Long-chain n-3 fatty acids specifically affect rat coagulation factors dependent on vitamin K: relation to peroxidative stress.

Fatty acids of marine origin have been shown to affect blood coagulation in the rat. In an attempt to gain insight into the mechanisms of this phenomenon, we studied the effects of dietary linseed and fish oils on the liver antioxidant status and plasma coagulation parameters in rats on a time-course basis. Dietary enrichment in eicosapentaenoic and docosahexaenoic acids resulted in strong hypocoagulation after only 1 week and a concomitant increase in liver lipid peroxidation and tocopherolquinone content. Enrichment in linolenic acid induced similar increases in lipid peroxidation and tocopherol catabolism but negligible alteration of coagulation. A significant correlation between plasma factor II coagulant activity and liver tocopherolquinone was found in fish oil- but not in linseed oil-fed rats. Although ingestion of tocopherolquinone led to high levels of this compound in the liver, it had only marginal effects on coagulation factors. Thus, it seems unlikely that this vitamin E metabolite could be involved in the lowering of vitamin K-dependent clotting factors through inhibition of gamma-glutamylcarboxylase. Rather, our results indicate that the effects of the n-3 fatty acids of fish oil on vitamin K-dependent coagulation factors are specific and independent of liver tocopherolquinone levels.

Lipid composition of erythrocytes and thrombocytes of a subantarctic seabird, the king penguin.

Phospholipid (PL) compositions and fatty acid (FA) patterns of PL were determined in the erythrocytes and blood thrombocytes of a seabird, the king penguin, living in the subantarctic area and feeding on prey rich in n-3 polyunsaturated FA. Results were compared between birds in three different physiological states (breeding and molting adults, chicks) to those reported for other birds. In erythrocytes, the ratios of cholesterol to PL and of sphingomyelin to phosphatidylcholine (PC) were lower than in other birds. The PL distribution was similar to those previously reported in the hen and pigeon. In contrast to other birds, cardiolipin levels were unexpectedly high (4%). Very long chain n-3 FA were abundant (13-27%) in phosphatidylethanolamine (PE), phosphatidylserine and PC, probably in relation to the natural diet of these birds. Among n-3 FA, 22:6n-3 was the most abundant in all PL (2-20%), whereas the highest levels of arachidonic acid were observed in PE (14%). In thrombocytes, the PL distribution and FA composition of the main PL (PC, PE) differed from those of erythrocytes, and in particular, levels of n-3 FA (9-12%) were 1.5-2 times lower. The highest levels of arachidonic acid were found in phosphatidylinositol (24%). The lipid profile of penguin erythrocytes could contribute to the efficiency of blood circulation and oxygen delivery in microvascular beds, thus favoring diving capacity of these animals. Our observations do not support the hypothesis of a common origin of avian thrombocytes and erythrocytes.

Cardiovascular morbidity and endothelial dysfunction in chronic haemodialysis patients: is homocyst(e)ine the missing link?

BACKGROUND: Haemodialysis patients exhibit an excessive burden of atherothrombotic disease, which is not explained adequately by traditional risk factors. Hyperhomocyst(e)inaemia, a consistent finding in uraemic patients, is now widely recognized as an independent risk factor for vascular disease. The aim of this study was to examine the hypothesis that hyperhomocyst(e)inaemia is associated with cardiovascular complications in dialysed patients. METHODS: In a cohort of 63 stable chronic haemodialysis patients, we examined the causal relationship between hyperhomocyst(e)inaemia and vascular endothelial and haemostatic function. All their markers were determined before and after an 8-week course of a 10 mg per day oral folate supplementation, a manoeuvre known to decrease hyperhomocyst(e)inaemia in uraemic patients. RESULTS: History of at least one cardiovascular atherothrombotic event was present in 47.6% of the haemodialysed patients, and radiographic evidence of vascular calcifications in 70%. Hyperhomocyst(e)inaemia was found in all patients, averaging 3.5-fold the upper limit of normal values (P<0.001), despite the lack of clinical and biological evidence of malnutrition. Fibrinogen, von Willebrand factor and plasminogen activator inhibitor type 1, but not endothelin 1, were significantly higher in haemodialysis patients than in controls. After adjustment for all variables, past history of cardiovascular events was independently associated with higher levels of homocyst(e)inaemia only (odds ratio (OR) 1.06; 95% confidence interval (CI) 1.01-1.12; P<0.026). The presence of aortic calcifications was independently and significantly associated with age (OR 1.37; 95% CI 1.07-1.75; P<0.025), homocyst(e)inaemia (OR 1.14; 95% CI 1.02-1.27; P<0.05) and fibrinogen concentration only (OR 9.74; 95% CI 1.25-75.2; P<0.05). None of the endothelial haemostatic factors was, however, related to homocyst(e)ine levels. Mid-term folate supplementation decreased plasma homocyst(e)ine levels significantly without achieving normal values. No significant change of endothelial-haemostatic markers was observed, however, despite the drop in plasma homocyst(e)ine. CONCLUSIONS: Hyperhomocyst(e)inaemia is associated with increased cardiovascular risk in haemodialysis patients. Folate supplementation was partially effective in lowering hyperhomocyst(e)inaemia, but its usefulness in terms of reduction in cardiovascular morbidity and mortality remains to be determined in prospective trials.

Antithrombotic effects of (n-3) polyunsaturated fatty acids in rat models of arterial and venous thrombosis.

The antithrombotic effects of dietary lipids were investigated in rat models of arterial and venous thrombosis. In the arterial model, thrombus formation was evaluated by determination of the occlusion time and the deposition of 111In-labeled platelets and 125I-labeled fibrinogen in a collagen-coated glass capillary inserted into an arterio-arterial shunt. Venous thrombosis was evaluated by measurement of the thrombus weight after administration of thromboplastin as a source of tissue factor and establishment of stasis in the vena cava. Diets were supplemented with saturated (SAT group) or (n-3) fatty acids, the latter being added either as MaxEPA oil (MaxEPA group), or as docosahexaenoic (DHA group) or eicosapentaenoic (EPA group) ethyl ester. Only the MaxEPA group displayed a prolonged occlusion time as compared with all other groups. Platelet accumulation, similar in the MaxEPA, EPA and DHA groups (13.3, 16.7 and 17.7 x 10(6) platelets/shunt, respectively), was significantly higher in the SAT group (25.3 x 10(6) platelets/shunt), while accumulation of fibrinogen-fibrin was similar whatever the group. There was a trend towards a lower venous thrombus weight in MaxEPA fed rats relative to those fed other diets. Our data indicate that the MaxEPA diet had antithrombotic effects in arterial and to a lesser extent venous thrombosis models, best attributed to its multiple targeting of platelets and coagulation.

Diets enriched in (n-3) fatty acids affect rat coagulation factors dependent on vitamin K.

The effects of dietary lipids on haemostasis were investigated in rats fed high fat diets enriched in saturated fatty acids (SAT), oleic acid (OLEIC), MaxEPA oil (MaxEPA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) and results were compared to those for rats fed standard chow (ST). Coagulant activities of factor IIc and factor VII-Xc were reduced by about 70% in the MaxEPA group and 50% in the EPA and DHA groups relative to the OLEIC, SAT and ST groups. Liver vitamin K levels were five times lower in the experimental groups than in the ST group, which would indicate an effect of high fat diets on vitamin K metabolism. However, only (n-3) fatty acids prolonged the prothrombin time. These components could act at the post-translational modification level of vitamin K-dependent plasma clotting factors. The changes in haemostatic factors found in the MaxEPA group were counteracted by vitamin K supplementation.

Simultaneous determination of homologues of vitamin E and coenzyme Q and products of alpha-tocopherol oxidation.

A sensitive procedure is described for the simultaneous determination of vitamin E and coenzyme Q homologues and alpha-tocopherol oxidation products using two-isocratic step high pressure liquid chromatography (HPLC) and electrochemical detection in the oxidative mode. Zinc-catalyzed reduction in a post-column reactor allows the detection of alpha-tocopherolquinone, epoxy-tocopherolquinone, and ubiquinones. This technique was used to quantify lipophilic antioxidants in the liver tissue of rats treated or not with alpha-tocopherolquinone and in a plant oil. Alpha-tocopherolquinone and its epoxide derivatives, formed from alpha-tocopherol during iron-catalyzed phospholipid peroxidation, were also determined in a liposome suspension. The high selectivity and sensitivity of the coulometric detection system enabled use of low oxidation potentials giving little baseline noise, while a fast isolation procedure and quantitative recoveries of all oxidized and reduced forms made it possible to measure a high ubiquinol/ubiquinone ratio in liver tissue. Administration of alpha-tocopherolquinone to rats did not alter the antioxidant status of the liver, despite strong accumulation of both this quinone and its reduced form, alpha-tocopherolhydroquinone. These results indicate the presence of an efficient reductase and suggest that it could contribute to the protection of cellular membranes from oxidative stress.

Epoetin alfa facilitates presurgical autologous blood donation in non-anaemic patients scheduled for orthopaedic or cardiovascular surgery.

Patients expected to need at least three units of blood for their elective cardiovascular or orthopaedic surgery, were allocated randomly to receive intravenous (i.v.) Epoetin alfa 600 IU kg-1 (n = 27), 300 IU kg-1 (n = 30) or placebo (n = 23), on days 1, 4 and 7. Provided haemoglobin > or = 11 g dL-1, one unit of blood was collected on days 1, 4, 7, 11 and 14. Iron supplementation was given throughout the study. Surgery was scheduled between days 18 and 21. Significantly more patients treated with Epoetin alfa (100% for 600 IU kg-1; 97% for 300 IU kg-1) were able to donate > or = 4 units of blood compared with placebo (78%) (P = 0.011 and P = 0.032). No significant differences were seen in total patient exposure to homologous blood (7.4%, 3.3% and 17.4%, respectively). Mean red cell volume donated (P = 0.005 for 600 IU kg-1; P = 0.158 for 300 IU kg-1 both vs. placebo) and production (P < 0.001 and P = 0.012, respectively) were dose related. Twenty-four patients became iron deficient. No differences in the incidence of adverse events were seen between the groups.

Diets rich in saturated n-9 and n-3 fatty acids differentially affect the fatty acid composition of phospholipids and function of rat platelets.

The aim of the present study was to determine whether dietary intake of monounsaturated or long chain n-3 fatty acids could be effective in lowering platelet responsiveness through modulation of platelet phospholipid composition. Rats were fed diets containing 20% fat with equal cholesterol and 13a-tocopherol contents. These diets were supplemented with saturated, oleic or n-3 fatty acids, n-3 polyunsaturated fatty acids being added either pure, as eicosapentaenoic and docosahexaenoic ethyl esters, or as MaxEPA oil. Dietary n-3 fatty acids did not affect the oxidation status of plasma lipids. Oleic acid- and saturated fatty acid-rich diets led to similar enrichment of platelet phospholipids in arachidonic acid and to comparable thromboxane A(2) generation on stimulation with collagen or thrombin. Platelets of n-3-fed groups were differently enriched in eicosapentaenoic and docosahexaenoic acids at the expense of arachidonic acid. These groups displayed similar thromboxane A(2) production, although levels were lower than those for groups fed with oleic- or saturated fatty acid-rich diets. Only the MaxEPA diet led to a reduction in platelet reactivity, measurable as a small decrease in the aggregation induced by collagen. This diet was also responsible for a high cholesteroUphospholipid ratio and low a-tocopherol content in platelets. Overall results indicated that (i) only MaxEPA reduced platelet reactivity and (ii) this effect was moderate and apparently unrelated to platelet arachidonic acid content, membrane cholesterol to phospholipid ratio or thromboxane A(2) production.

Experimental thrombosis on a collagen coated arterioarterial shunt in rats: a pharmacological model to study antithrombotic agents inhibiting thrombin formation and platelet deposition.

A rat thrombosis model was developed to assess the efficacity of antithrombotic drugs. It had the following characteristics: controlled hemodynamic and rheological conditions corresponding to arterial flow, a collagen coated surface as a relevant thrombogenic stimulus, a method of measurement allowing dynamic monitoring of thrombus formation and the possibility to assess the thrombus structure. A shunt composed of polyethylene and silicone catheters, including in the middle of the shunt a collagen coated glass capillary, was inserted between the two primitive carotids of the rat. The duration of patency of the shunt was recorded using a thermic probe fixed on its central part. In this model, the patency of the shunt was 539 +/- 55 s. Platelet and fibrinogen-fibrin accumulation in successive one centimeter segments along the shunt were measured using 111In labeled platelets and 125I labeled fibrinogen. Platelet accumulation occurred on the collagen coated surface and at the junctions between the different components of the shunt, where flow was disturbed. The effects of four antithrombotic agents were measured: aspirin, clopidogrel, heparin and r-hirudin. Clopidogrel, heparin and hirudin significantly prolonged patency duration of the shunt, whereas aspirin was inactive. Aspirin did not reduce platelet or fibrinogen-fibrin accumulation on the collagen coated surface. Platelet accumulation on the collagen surface was significantly lower in the clopidogrel group (50 mg/kg) than in the group treated with heparin (500 U/kg), demonstrating the direct antiplatelet effect of clopidogrel. Hirudin at doses giving similar values of APTT as heparin (500 U/kg) prolonged the occlusion time to over 2 h while the heparin occlusion time was only 20 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Competitive adsorption of human immunoglobulin G and albumin: consequences for structure and reactivity of the adsorbed layer.

The affinity of polyclonal anti-IgG for human IgG adsorbed on silica surfaces was investigated by two complementary techniques, scanning angle reflectometry and 125I radiotracing. Special attention was paid to compare the reactivity of IgG adsorbed directly or by exchange with already adsorbed albumin. In particular it was shown that (i) in the first case (direct adsorption) the reaction between anti-IgG and adsorbed IgG was in the ratio 1:1 and (ii) in the second case (adsorption by exchange) there was no reaction.

Old and new natural products as the source of modern antithrombotic drugs.

Natural compounds have been the first historical source of antithrombotic compounds (heparin, vitamin K antagonists, streptokinase, urokinase); molecules extracted from plants or animals still provide some of the most original and promising approaches for the discovery of new drugs in this class. In this review, we will briefly describe three examples of current research trends that could lead to the development of new antithrombotic drugs of natural origin. Flavonoids have been shown to be inhibitors of cyclic nucleotide phosphodiesterase; this enzymatic activity is one of the main mechanisms of inhibition of aggregation of blood platelets by flavonoids. Some of these compounds could represent templates for the development of new inhibitors of platelet activation. Garlic (Allium sativum) has been shown to inhibit platelet aggregation in vivo and in vitro; a number of active principles has now been identified and their mechanisms of action are currently being explored. An ancient remedy, the medicinal leech (Hirudo medicinalis), has been found to contain several potent anticoagulant proteins. Among them, hirudin, a polypeptide of 65 amino acids, has been identified as one of the most potent inhibitors of thrombin. The production of sufficient amounts of hirudin through molecular biology techniques has now allowed the performance of clinical trials. These three examples show that careful consideration of biochemical, ethnopharmacological, or toxicological properties of natural products can still constitute a valuable basis for the development of new drugs.

Old and new natural products as the source of modern antithrombotic drugs.

Natural compounds have been the first historical source of antithrombotic compounds (heparin, vitamin K antagonists, streptokinase, urokinase); molecules extracted from plants or animals still provide some of the most original and promising approaches for the discovery of new drugs in this class. In this review, we will briefly describe three examples of current research trends that could lead to the development of new antithrombotic drugs of natural origin. Flavonoids have been shown to be inhibitors of cyclic nucleotide phosphodiesterase; this enzymatic activity is one of the main mechanisms of inhibition of aggregation of blood platelets by flavonoids. Some of these compounds could represent templates for the development of new inhibitors of platelet activation. Garlic ( ALLIUM SATIVUM) has been shown to inhibit platelet aggregation IN VIVO and IN VITRO; a number of active principles has now been identified and their mechanisms of action are currently being explored. An ancient remedy, the medicinal leech ( HIRUDO MEDICINALIS), has been found to contain several potent anticoagulant proteins. Among them, hirudin, a polypeptide of 65 amino acids, has been identified as one of the most potent inhibitors of thrombin. The production of sufficient amounts of hirudin through molecular biology techniques has now allowed the performance of clinical trials. These three examples show that careful consideration of biochemical, ethnopharmacological, or toxicological properties of natural products can still constitute a valuable basis for the development of new drugs.

ADP plays a key role in thrombogenesis in rats.

The relative importance of ADP, arachidonic acid metabolites and serotonin as thrombogenic factors was evaluated in rats by comparing, after oral administration, the effects of two inhibitors of ADP-induced platelet aggregation (ticlopidine and PCR 4099), three cyclo-oxygenase inhibitors (aspirin, triflusal and indobufen) and a selective serotonin 5HT2 receptor antagonist (ketanserin) on platelet aggregation, in four platelet-dependent thrombosis models and on bleeding time. Platelet aggregation induced by ADP and collagen was completely inhibited by ticlopidine and PCR 4099 whereas only the collagen aggregation was reduced by the cyclo-oxygenase inhibitors. Ketanserin or a depletion of platelet serotonin by reserpine did not affect platelet aggregation. Ticlopidine and PCR 4099 greatly prolonged rat tail transection bleeding time. This is probably related to their known ability to inhibit ADP-mediated platelet aggregation. In contrast, the cyclooxygenase inhibitors did not affect bleeding time at all. Reserpine and ketanserin prolonged bleeding time by interfering with the action of serotonin on the vascular wall. Ticlopidine and PCR 4099 were very potent antithrombotics in all the models. Aspirin, only at a high dose, inhibited poorly thrombus formation on a silk thread in an arterio-venous shunt, suggesting that the inhibition of cyclo-oxygenase was not responsible. Triflusal was inactive in all models while indobufen slightly reduced thrombus formation in the silk thread and metallic coil models. Ketanserin and reserpine reduced thrombus only in the metallic coil model. Thrombus formation was greatly reduced in fawn-hooded rats, which lack ADP in their platelet dense granules because of a genetic storage pool deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyindolinic Alkaloids from Psychotria forsteriana. Potent Inhibitors of the Aggregation of Human Platelets.

Quadrigemine B, quadrigemine A, isopsychotridine C and psychotridine, isolated from PSYCHOTRIA FORSTERIANA, are potent inhibitors of the aggregation of washed human platelets induced by ADP, collagen or thrombin. The four compounds are active in the 1-10 microM range. The quadrigemine-type alkaloids do not increase the level of platelet cyclic AMP, either alone or in the presence of 20 nM prostaglandin E, (PGE (1)), an activator of adenylate cyclase. The characteristics of the pharmacological action of these compounds suggest that they act at a later stage in platelet activation, possibly through an interaction with cytoskeletal proteins.