Aggregation and thromboxane B2 formation in platelets and vascular prostacyclin production from genetically obese rats

Obesity, diabetes, hyperlipidaemia and age are conditions predisposing to atheroscleorosis and arterial occlusion. Recently it has been claimed that increased synthesis of thromboxane A₂ by platelets and decreased synthesis of prostacyclin (PGI₂) by blood vessels play an important role. The “Zucker” rat, a genetically obese animal with hyperlipidaemia, hyperinsulinaemia and normoglycaemia was used to study platelet aggregation, thromboxane (TXB₂) production and aortic PGI₂ synthesis. Two age groups (6–8 months and 14–16 months old) and their homozygote lean controls were used. In the obese rats no increased aggregation was found with ADP, arachidonic acid and collagen. On the contrary platelets from young fatty rats were less sensitive to ADP than platelets from lean young animals. An increase in platelet sensitivity to aggregating agents with age was observed, especially in the obese rats. TXB₂ measured in platelet rich plasma after exposure to ADP, arachidonic acid, arachidonic acid plus ADP and collagen was similar in the fatty and lean animals.Production of PGI₂ from incubated aortic rings was lowest in young lean animals. No differences existed between the other groups of rats studied. Insulin added to aortic rings had no influence on PGI₂ production. It is concluded that age rather than obesity, hyperlipidaemia or hyperinsulinaemia may cause platelet hyperresponsiveness to aggregating agents. Thromboxane and plateletaggregation do not closely correlate. PGI₂ production is not reduced by metabolic alterations, thought to predispose to atherosclerosis.     

Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts.

The interaction of human platelets with one another and with the blood vessel wall is thought to be regulated in part by a balance between two arachidonic acid metabolites: thromboxane A₂, synthesized by platelets, and prostacyclin (PGI₂), synthesized by the vessel wall. We have studied the ability of cultured human vascular cells to synthesize PGI₂ from arachidonic acid. Four strains of human arterial smooth muscle cells synthesized a mean of 1.36 ng PGI₂ per 10⁵ cells, with a range of 0.2–5.3 ng PGI₂ per 10⁵ cells among the different strains. Human umbilical vein endothelial cells synthesized a mean of 7.16 ng PGI₂ per 10⁵ cells with a range of 2.3–14.0 ng per 10⁵ cells. In contrast, cultured human diploid skin fibroblasts synthesized only 0.27 ng PGI₂ per 10⁵ cells with a range of 0.05–0.6 ng per 10⁵ cells. When cultured cells were mixed with platelets, PGI₂ synthesis from added arachidonate was reduced rather than stimulated. Thus the major precursor cyclic endoperoxides utilized for PGI₂ synthesis are formed within the cells and not from endoperoxides synthesized by platelet cyclooxygenase. Aspirin has been proposed as an anti-thrombotic agent. Aspirin could be ineffective, however, if it inhibited not only platelet cyclooxygenase but that of vessel wall cells as well. Measurement of the rate constant or potency for aspirin inhibition of PGI₂ synthesis in cultured cells indicates that the cyclooxygenase in both cell types of the blood vessel wall is 14–44 fold less sensitive to aspirin inactivation than that in platelets, and appropriate levels of aspirin can selectively block human platelet thromboxane A₂ synthesis without compromising the capacity of the vasculature to produce PGI₂.     

Ovarian hormones inhibit the release of prostacyclin-like material from isolated rat uterus

The influence of sex steroids on the production of prostacyclin (PGI₂) like material by the isolated rat uterus incubated in a buffer medium was explored by monitoring its ability to inhibit ADP-induced platelet aggregation. Chopped uterine strips from rats in natural estrus can generate an unstable substance that inhibits platelet aggregation and suggest to be prostacyclin. This capacity was significantly enhanced in preparations from spayed animals. The injection of 17-beta estradiol; progesterone or both diminished the production of the prostacyclin-like material by the uterus from ovariectomized rats. The already existing notion that ovarian steroids are able to regulate the synthesis of stable prostaglandins is discussed together with the present results suggesting in addition a depressive effect of sex hormones on the uterine PGI₂ synthetase system.     

Modulation of platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount fo thromboxane A₂ (TxA₂) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produce more TxA₂ than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI₂) than arterial tissue from vitamin E-supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, $\text{in}$$\text{vivo}$, inhibits platelet aggregation both by lowering platelet TxA₂ and by raising arterial PGI₂.     

Production of prostacyclin-like substance in stroke-prone and stroke-resistant spontaneously hypertensive rats

Activities of aortae to produce prostaglandin (PG) I₂-like substance in stroke-prone spontaneously hypertensive rats (SHRSP), stroke-resistant SHR (SHRSR) and normotensive control rats from the Wistar-Kyoto (WK) colony were compared. PGI₂-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and the amount produced was estimated by comparison of its anti-aggregatory activity with that produced by known amounts of the sodium salt of synthetic PGI₂. Before the development of stroke, amounts of this substance generated in SHRSP and SHRSR were significantly higher than those in WK rats (p<0.01 and p<0.02, respectively). Remarkably reduced capacity to generate PGI₂-like substance was observed in some SHRSP after the development of stroke.     

Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA; C20:5, ω-3) on PGI2-like substance production by rat aorta

A highly purified ethyl ester of EPA (EPAEE) (74%) was manufactured from sardine oil. Sixty mg/kg/day of EPAEE was given orally to male Wishar rats for 8 weeks. No side effect or toxicity from the administration of EPAEE was observed. Plasma EPA concentration and the ratio of EPA to arachidonic acid were significantly increased, compared with control Wistar rats. An enhancement of PGI₂-like substance production by aortas obtained from rats fed EPAEE was noted. Conversion of EPA to Λ¹⁷-6-keto-PGF_1α, a stable metabolite of PGI₃, could not be detected by an incubation study of ¹⁴C-EPA and aortas either from rats fed EPAEE or from control rats. Therefore, PGI₂-like substance produced by rat aorta is most likely to be PGI₂. itself and not PGI₃.     

Generation of prostacyclin-like substance and lipid peroxidation in vitamin E-deficient rats

Endogenous generation of prostacyclin (PGI₂)-like substance and lipid peroxidation were studied in the aorta of rats fed on vitamin E-deficient diet and/or vitamin E-supplemented one for 4 to 10 months after they were weaned at 4 weeks. PGI₂-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and was estimated by comparison of its antiaggregatory activity with that produced by known amounts of synthetic PGI₂. Thiobarbituric acid-reacting substance (TBARS) was determined as an indicator of lipid peroxidation. The generation of PGI₂-like substance was significantly reduced in rats fed on vitamin E-deficient diet for 8 and 10 months as compared with that in the animals fed on vitamin E-supplemented one for the same period (p<0.001). Mean concentration of TBARS in the aortae of rats fed on vitamin E-deficient diet for 10 months was significantly higher than that of the animals fed on vitamin E-supplemented diet for the same feeding period (p<0.001). These alterations in the aortae of rats fed on vitamin E-deficient diet were corrected by feeding them on vitamin E-supplemented diet for subsequent 2 months.     

Prostacyclin and thromboxane A2 release in isolated rat lungs

The influence of platelets and platelet membranes on the generation of prostacyclin (PGI₂) and thromboxane A₂(TXA₂) by isolated rat lung and porcine aortic endothelial cell, as measured by RIA of their stable end-producs, 6-oxo-PGF_1α and TXB₂ respectively, was studied. After introduction of either aspirin-treated platelets or membranes from aspirin-treated platelets to the perfusate, 1 5-fold increase in the amount of 6-oxo-PGF_1α and TXB₂ in the perfusate was observed. Treatment of the lung with aspirin produced a 50% reduction in the platelet-stimulated release of PGI₂ and TXA₂. Treatment of the lung with the phospholipase inhibitor, mepacrine, significantly reduced the platelet-stimulated release of PGI₂ and TXA₂. Incubation of endothelial cells with untreated platelet membranes did not alter the generation of PGI₂. These results suggest that platelet-stimulated release of PGI₂ and TXA₂ occurs via mechanical stimulation of phospholipase A₂, liberating arachidonic acid.     

Human platelet rich plasma and human serum protects from inactivation the antiaggregatory capacity of prostacyclin-like material (PGI2) produced by the rat stomach fundus

Prostacyclin (PGI₂) synthetizing capacity of rat stomach fundus in Krebs-Ringer-Bicarbonate (KRB); human platelet rich plasma (PRP) or human serum (HS), was explored. The basal production of PGI₂-like material was similar in the three media, suggesting the absence of any special substance in plasma or serum able to modify prostacyclin synthesis from tissue substrate. On the other hand it was also documented that in PRP and in HS the antiaggregatory activity of the PGI₂-like material declined in its capacity less than 50% following 60 minutes of incubation at 37 °C, whereas it almost dissapeared when incubated in KRB. Possible explanations underlying such finding are discussed.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO₂) exposure on prostacyclin (PGIP₂) synthesis in the rat lung and thromboxane A₂ (TXA₂) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO₂ for 1, 3, 5, 7 and 14 days. PGI₂ synthesizing activity of homogenized lung decreased. The damage of PGI₂ synthesizing activity reaches its maximum at 3 days. At 14 days, PGI₂ synthesizing activity returned to the normal level. The activity of PGI₂ synthetase decreased significantly. The formation of lipid peroxides due to NO₂ exposure may cause the depression of PGI₂ synthesizing activity of lung. On the other hand, platelet TXA₂ synthesizing activity increased. This increased TXA₂ synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO₂ exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO₂ exposure. These results indicate that the depression of PGI₂ synthesizing activity lung by NO₂ exposure cause an increase in TXA₂ synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO₂ exposure.     

Release of prostaglandin I2-like activity from the rat aorta: Effect of captopril,furosemide, and sodium

The effect of captopril, furosemide, indomethacine and intake of sodium on the production of PGI₂-like material was studied in the rat aorta. Release of PGI₂-like material from these vessels was estimated by its ability to inhibit ADP-induced vessels was estimated by its ability to inhibit ADP-induced platelet aggregation. Pretreatment with indomethacin (15 mg/kg/day) reduced the capacity of the aorta to release PGI₂-like material. Pretreatment with captopril (10 mg/kg/day) had no effect. Intravenous furosemide (60 μg/ml plasma volume) increased the capacity of the aorta to inhibit by 28% (p<.025). The inhibitory capacity of aorta removed from rats on a low sodium diet did not differ from those on a high sodium diet. We conclude that the action of furosemide in reducing vascular tone may be related to stimulation of PGI₂ synthesis in blood vessels whereas the effect of captopril and sodiumin in reducing vascular tone may involve a mechanism unrelated to PGI₂ synthesis or may involve the synthesis of a prostaglandin other than PGI₂.     

Activated Platelets from Diabetic Rats Cause Endothelial Dysfunction by Decreasing Akt/Endothelial NO Synthase Signaling Pathway

Diabetes is associated with endothelial dysfunction and platelet activation, both of which may contribute to increased cardiovascular risk. The purpose of this study was to characterize circulating platelets in diabetes and clarify their effects on endothelial function. Male Wistar rats were injected with streptozotocin (STZ) to induce diabetes. Each experiment was performed by incubating carotid arterial rings with platelets (1.65×10⁷ cells/mL; 30 min) isolated from STZ or control rats. Thereafter, the vascular function was characterized in isolated carotid arterial rings in organ bath chambers, and each expression and activation of enzymes involved in nitric oxide and oxidative stress levels were analyzed. Endothelium-dependent relaxation induced by acetylcholine was significantly attenuated in carotid arteries treated with platelets isolated from STZ rats. Similarly, treatment with platelets isolated from STZ rats significantly reduced ACh-induced Akt/endothelial NO synthase signaling/NO production and enhanced TXB₂ (metabolite of TXA₂), while CD61 (platelet marker) and CD62P (activated platelet marker) were increased in carotid arteries treated with platelets isolated from STZ rats. Furthermore, the platelets isolated from STZ rats decreased total eNOS protein and eNOS dimerization, and increased oxidative stress. These data provide direct evidence that circulating platelets isolated from diabetic rats cause dysfunction of the endothelium by decreasing NO production (via Akt/endothelial NO synthase signaling pathway) and increasing TXA₂. Moreover, activated platelets disrupt the carotid artery by increasing oxidative stress.     

The effects of a 5-lipoxygenase inhibitor, AA-861, on PGI2-like substance production in guinea-pig lungs

To determine the effects of AA-861 on PGI₂ production in guinea-pig lungs, 3 g of guinea-pig lung was chopped in 4 ml of buffer (control group), in buffer with 4 μg/ml indomethacin (indomethacin group) and in buffer with 2.5 × 10⁻⁵M AA-861 (AA-861 group). The chopped lungs were incubated for 30 min. 250 μl of incubation medium from each group was assessed before and after 3, 5, 10, 15, 20, 25 and 30 min of incubation. The incubation medium was centrifuged and the supernatant was tested for a PGI₂-like substance (PGI₂) by platelet aggregation inhibition. PGI₂ was produced mainly during the initial 3–5 min of incubation and was decreased thereafter. PGI₂ production was almost completely inhibited in the indomethacin group at all of the incubation times and was partially inhibited in the AA-861 group during the initial 3–5 minutes. Endogenous 5-lipoxygenase products generated in the early stages of incubation seem to be involved in PGI₂ production in guinea-pig lungs.     

Modulation of the platelet thromboxane A2 and aortic prostacyclin synthesis by dietary selenium and vitamin E

Vitamin E and selenium (Se) interact synergistically as an important antioxidant defense mechanism. Se, an essential component of glutathione peroxidase (GSH-Px) and vitamin E decompose fatty acid hydroperoxides and hydrogen peroxides generated by free radical reactions. Vitamin E and GSH-Px may modulate arachidonic acid metabolism and the activity of cyclooxygenase enzymes by affecting peroxide concentration. The balance between arterial wall prostacyclin (PGI₂) production and platelet thromboxane (TX)A₂ directly influences platelet activity. In order to elucidate the differential role of dietary vitamin E and Se in aortic PGI₂ and platelet TXA₂ synthesis, 1-mo-old F344 rats were fed semipurified diets containing different levels of vitamin E (0, 30, 200 ppm) and Se (0, 0.1, 0.2 ppm) for 2 mo. Thromboxane B₂ (TXB₂) and 6-keto-PGF₁α, were measured by radioimmunoassay (RIA) after incubation of whole blood and aortic rings at 37°C for 10 and 30 min, respectively. Vitamin E deficiency reduced plasma vitamin E to 5–17% of control-fed rats, and supplementation increased it to 53% of the control-fed rats. Se supplementation in vitamin E-supplemented animals increased plasma GSH-Px by 17%, compared to vitamin E-deficient rats. Se and vitamin E supplementation did not have a similar effect on TXB₂ and PGI₂ synthesis. Se deficiency did not alter platelet TXB₂ synthesis, but significantly decreased aortic PGI₂ synthesis. It was necessary to supplement with both antioxidants in order to increase, PGI₂ synthesis. Se and vitamin E deficient groups had a higher TXB₂/PGI₂ ratio (0.17±0.08) compared to Se- and vitamin E-supplemented groups (0.03±0.01). These results confirm previous reports in humans and animals and are in accordance with epidemiological data indicating an inverse relationship between plasma Se and platelet aggregation. Thus, further suggesting that vitamin E and Se may have a specific role in controlling TXA₂ and PGI₂ synthesis.     

Microsomal preparations of normal bovine iris-ciliary body generate prostacyclin-like but not thromboxane-A2-like activity

Indomethacin-treated bovine iris-ciliary body microsomes (IBIM) have been studied for their ability to convert PG endoperoxides into either thromboxance-A₂ (TxA₂)-like or prostacyclin (PGI₂)-like activity. The biological activity of the ocular tissue microsomes were compared with either indomethacin-treated human platelet microsomes (for TxA₂-like activity) or rabbit aorta microsomes (for PGI₂-like activity) under appropriate incubation conditions. No evidence could be found for the formation of TxA₂-like activity from PG endoperoxides by the IBIM. In contrasts, when the IBIM were incubated with PGH₂ for 1 min at 22°C without cofactors, PGI₂-like activity was produced, causing profound relaxation of the isolated dog coronary artery preparation without contracting the rabbit aorta and inhibiting arachidonic acid-induced platelet aggregation. Equivalent quantities of boiled IBIM failed to aleter the biological activity of PGH₂ under identical conditions. Tranylcypromine (500 μg/ml) completely abolished the appearance of PGI₂-like activity. Furthermore, the PGI₂-like activity found was stable for 10 min at 22°C at pH 8.5 but completely lost under similar conditions at pH 5.5. It is concluded than microsomal preparations of normal bovine iris-ciliary body can synthesize PGI₂-like activity in substantial amounts but not TxA₂-like activity.     

Dietary administration of eicosapentaenoic and linolenic acid increases arterial blood pressure and suppresses vascular prostacyclin synthesis in the rat

The role of the ‘prostacyclin-thromboxane system’ in the regulation of arterial blood pressure was investigated in rats receiving diets which contained different amounts of eixosapentaenoic (EPA) and linolenic acid (LNA). Forty rats were divided into five groups of 8 animals, each group receiving 25 energy (en) % as fat. All diets contained equal amounts of linoleic acid (5 en%) and oleic acid (5 en%). In the control group I, the remaining 15 en% of fat were given as saturated fat. Two groups of animals received cod liver oil as a source for EPA in amounts of 2.5 (group II)_and 5 en% (group III) while the two remaining groups were given diets supplemented with linseed oil as a source for LNA in amounts of 2.5 (group IV) and 5 en% (group V), respectively. After six weeks of feeding period the animals were sacrificed and portions of their isolated aorta incubated in Tris buffer (pH 9.3) for determination of prostacyclin (PGI₂)-like activity. Arterial blood pressure was uncharged in group I animals, but significantly increased in all rats receiving dietary EPA or LNA supplements. This rise is arterial blood pressure was associated with a marked suppression of the appearance of PGI₂-like activity in the incubation buffer while platelet thromboxane release during blood clotting was unchanged. Our results show that dietary adminis- tration of EPA and LNA increases arterial blood pressure in the rat and that this effect is associated with a suppressed generation of vasodilator prostacyclin by vascular tissue.     

Circulatory and platelet actions of 6,9-thiaprostacyclin (PGI2-S) in the cat.

A new analog of prostacyclin, 6,9-Thiaprostacyclin was infused intravenously in pentobarbital anesthetized cats in order to determine its hemodynamic and anti-platelet aggregating properties. At an infusion rate of 0.01 μmoles/kg/min, PGI₂-S moderately decreased arterial blood pressure without altering heart rate of superior mesenteric artery flow or platelet aggregation responses to ADP. However, at 0.05 μmoles/kg/min, PGI₂-S significantly reduced arterial blood pressure and significantly increased heart rate, and superior mesenteric artery flow. Moreover, at 0.05 μmoles/kg/min, PGI₂-S inhibited ADP platelet aggregation by 80%. PGI₂-S may be a useful agent in circulatory shock.     

Fish oil administration as a supplement to a corn oil containing diet affects arterial prostacyclin production more than platelet thromboxane formation in the rat

The administration to male rats of 5 en % fish oil (FO) as supplement to a diet containing 5 en % corn oil (CO), selectively and markedly decreased arterial parameters (6-keto-PGF_1α formation and platelet antiaggregatory activity) assessed in isolated aortic segments perfused with autologous platelet rich plasma (PRP). Platelet parameters (ADP-induced aggregation, TxB₂ formation in thrombin-stimulated PRP and sensitivity to exogenous PGI₂) were instead minimally affected. Eicosapentaenoic acid (EPA, 20:5 n-3) did not accumulate in plasma, platelet and aorta lipids and arachidonic acid (AA, 20:4 n-6) levels declined markedly only in the plasma compartment. When FO was given alone at the same 5 en % level, both arterial and platelet parameters were similarly affected. EPA accumulated in plasma cholesterol esters and was present in appreciable concentrations also in platelets and aortic walls. AA levels declined markedly in plasma lipids and appreciably also in platelet and aorta lipids. It is concluded that a) arterial and platelet parameters are differentially affected by FO administration depending upon the presence of n-6 polyunsaturated fatty acids in the diet, b) 6-keto-PGF_1α production by arterial tissues does not seem to be related to changes of PG precursor fatty acid levels in the phospholipid fraction.     

Prostanoid synthesis by human umbilical artery

A discrepancy between published values of PGI₂ production by human umbilical artery measured by platelet bioassay, compared with values of 6-oxo-PGF_1α by radioimmunoassay, raised the possibility that another anti-aggregatory prostanoid was produced by this tissue. To test this hypothesis, umbilical artery rings were incubated in buffer and PGI₂ determined by platelet bioassay and by a more specific radioimmunoassay based on comparison of 6-oxo-PGF_1α in hydrolysed and non-hydrolysed samples. 6-oxo-PGF_1a, PGF_2α and TXB₂ were also measured by gas chromatography negative ion chemical ionisation mass spectrometry. PGI₂ concentrations by radioimmunoassay and bioassay were significantly correlated (r = 0.92, p < 0.01). There was no difference between them, disproving the presence of an additional antiaggregatory substance. PGI₂ production determined by bioassay (mean 1.21 ng/mg wet weight/h, range 0.59–1.53 ng/mg/h) differed from previously reported values (range 70–325 ng/mg/h). 6-oxo-PGF_1α concentrations were confirmed by gas chromatography negative ion chemical ionisation mass spectrometry. Previous determinations of PGI₂ production by this tissue overestimated it by approximately 100 times.     

Prostacyclin inhibits 5-hydroxytryptamine release but stimulates thromboxane synthesis during cardiopulmonary bypass

The antiaggregating agent prostacyclin (PGI₂) was infused into ten dogs during cardiopulmonary bypass (CPB) to minimize thrombocytopenia and platelet dysfunction. The animals were anesthetized, placed on mechanical ventilation and underwent thoracotomy. After heparinization with 300 u/kg, animals were assigned to control (n=5) or PGI₂ treated groups (n=5). Thoracotomy and then CPB decreased platelet numbers to below 30, 000/mm³ (p < 0.05) and fibrinogen to less than 150 mg/dl (p < 0.05). PGI₂ at 100 ng/kg·min was infused for the 2 h period of CPB. PGI₂ infusion did not prevent these changes, but did prevent platelet serotonin release. In the control group after CPB, platelet serotonin fell from the baseline value of 1.11 μg/10⁹ to 0.35 μg/10⁹ platelets (p < 0.05). In contrast, PGI₂ treatment resulted in a serotonin increase to 2.27 μg/10⁹ platelets (p < 0.05). Thromboxane B₂ concentrations of platelets and plasma rose during CPB (p < 0.05). Surprisingly, PGI₂ infusion accentuated this rise in platelet and plasma thromboxane B₂ (p < 0.05). These data indicate that during CPB, an infusion of PGI₂: 1) does not prevent thrombocytopenia; 2) increases platelet serotonin uptake despite, 3) an associated rise in platelet and plasma thromboxane B₂.