6 mm Hg, p<0.05) and diastolic (-5 mm Hg, p<0.05) BP significantly decreased during the n-3 PUFA ethyl ester supplementation. No further effect was observed at 4 months with a return to baseline values during the recovery period. These data indicate that 4 g/day of highly purified EPA + DHA ethyl esters are able to favorably affect BP in mild hypertensives.

n-3 PUFA supplementation, monocyte PCA expression and interleukin-6 production

n-3 polyunsaturated fatty acids (PUFA) can affect several monocyte functions and the biochemistry of blood cells, thus possibly influencing the initiation of thrombosis, inflammatory disease and atherosclerosis. In this study, we have investigated the effect of dietary supplementation with n-3 PUFA ethyl esters on procoagulant activity (PCA) and interleukin-6 (IL-6) production by human mononuclear cells. Nine healthy volunteers received 4 g/d of n-3 PUFA ethyl esters (4 x 1 g capsules with at least 85% eicosapentaenoic + docosahexaenoic acid ethyl esters) for 18 weeks. Before and at the end of the treatment, mononuclear cells were obtained from peripheral citrated blood by Ficoll-Hypaque density gradient centrifugation. Cellular suspensions (10(7) cells/ml) were incubated at 37 degrees C for 4 h in the absence and presence of lipopolysaccharide (10 micrograms/ml); PCA was determined by one-stage clotting assay and IL-6 concentrations were assayed in supernatants by specific ELISA. After 18-week treatment, both unstimulated and stimulated monocyte PCA were significantly reduced by 66% and 63%, respectively (P < 0.01). Similarly, a significant inhibitory effect by n-3 PUFA treatment on basal and LPS-stimulated IL-6 monocyte production was observed (50% and 46%, respectively, P < 0.05). These data indicate that 18-week n-3 PUFA supplementation may influence monocyte activities, which play a specific role in atherosclerosis and its thrombotic complications.

D-Dimer plasma levels during normal pregnancy measured by specific ELISA

A progressive increase in D-dimer plasma levels together with an increase in fibrinogen has been previously reported during normal pregnancy. However, significantly different D-dimer levels have been observed in different assays, due to different specificity of the antibodies employed. The aim of this study was to verify the increase in fibrin degradation product levels during normal pregnancy, using a recently introduced specific D-dimer ELISA. We determined D-dimer (ELISA) and fibrinogen (clotting method) plasma levels in 63 normal pregnant women, during three different periods of pregnancy (A, 7–20 weeks; B, 21–30 weeks; C, > 30 weeks). During period A, D-dimer plasma levels (range 2–103 ng/ml) showed an insignificant increase compared with a control group of non-pregnant women (range 2–73 ng/ml). During periods B and C, we observed an increase in D-dimer level (P<0.0001) compared with period A, with a significant correlation between D-dimer levels and gestational age (P<0.0001). Period A fibrinogen levels (range 3.24–6.43 g/l) were significantly higher (P<0.0001) than in controls (range 2.31–4.71 g/l), with a further increase in periods B and C. In conclusion, we confirmed a progressive increase in plasma concentrations of fibrin degradation product during normal pregnancy, but D-dimer levels were significantly lower than those reported in the literature for other ELISAs.

Effect of n-3 fatty acid ethyl ester supplementation on fatty acid composition of the single platelet phospholipids and on platelet functions

Twenty healthy male volunteers were randomly assigned to receive either four 1-g capsules of n-3 polyunsaturated fatty acids (PUFA) ethyl esters or four 1-g capsules of olive oil (as placebo) for a period of 4 months, followed by a 3-month wash-out period. Fatty acids of platelet phospholipid fractions, platelet aggregation, and thromboxane B2 (TXB2) formation were analyzed at 0, 2, and 4 months of treatment and at 1, 2, and 3 months of wash-out. During n-3 PUFA supplementation, accumulations of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic (DHA) acids were markedly increased after 2 months, with slight differences in further accumulation up to 4 months among the various phospholipid fractions. Significant decreases in platelet sensitivity to collagen, serum TXB2 levels, and urinary TXB2 metabolites were also observed following n-3 PUFA treatment. During the first and second month of wash-out, slight differences were observed in changes of various fatty acids among different phospholipid fractions, but after 3 months of wash-out, alterations were no longer detectable with respect to pretreatment values. After 3 months of wash-out, platelet function parameters also were returned to baseline. Thus, both platelet lipids and function are influenced by n-3 PUFA ethyl ester supplementation, and significant alterations are still detectable after 2 months of wash-out.

Platelet activation and platelet lipid composition in pulmonary cancer

In order to investigate the possible mechanisms underlying platelet functional changes in patients affected by neoplasms, platelet lipid composition, plasma beta-thromboglobulin (Beta-TG) and serum thromboxane B2 (TXB2) were investigated in 16 male patients affected by pulmonary carcinoma and in 16 comparable control subjects. In patients high levels of plasma Beta-TG (67 +/- 9 versus controls 14 +/- 4 ng/ml, p < 0.001) and serum TXB2 (434 +/- 56 versus 223 +/- 48 ng/ml, p < 0.001) were observed. Also platelet lipid composition was found altered in patients with respect to controls (lower percent levels in n-3 fatty acids and in linoleic acid esterified in the main platelet phospholipid fractions: at least p < 0.05). These results indicate that in vivo platelet activation is detectable in neoplastic patients and it is associated with alterations in platelet lipid composition. In the light of the important role played by membrane lipids in platelet functions related to thrombosis and haemostasis we conclude that platelet lipid changes could cooperate in platelet activation and increased thrombotic risk so frequently observed in neoplastic disease.

Physical exercise and hemostasis

SummaryA number of hemostatic changes involving platelets, coagulation and fibrinolysis have been reported after acute physical exercise. Results have sometimes been controversial, due to differences in subjects investigated, type of exercise and methods used for hemostatic evaluation. On the whole, physical exercise has been shown to induce: (1) increases in platelet number and activity, (2) activation of coagulation leading to a slight but significant thrombin generation and (3) activation of fibrinolysis. These changes are short lasting. Less known are hemostatic changes induced by exercise training programs: a few data are available on the effects on platelets and coagulation, whereas studies performed on fibrinolysis show a decrease in plasminogen activator inhibitor-1 levels at rest and an increase in fibrinolytic capacity after training.

No changes in PAI-1 levels after four-month n-3 PUFA ethyl ester supplementation in healthy subjects

Recent studies have indicated that diets rich in fish or supplemented with fish oils may increase PAI-1 plasma levels. However, this finding has not been consistent and could be related, at least in part, to the type of supplementation. Aim of this study was to investigate the effects of medium-term treatment with n-3 polyunsaturated fatty acid (PUFA) ethyl esters on fibrinolysis. Twenty normolipemic healthy male subjects (age 27 to 41 yrs) were randomly assigned to receive either 4 x 1 g capsules of n-3 PUFA ethyl esters (ESAPENT, Farmitalia-Carlo Erba, Milan, Italy) or 4 x 1 g capsules of olive oil (as placebo) for 4 months in a double blind study. Blood samples for lipid and hemostatic studies were obtained at 0, 2, and 4 months of treatment and 1, 2 and 3 months of wash-out. Plasma lipids, fibrinolytic system, lipoprotein (a)-Lp(a)-, fibrinogen (Fbg) and prothrombin activation fragment 1+2 (F1+2) were assayed. No changes in these parameters were observed in the group of ten subjects treated with olive oil. After n-3 PUFA supplementation no significant alterations were found in plasma lipids, even if a trend to lower triglyceride and Lp(a) levels was detectable. No changes in either PAI-1 activity or PAI-1 antigen levels or F1+2 plasma levels were observed. A trend to lower Fbg levels was found after n-3 PUFA, but changes were not statistically significant. The results of this study indicate that a 4-month treatment with 4 g daily n-3 PUFA ethyl esters does not affect PAI-1 plasma levels.

D-Dimer in intra-uterine growth retardation and gestational hypertension

Pregnancy is sometime related to thromboembolic complications (1) and alterations in different hemostatic parameters have been reported in pregnancy (2-4). In particular, a progressive increase in fibrinogen and D-dimer levels occurs during normal pregnancy (5-9). D-dimer levels may be predictive for some complications such as preeclampsia (10) and they have been also reported to be useful for diagnosis of abruptio placentae (6). However, it remains to be established if common ELISA for D-dimer are able to discriminate pathologic samples in conditions such as intrauterine growth retardation (IUGR) or gestational hypertension (GH). Aim of the present study has been to evaluate the behavior of D-dimer in pregnant women with IUGR and GH.

Relationship between plasma and platelet phospholipid fatty acid composition in healthy subjects

In order to furtherly clarify the mechanisms regulating the fatty acid composition of platelet phospholipids the relationships between the fatty acid composition of major phospholipid fractions from plasma and platelets were investigated in 30 healthy male subjects. Strict correlations between all but two plasma and platelet fatty acids were observed for phosphatidylcholine (PC) (r = 0.51-0.95, at least P < 0.01), whereas only poor correlations were found for the other fractions. These results suggest that the direct transfer of PC molecules from plasma to platelet membrane is a pivotal mechanism for renovation of platelet PC fatty acids, while other mechanisms appear to play a major role for renovation of other phospholipid fractions of the platelet membrane.