Prostacyclin stimulation of the activation of blood coagulation factor X by platelets.

When platelets were incubated with prostacyclin, prostaglandin E1, or prostaglandin D2 at concentrations insufficient to increase the level of adenosine 3',5'-monophosphate (cyclic AMP), coagulation factor X was activated by a platelet cysteine protease. Prostacyclin or prostaglandin E1 at higher concentrations increased the cyclic AMP level and inhibited the activation of factor X by platelets. Inhibition of platelet adenylate cyclase by 2',5'-dideoxyadenosine allowed the activation of the protease at higher concentrations of the autocoids. Prostaglandins A1, A2, B1, B2, E2, F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, which do not affect platelet cyclic AMP level, did not stimulate the protease.

Binding of prostaglandin E1 to human erythrocyte membrane.

Prostaglandin E1 is known to alter the structural and functional characteristics of red blood cells, yet, little is understood about the membrane receptors mediating this process. We therefore studied the binding of tritium-labeled prostaglandin E1 to the intact human erythrocyte membrane and demonstrated that the interaction is highly specific, rapid, saturable and reversible. Scatchard analysis of prostaglandin E1 binding to the membrane preparations showed the presence of two independent classes of prostaglandin E1 binding sites which differed in their affinity for the autacoid. The high-affinity class had Kd = 3.6 X 10(-9) M and the low-affinity class had Kd = 5.6 X 10(-5) M. The optimum pH for the binding of [3H]prostaglandin E1 to the erythrocyte membrane was found to be around 7.5 and maximum specific binding occurred at a concentration of 5 mM Mg2+ in the incubation mixture. [3H]Prostaglandin E1 bound to the membrane preparation could not be displaced by GTP or by its stable derivative Gpp[NH]p. However, prostaglandin E1 bound to the erythrocyte membrane preparation could be rapidly displaced by cyclic AMP. The IC50 (concentration of the nucleotide displacing 50% bound [3H]prostaglandin E1 from the membrane) was 75 nM. Other adenine nucleotides or cyclic GMP could not substitute for cyclic AMP. Unlike the right-side-out erythrocyte membrane, the inside-out membrane preparations do not bind [3H]prostaglandin E1. Treatment of right-side-out erythrocyte membrane preparation with neuraminidase markedly decreases the binding of prostaglandin E1. Incubation of the erythrocyte membrane preparation with trypsin resulted in total loss of the binding activity. These results indicate that the prostaglandin E1 binding sites located on the cell surface and sialic acid residues are required for prostaglandin E1 binding to the human erythrocytes. These results also indicated that the binding sites are glycoprotein in nature.

Purification and characterization of fatty acid-binding proteins from brown adipose tissue of the rat.

Fatty acid-binding proteins (FABPs) have been identified and purified from interscapular brown adipose tissue of the rat. The proteins were characterized and their properties compared with the FABP present in white adipose tissue. FABP was purified to electrophoretic homogeneity from brown adipose tissue by a procedure involving precipitation with 70% ammonium sulphate, followed sequentially by ion-exchange chromatography and gel filtration chromatography. The purified fraction migrated as a single band on SDS-PAGE with an apparent molecular mass of 14,200. Scatchard analysis of [14C]oleate-binding to purified FABP gave a Kd value of approx. 0.80 +/- 0.02 microM and a maximal binding of 0.65 +/- 0.03 mol per mol of protein; these values were similar to that found with the FABP purified from white fat. The FABP concentration in brown adipose tissue was almost twice that of FABP in white adipose tissue. Fatty acid analysis of FABP from brown adipose tissue revealed that the intrinsic arachidonic acid content was proportionately higher than that present in FABP of white adipose tissue. Isoelectric focusing of delipidated FABP indicated that it existed with two charge isoforms (pI 6.85 and 7.35). The purified FABP additionally emerged in two peaks (FABP-I and FABP-II) from a reverse phase HPLC column. Amino acid analysis showed that Gly, Thr, and Ser residues in FABP-I were almost twice as high as in FABP-II. The N-terminals of both FABP-I and -II were not blocked. These components have been partially sequenced and showed a sequence homology only between 25-31 residues from the N-terminal. Further studies are required to elucidate the precise function of the two different isoforms of FABP in brown adipose tissue.

Characterization of prostaglandin E2 receptors expressed on human monocytic leukaemic cell line, U937.

Prostaglandin E2 (PGE2) receptors of a human monocytic leukaemic cell line, U937 cells, have been identified. [3H]PGE2 binding to these cells was found to be saturable and highly specific. Scatchard analysis of binding data revealed a non-linear plot indicating the presence of two independent classes of binding sites with different affinities and capacities. The high-affinity class had Kd1 = 3.1 nM and binding capacities n1 = 0.6 fmol/10(6) cells, whereas the low-affinity class had Kd2 = 137 nM and capacities n2 = 16 fmol/10(6) cells. Incubation of U937 cells with 3 microM PGE2 stimulated a 15-fold increase in cAMP formation compared to basal levels. Prior exposure of these cells with 10 microM PGE2 for 60 min induced both homologous and heterologous desensitization of adenylate cyclase activity. PGE2 (3 microM) or histamine (100 microM) showed reduced stimulation of cAMP formation in these desensitized cells compared to controls. The desensitized cells also showed 80% reduction of specific PGE2 binding compared to control cells. Our data suggest that U937 cells have PGE2 receptors which are linked to the adenylate cyclase system.

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process.

The interaction between interleukin IL-1 alpha and PGE2 on P388D1 cells has been investigated. Preincubation of murine macrophage-like cells, P388D1, with IL-1 alpha (0-73 pM) reduced the binding of PGE2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1 alpha decreased the PGE2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 +/- 0.02 to 0.12 +/- 0.01 fmol/10(6) cells for the high affinity receptor binding sites and from 2.41 +/- 0.12 to 1.51 +/- 0.21 fmol/10(6) cells for the low affinity receptor binding sites). However, the dissociation constants of the receptors of the IL-1 alpha-treated cells remained unchanged. Inhibition of PGE2 binding by IL-1 alpha did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1 alpha inhibits the binding of PGE2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE2.

Plasma membrane fatty acid-binding protein (FABPpm) of the sheep placenta.

Fatty acid-binding protein (FABPpm) has been identified and characterised from sheep placental membranes. Binding of [14C]oleate to placental membranes was found to be time- and temperature-dependent. Addition of a 20-fold excess unlabelled oleic, palmitic, or linoleic acid reduced the binding of [14C]oleate to the membranes to around 50% of total binding, whereas D-alpha-tocopherol at similar concentrations did not affect [14C]oleate binding. This indicates that the binding sites are specific to fatty acids. Specific binding of [14C]oleate was reduced by heat denaturation or trypsin digestion of the membranes, suggesting that the fatty acid-binding sites are protein in nature. FABPpm was then solubilised from sheep placental membranes, and subsequently purified to electrophoretic homogeneity using an oleate-agarose affinity column. The purified FABPpm had an apparent molecular mass of 40 kDa, as determined by SDS-PAGE and by gel permeation chromatography. The [14C]oleate-binding activity of the purified protein was also confirmed by PAGE followed by autoradioblotting. The specific binding for oleate was around 1.5 nmol per mg of membrane protein. Our data indicate the presence of FABPpm in sheep placental membranes.

Regulation of insulin receptor activity of human erythrocyte membrane by prostaglandin E1.

Incubation of human erythrocyte membrane with low concentration of prostaglandin E1 or prostacyclin increased the binding of 125I-labeled insulin to the membrane. The binding of the radioiodinated hormone was maximally stimulated at 3 nM prostaglandin E1 and the use of higher concentrations (above 8 nM) of the autacoid tended to reverse its own effect at lower concentrations. While prostaglandins A1, A2, B1, B2, D2, F1 alpha, F2 alpha or 6-keto-prostaglandin F1 alpha had no effect on the binding of insulin to the erythrocyte membrane, prostaglandin E2 at similar concentrations decreased the binding of the hormone. The effect of prostaglandin E1 on the increased binding of the insulin was found to be reversible and depended on the occupancy of the autacoid molecules on the membrane and showed positive cooperativity. Scatchard analysis of the binding of 125I-labeled insulin to the erythrocyte ghosts indicated that in the presence of the autacoid, the binding capacity of the insulin receptor increased 2-fold (from 207 to 424 fmol/mg protein) without any change in the ghosts affinity for the ligand (Kd 2.4 X 10(-9) versus 2.49 X 10(-9) M). As a consequence of increased binding of insulin to the erythrocyte membrane in the presence of prostaglandin E1 (3.0 nM), the optimal concentration of the peptide hormone for the maximal reduction of the membrane microviscosity decreased from approx. 1.6 to approx. 0.4 nM. Addition of prostaglandin E1 alone at the above concentration to the assay mixture had no effect on the membrane microviscosity.

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.

Control of erythrocyte membrane microviscosity by insulin.

The human erythrocyte membrane binds insulin through high-affinity, low-capacity binding sites (dissociation constant Kd1 2.45 X 10(-9)M; capacity n1 207 fmol/mg protein) and low-affinity, high-capacity binding sites (Kd2 0.63 X 10(-6) M; n2 37 pmol/mg protein). Treatment of the erythrocyte membrane or the intact cells with the physiological concentration of insulin, which is within the range of Kd value of the high-affinity sites, results in a significant reduction of the membrane microviscosity and the filtration time of the intact cells. Use of supraphysiological concentrations of the hormone reverses the effect of the lower concentration of insulin on the membrane microviscosity and the filtration time.

Characterisation of triacylglycerol hydrolase activities in human placenta.

Triacylglycerol hydrolase activities were characterised in homogenates, cytosol, and microvillous membranes (MVM) of human placenta. Homogenates of placenta exhibited three distinct triacylyglycerol hydrolase activities with pH optima 4.5, 6.0 and 8. 0. On further fractionation, placental cytosol exhibited both acid cholesterol ester hydrolase (pH 4.5) and hormone sensitive lipase (pH 6.0) activities, whereas purified placental MVM exhibited two distinct triacylyglycerol hydrolase activities; a minor activity at pH 8.0 and a second major activity at pH 6.0. Triacylglycerol hydrolase activity at pH 8.0 of MVM appeared to be lipoprotein lipase (consistent with criteria such as serum stimulation and salt inhibition), whereas at pH 6.0 the activity was unique in that it was almost abolished by serum, but was not affected by high NaCl concentrations. Our data, for the first time, demonstrate that human placental MVM, in addition to lipoprotein lipase, contain a newly identified triacylglycerol hydrolase activity at pH 6.0.

Plasma membrane fatty acid-binding protein (FABPpm) is exclusively located in the maternal facing membranes of the human placenta.

We reported earlier the presence of a 40 kDa plasma membrane fatty acid-binding protein (FABPpm) in human placenta. This protein is thought to be involved in the sequestration of unesterified free fatty acids bound to albumin from the maternal plasma for delivery to the fetus. However, its location in human placental syncytiotrophoblasts is not known. These cells are bipolar; one side facing maternal circulation (microvillous membranes), and the other side facing fetal circulation (basal membranes). Therefore, it is important to resolve the location of this protein in trophoblast membranes in order to understand fatty acid transport and metabolism in human placenta. Isolated plasma membranes vesicles were prepared respectively from the maternal facing microvillous and fetal facing surface of the human full-term placental syncytiotrophoblast. Using these membrane preparations, fatty acid binding activity, the polyacrylamide gel electrophoresis radiobinding assay for FABPpm, and Western blot analysis of FABPpm were carried out to determine the location of this protein in these membranes. Based on the above studies we conclude that the FABPpm is located exclusively in the microvillous membranes. Since FABPpm may be responsible for FFA uptake, its location in the microvillous membranes favours the unidirectional flow of maternal FFA to the fetus.

Transport mechanisms for long-chain polyunsaturated fatty acids in the human placenta.

To understand the placental role in the processes responsible for the preferential accumulation of maternal long-chain polyunsaturated fatty acids (LCPUFAs) in the fetus, we investigated fatty acid uptake and metabolism in the human placenta. A preference for LCPUFAs over nonessential fatty acids has been observed in isolated human placental membranes as well as in BeWo cells, a human placental choriocarcinoma cell line. A placental plasma membrane fatty acid binding protein (p-FABP(pm)) with a molecular mass of approximately 40 kDa was identified. The purified p-FABP(pm) preferentially bound with essential fatty acids (EFAs) and LCPUFAs over nonessential fatty acids. Oleic acid was taken up least and docosahexaenoic acid (DHA) most by BeWo cells, whereas no such discrimination was observed in HepG2 liver cells. Studies on the distribution of radiolabeled fatty acids in the cellular lipids of BeWo cells showed that DHA is incorporated mainly into the triacylglycerol fraction, followed by the phospholipid fraction; the reverse is true for arachidonic acid (AA). The greater cellular uptake of DHA and its preferential incorporation into the triacylglycerol fraction suggests that both uptake and transport modes of DHA by the placenta to the fetus are different from those of AA. p-FABP(pm) antiserum preferentially decreased the uptake of LCPUFAs and EFAs by BeWo cells compared with preimmune serum. Together, these results show the preferential uptake of LCPUFAs by the placenta that is most probably mediated via the p-FABP(pm).

The effects of structurally defined triglycerides of differing fatty acid composition on postprandial haemostasis in young, healthy men.

The aim of this study was to investigate whether a number of key haemostatic factors were altered when healthy young individuals were challenged with a fat load of physiological size contained within a meal composed of normal ingredients and whether this response was modified when the fatty acid composition of the meal was altered radically. Eight healthy male volunteers each randomly consumed four meals which were identical in terms of gross nutritional content (41% of energy provided as fat, 17% as protein and 42% as carbohydrate) but which differed in fatty acid composition. To reduce the possible influence of fatty acid position within the triglyceride molecule on lipid absorption and subsequent metabolic effects, the structural integrity of 91% of fat (test triglycerides such as 1,3 distearoyl-2-oleoyl glycerol (S-O-S), trioleine (O-O-O), and 1,3 dilinoleoyl-2-oleoyl glycerol, (L-O-L)) in the meals was controlled so that the principal fatty acid in the sn-2 position was oleic acid (18:1n-9). Meals rich in either a test triglyceride or a control oil provided 44+/-6 g of fat. No significant alterations from fasted values of elevated plasma factor VII coagulant activity (FVIIc) or F1 + 2 were observed. FVIIA varied significantly over the postprandial time course; however, when expressed as a percentage of the fasting value, the FVIIa responses to O-O-O and L-O-L differed significantly but this was not evident when the absolute values were analysed. Similarly, no difference in plasma fibrinopeptide A (FPA) concentrations were evident. After all four meals, chylomicron contained proportionately more palmitic acid and generally less oleic acid than the ingested lipids. This study clearly demonstrates that postprandial haemostatic responses of young healthy individuals to a physiological fat load are minimal, (irrespective of triglyceride structure).

Further characterization of a novel triacylglycerol hydrolase activity (pH 6.0 optimum) from microvillous membranes from human term placenta.

We recently identified the presence of two distinct triacylglycerol hydrolases with pH optima of 6.0 and 8.0 in human placental microvillous membranes (MVM). The TAG hydrolase with a pH optimum of 8.0 has properties similar to lipoprotein lipase, whereas TAG hydrolase with a pH optimum of 6.0 still to be fully characterized. In order to understand the functional and structural relationships between these two TAG hydrolases of MVM we have further investigated their biochemical and molecular properties. The presence of oleic acid inhibited TAG hydrolase activity with a pH optimum of 8.0 by 60 per cent whilst it had very little effect on the pH 6.0 TAG hydrolase activity. K(m)values for TAG hydrolases at pH 6.0 and pH 8. 0 optima were 170.6 and 9.83 nmol triolein, respectively, whereas the corresponding V(max)values were 0.32 and 0.037 nmol oleic acid/min mg/protein. Treatment of MVM with phenylmethylsulphonofluoride or protamine had no effect on TAG hydrolase at pH 6.0 whereas both decreased activity at pH 8.0, by 70 per cent and 52 per cent, respectively (P< 0.05), compared with control. p-Chloromercuribenzoate inhibited both TAG hydrolase activities by 25-30 per cent whereas iodoacetate inhibited TAG hydrolase activity with optimum pH 8.0 by 74 per cent and the activity at pH 6.0 by 28 per cent. Unlike the TAG hydrolase activity at pH 8.0, the activity at pH 6.0 was not affected by heparin. TAG hydrolase activity at pH 6.0 was significantly decreased compared with that of pH 8.0 optimum TAG hydrolase activity in smokers placenta. A threefold increase in pH 6.0 TAG hydrolase activity was observed following differentiation, whereas membrane associated TAG hydrolase activity with optimum pH 8.0 did not change. The TAG hydrolase with optimum pH 6.0 was subsequently purified from MVM to almost 1000-fold enrichment of the activity over the starting material. The final preparation however, still contained three distinct protein bands (90, 70 and 45 kDa). When extracted from non-denaturing polyacrylamide gels, the 70 kDa protein was the only protein to have TAG hydrolysing activity and had a pH optimum of 6.0. Labelling of samples with [(14)C]tetrahydrolipstatin also confirmed that the TAG hydrolase active protein was a 70 kDa protein. In conclusion, we report that there is a 70 kDa TAG hydrolase with optimum pH 6.0 in human placental MVM which is quite distinct from placental lipoprotein lipase.

Detection and cellular localization of plasma membrane-associated and cytoplasmic fatty acid-binding proteins in human placenta.

The aim of this study was to investigate location and the types of membrane-associated and cytoplasmic fatty acid-binding proteins in human placental trophoblasts using monospecific polyclonal antibodies. Western blot analysis demonstrated the presence of multiple membrane and cytoplasmic fatty acid transport/binding proteins in human placenta. In addition to previously reported placental membrane fatty acid-binding (p-FABPpm, 40 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transport protein (FATP, 62 kDa) were detected in both microvillous and basal membranes of the human placenta. Among the cytoplasmic proteins, heart (H) and liver (L) type FABP were detected in the cytosol of the human placental primary trophoblasts as well as in human placental choriocarcinoma (BeWo) cells. The immunoreactivity of epidermal type (E)-FABP was not detected in trophoblasts or BeWo cells despite its presence in human placental cytosol. Location of FAT and FATP on the both sides of the bipolar placental cells may favour transport of free fatty acids (FFA) pool in both directions i.e. from the mother to the fetus and vice versa. However, p-FABPpm, because of its exclusive location on the microvillous membranes, may favour the unidirectional flow of maternal plasma long-chain polyunsaturated fatty acids present in the FFA pool to the fetus, due to binding specificity for these fatty acids. Although the roles of these proteins in placental fatty acid uptake and metabolism are yet to be understood fully, their complex interaction may be involved in the uptake of maternal FFA by the placenta for delivery to the fetus.

Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes.

We investigated the fatty acid distribution in guinea pig alveolar apical membranes at different developmental stages. Fatty acid composition of the purified membranes isolated from guinea pig fetuses (at 65 day, term=68 day), neonates (day 1) and adult males was determined. The levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in the adult guinea pig alveolar apical membrane phosphatidylethanolamine (PE) fraction (9. 3+/-2.2 and 2.9+/-1.0%, respectively) while in other phospholipids (PL) fractions their levels were low or absent (P<0.01). Furthermore, levels of AA and DHA in the PE fraction of apical membrane increased significantly from fetal (6.6+/-3.0 and 0.8+/-0.4%, respectively) to neonatal life (10.3+/-1.5 and 3.0+/-0.8%, respectively). Increase in the level of DHA (almost four-fold) was much more pronounced than that of AA (P<0.05). As for guinea pig alveolar membranes, EPA and AA were mostly present in the PE fraction in pulmonary adenocarcinoma derived cells (A549 cells), a parallel model of type II pneumocytes, with the levels of AA around three-fold greater than that of EPA, Binding of radiolabelled fatty acids to A549 cells showed no significant differences between the maximum uptake achieved for different fatty acids (AA, 1.7+/-0.2, EPA, 2.3+/-0.3, LA, 1.7+/-0.2, OA, 2.0+/-0.2nmol/mg protein, P>0.5). Once the fatty acids were taken up by these cells AA was mostly identifiable in the monoacylglycerol (MAG) fraction, whereas EPA was equally distributed between the MAG and PL fractions. Oleic acid was mainly present in the triglyceride (TAG) fraction whereas LA was evenly distributed between the TAG, MAG, and PL fractions. Our data demonstrate a preferential distribution of AA and DHA in PE fractions of alveolar apical membranes during development.

alpha-Tocopherol binding activity of red blood cells in smokers.

Despite high plasma levels of vitamin E, red blood cell membranes contain relatively low levels of vitamin E. This suggests the existence of a selective vitamin E uptake/regeneration system in human red blood cell membranes. alpha-Tocopherol binding sites on human red blood cells are thought to be involved in the uptake of alpha-tocopherol from the plasma. To understand the role of the uptake system we have compared the alpha-tocopherol content and binding activity of red blood cells from smokers and non-smokers. The specific binding of [3H] alpha-tocopherol to pure red blood cell preparations from smokers (n = 7, 28.4 +/- 2.8 years) was 30.6 +/- 3.2 fmoles per 3 x 10(8) red blood cells and for non-smokers (n = 17, 27.9 +/- 1.3 years) was 41.7 +/- 3.7 fmoles per 3 x 10(8) red blood cells. Thus alpha-tocopherol uptake activity was significantly lower in smokers (P = 0.05). Red blood cells from smokers contained less (1.8 +/- 0.4 micrograms/gHb) alpha-tocopherol than non-smokers (2.8 +/- 0.3 micrograms/gHb), (P < 0.05), despite plasma levels of alpha-tocopherol being similar: 12.9 +/- 0.8 microM in non-smokers vs. 12.7 +/- 0.5 microM in smokers. However, adjusting plasma alpha-tocopherol for total plasma cholesterol plus triacylglycerols showed alpha-tocopherol levels were higher (P < 0.01) in non-smokers (2.84 +/- 0.10 mumol alpha-tocopherol/ mmol [cholesterol+triacylglycerol]) than in smokers (2.36 +/- 0.11 mumol alpha-tocopherol/mmol [cholesterol+triacylglycerol]). The reduced alpha-tocopherol levels in red blood cells from smokers may be due to impairment of alpha-tocopherol uptake activity. The reduced levels of alpha-tocopherol in smokers red blood cells was not associated with any changes in cell membrane fluidity. At present it is not known whether supplementation of smokers with vitamin E would normalise the alpha-tocopherol uptake activity of red blood cells.

A residential study comparing the effects of diets rich in stearic acid, oleic acid, and linoleic acid on fasting blood lipids, hemostatic variables and platelets in young healthy men.

Dietary fat is known to influence the variables of blood coagulation and fibrinolysis associated with vascular disease. However, the role of fat content and/or fat composition of the diet in this regard is still not well understood. In the present study, we investigated the effects of three isoenergic diets of differing fat composition in nine healthy young men in a strictly controlled residential study. Subjects consumed the three experimental diets for periods of 2 weeks each, separated by a washout period of at least 5 weeks in a randomized crossover design. The diets provided 38% of total energy intake as fat, 45% as carbohydrate, and 17% as protein, and differed only with respect to the fatty acid composition (stearic acid-rich diet: 34.1% stearic acid, 36.6% oleic acid; oleic acid-rich diet: 65.8% oleic acid; linoleic acid-rich diet: 36.5% linoleic acid, 38% oleic acid). Blood samples were collected at the beginning and at the end of each dietary period from fasted subjects for determination of factor VII coagulant activity (FVIIc), activated factor VII (FVIIa), factor VII antigen (FVIIag), tissue plasminogen activator (tPA) activity, plasminogen activator inhibitor type 1 (PAI-1) activity, fibrinogen, prothrombin fragment 1+2 (F(1+2)), and plasma lipids. There were no significant differences between diets in fasting plasma concentrations of FVIIc, FVIIa, FVIIag, fibrinogen, F(1+2), PAI-1 activity, and tPA activity. Plasma concentrations of lipids (high density lipoproteins, low density lipoproteins, triacylglycerols, and total cholesterol) were also unaffected. Although there were no changes in platelet aggregation response and membrane fluidity observed in any of the diets, increased anti-aggregatory prostaglandin E(1) binding to platelet membranes was observed only in the case of linoleic acid-rich diet. In conclusion, diets with very different fatty acid compositions, at 38% of energy as fat intake, did not significantly influence blood coagulation, fibrinolysis, or blood lipids in the fasting state in young healthy men.

Interaction of receptors for prostaglandin E1/prostacyclin and insulin in human erythrocytes and platelets.

Prostaglandin E1/I2 and insulin receptors of human erythrocyte and platelet are capable of modulating each other's activity. This modulation of the receptor activity and number in one system by a second receptor system in human platelet and erythrocyte seems to be beneficial. Insulin increases the PGE1 binding to platelets and thereby enhances the platelet antiaggregatory action of prostaglandin by increasing cyclic AMP levels. Similarly, PGE1 increases insulin binding to human erythrocyte, and thereby reduces the optimum concentration of insulin for a maximal reduction in membrane microviscosity. During ischemia the reduced response of platelets to the inhibitory effect of PGE1 or PGI2 relates to the impaired PGE1/I2 receptor activity. Treatment of these platelets with insulin at physiological concentrations can normalise the PGE1/I2 receptor activity. This review focuses on the relationship between the two receptor systems in human blood cells.

Placental membrane fatty acid-binding protein preferentially binds arachidonic and docosahexaenoic acids.

To elucidate further the role of placental membrane fatty acid-binding protein (p-FABPpm) in preferential transfer of maternal plasma long chain polyunsaturated fatty acids (LCPUFA) across the human placenta, direct binding of the purified protein with various radiolabelled fatty acids (docosahexaenoic, arachidonic, linoleic and oleic acids) was investigated. Binding of these fatty acids to the protein revealed that p-FABPpm had higher affinities and binding capacities for arachidonic and docosahexaenoic acids compared with linoleic and oleic acids. The apparent binding capacities (Bmax) values for oleic, linoleic, arachidonic and docosahexaenoic acids were 2.0 +/- 0.14, 2.1 +/- 0.17, 3.5 +/- 0.11, 4.0 +/- 0.10 mol per mol of p-FABPpm whereas the apparent dissociation constant (Kd) values were 1.0 +/- .0.07, 0.73 +/- 0.04, 0.45 +/- 0.03 and 0.4 +/- 0.02 microM, respectively (n=3). In the case of human serum albumin, the Kd and Bmax values for all fatty acids were around 1 microM and 5 mol/mol of protein, respectively. These data provide direct evidence for the role of p-FABPpm in preferential sequestration of maternal arachidonic and docosahexaenoic acids by the placenta for transport to the fetus by virtue of its preferential binding of these fatty acids.