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The relationship between the antithrombotic and antiplatelet effects of aspirin is complex, since aspirin influences other systems that protect against thrombosis as well as inhibiting platelet function. We investigated possible cumulative effects of low-dose aspirin on vascular production of prostacyclin in patients with documented atherosclerotic cardiovascular disease. Candidates for coronary artery vein graft bypass ingested 20 mg of aspirin daily during the week before surgery, and platelet aggregation, platelet formation of thromboxane A2 (TXA2), aortic and saphenous vein production of prostacyclin (PGI2), and hemostatic status were measured at the time of the bypass surgery. Low-dose aspirin markedly inhibited platelet aggregation responses and reduced TXA2 generation by greater than 90%, effects similar to those observed with much higher doses of aspirin. Both aortic and saphenous vein production of PGI2 were inhibited by 50% compared with PGI2 produced by vascular tissues of control subjects who received no aspirin preoperatively (51 +/- 10 pg 6-keto-PGF1 alpha/mg aortic wet weight [mean +/- SEM] in aspirin-treated subjects vs 130 +/- 16 pg/mg in control subjects, and 71 +/- 8 pg/mg saphenous vein wet weight vs 131 +/- 17 pg/mg). Blood loss at surgery was not significantly increased by preoperative low-dose aspirin as measured by chest tube drainage (754 +/- 229 ml in aspirin-treated subjects vs 645 +/- 271 ml in control subjects), hematocrit nadir (31.2 +/- 1.9% vs 31.8 +/- 1.7%), or transfusions (2.2 +/- 1.3 units of red blood cells vs 2.2 +/- 1.7 units).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of platelet physiology, biochemistry, ultrastructure, and function in a young woman without history of hemorrhagic problems revealed that her platelets were deficient in cyclooxygenase activity. Her citrate platelet-rich plasma (C-PRP) responded monophasically when stirred with aggregating agents in the same manner as aspirin-treated normal C-PRP, but could be irreversible aggregated by high concentrations of thrombin, collagen, and ADP. Her platelets did not aggregate when stirred with AA at concentrations as high as 2 mM. Ultrastructure and levels of serotonin and adenine nucleotides wer normal. Amounts of 14C-AA released after stirring with thrombin were similar to normal cells. However, evaluation of prostaglandin synthesis after stirring with 14C-AA revealed no evidence of endoperoxide or thromboxane production, although products of the lipoxygenase pathway were produced in normal amounts. Aggregation in response to AA was completely corrected after mixing with 10% normal C-PRP. However, equal volumes of her C-PRP and normal aspirin-treated C-PRP did not respond to AA, whereas 10% normal platelets combined with aspirin-treated cells corrected aggregation to AA. Since epinephrine pretreatment corrects the response of dog platelets that are not aggregated by AA, we evaluated the influence of epinephrine on her platelets. Preexposure to 5 micro M epinephrine, a concentration that gave only primary waves of aggregation, resulted in normalization of her response to AA, even though correction was not associated with the generation of endoperoxides or thromboxanes. The results may explain why patients with platelet cyclooxygenase deficiency have mild or absent bleeding symptoms.

SummaryLow doses of enteric-coated aspirin were administered orally to pigs. Plasma aspirin concentrations measured in blood obtained simultaneously from permanent catheters in a systemic artery and portal vein for 6 hours after dosage showed a large variation in the plasma aspirin concentration : time profile between pigs. After 50 mg single dose the ratio of the arterial : portal area under the plasma concentration versus time curve (AUC) was 0.63 ± 0.08 (mean ± SE, n = 6). In three pigs which received all three dosage regimens, the arterial : portal AUC ratios were 0.48 ± 0.05 after 50 mg single dose, 0.52 ± 0.02 after 100 mg single dose and 0.47 ± 0.02 after 100 mg daily for 1 week. Platelet aggregation in response to sodium arachidonate (1.65 mM) was completely abolished after chronic aspirin administration of 100 mg daily. Thromboxane production (pg/ 106 platelets) induced by this stimulus decreased from 536 ± 117 before aspirin to 57 ± 14 after aspirin (mean ± SE, n = 4; p = 0.03). Aortic prostacyclin synthesis, measured as 6-keto PGF1α (ng/disc after 10 min incubation), was 1.66 ± 0.28 (mean ± SE, n = 4) in untreated pigs and 0.95 ± 0.25 (n = 5) in treated pigs (p = 0.07). Results from this study support the idea that a difference between aspirin concentrations in the portal and systemic circulations can be achieved. Whether this can be translated into a clinically useful differential effect on the vessel wall compared to the platelet remains to be determined.

Previous studies from our laboratory have demonstrated a critical role for ferrous heme in prostaglandin synthesis. Based upon these studies, we proposed a model for heme-arachidonic acid interaction and demonstrated that compounds which interfere with this interaction inhibit arachidonic oxidation by ferrous heme. In this study, we have examined the effect of four different inhibitors for their effect on platelet arachidonic acid metabolism and function. The compounds studied were an iron chelator, 2,2'-dipyridyl, the cyclooxygenase inhibitors, Ibuprofen and aspirin, and a polyenoic acid, docosahexaenoic acid. All four compounds at approximately 100 microM concentration blocked the second wave of platelet aggregation in response to epinephrine or adenosine diphosphate. They were equally potent in inhibiting 14C-arachidonic conversion by platelets to thromboxane. However, inhibition of platelet thromboxane production and function by dipyridyl and DHA was reversible. Removal of these compounds from the medium restored platelets ability to respond to agonists and generate products through the cyclooxygenase pathway. The inhibitory effect of Ibuprofen and aspirin on cyclooxygenase activity could not be reversed by washing the platelets. However, Ibuprofen treated platelets aggregated when stirred with arachidonate in a normal way. No such response could be elicited from aspirin treated platelets. All compounds (except DHA) interfered with heme-arachidonic acid interaction in a cell-free system and prevented arachidonic acid oxidation. Results of our studies suggest a common mechanism of action for these different classes of compounds. In spite of the common mechanism, each class of drug seems to have a relatively different effect upon platelet cyclooxygenase and function.

Ro 21-7634 has previously been shown to inhibit histamine and SRS-A release from actively-sensitized guinea pig lung fragments upon antigen challenge. In the studies described herein, it was observed that Ro 21-7634 does not decrease SRS-A release but instead acts to inhibit the synthesis of this mediator. This was confirmed by studying SRS-A synthesis in vitro in rat peritoneal cells after challenge with ionophore A23187. In the peritoneal cell system, Ro 21-7634 exhibited an IC50 of 500 microM, in comparison with 5.8,11,14-eicosatetraynoic acid, phenidone and BW755C (IC50's of 2, 100, and 100 microM, respectively). When studied at 10(-4) and 10(-3) M in perfused guinea pig lung, Ro 21-7634 inhibited antigen-induced thromboxane A2 production by 68 and 96%, respectively. In this system, antigen is believed to induce thromboxane A2 production through the release of histamine and SRS-A from lung tissue. These mediators then interact at receptor sites in the lung parenchyma to induce thromboxane A2 synthesis. Ro 21-7634 could thus be inhibiting thromboxane A2 production by preventing the release of histamine and synthesis of SRS-A in the perfused lung system. Such a mechanism is suggested by the fact that although Ro 21-7634 was effective in inhibiting antigen-induced thromboxane production, it was ineffective in inhibiting thromboxane A2 production induced in the guinea pig lung system by the direct perfusion of histamine or SRS-A through the lung.

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI2) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 micrograms ml(-1)). Both 15-HPAA (1-20 micrograms ml(-1) min (-1)) and 13-hydroperoxy linoleic acid (13-HPLA, 20 micrograms ml(-1) min(-1)) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI2 (5 micrograms ml(-1) min (-1)) or 6-oxo-prostaglandin F1alpha (6-oxo-PGF1alpha, 5 micrograms ml(-1) min(-1)). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI2 (10 ng - 10 microgram ml(-1) min(-1)) but was inhibited by PGE2 (5 and 10 micrograms ml(-1) min (-1)). Antiserum raised to 5,6-dihydro prostacyclin (PGI1) in rabbits, which also binds PGI2, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).

Bleeding time, platelet thromboxane B2 production and megakaryocyte nuclear DNA concentration were measured in rabbits recovering from thrombocytopenia caused by a single injection of anti-platelet serum. Similar measurements were made on rabbits in a steady state of normal platelet production. The effects of a sustained state of thrombocytopenia on megakaryocyte DNA concentration were investigated by repeated daily injections of anti-platelet serum. It is shown that bleeding time depends on both platelet count and mean platelet volume. Furthermore changes in mean platelet volume appear to play a more important role in haemostasis than changes in platelet count. The mean megakaryocyte nuclear DNA concentration is significantly increased after 24 hours of thrombocytopenia and continues to increase as thrombocytopenia is sustained. Thromboxane B2 production/unit volume of platelet is increased in platelets produced after 24 hours of thrombocytopenia compared with platelets produced in normal steady state function. As a consequence platelets produced in response to thrombocytopenia not only have a larger mean platelet volume but are also more reactive. Mean platelet volume, as well as platelet count, should be considered as an index of haemostasis and its dysfunction, thrombosis.

Platelet aggregation and thromboxane B2 production, in response to adenosine diphosphate, were significantly impaired in 7 undialyzed or inadequately dialyzed patients with renal failure when compared to adequately dialyzed individuals or normal subjects. In 1 patient, platelet aggregation and thromboxane synthesis were corrected after adequate hemodialysis. The defect in both platelet aggregation and thromboxane production was induced in normal platelets incubated with uremic platelet-poor plasma. These findings suggest that the uremic platelet defect may be due, in part, to a plasma factor that inhibitors platelet thromboxane synthesis. Further, adequate dialytic therapy may reverse this defect.

Preeclampsia is associated with increased thromboxane and decreased prostacyclin production by the placenta. Low-dose aspirin can selectively inhibit thromboxane production in the adult circulation, but its effects on placental vascular production of thromboxane and prostacyclin are incompletely understood. We therefore studied the effects of low-dose aspirin on the production rates of prostacyclin and thromboxane, with and without vasoconstricting doses of angiotensin II, in human placental arteries. Chorionic plate arteries were incubated and samples were assayed for thromboxane and prostacyclin by radioimmunoassay of their stable metabolites. Production rates for prostacyclin were similar in the control, aspirin, angiotensin II, and angiotensin II plus aspirin groups. Mean (+/- SEM; n = 8) thromboxane production rates in the aspirin (1.4 +/- 0.5 pg/mg/hr) and angiotensin II plus aspirin (2.9 +/- 0.6 pg/mg/hr) groups were significantly lower (p less than 0.05) than values in the control (8.6 +/- 2.7 pg/mg/hr) and angiotensin II (6.7 +/- 1.3 pg/mg/hr) groups. We conclude that low-dose aspirin significantly decreases production of thromboxane in placental arteries both with and without vasoconstricting doses of angiotensin II.

To determine whether the amount of cyclooxygenase metabolites correlates with the development of lupus nephritis, intrarenal eicosanoid production was measured in autoimmune mice. Disease progression was related to the renal biosynthesis of prostaglandin (PGE2), prostacyclin (6 keto PGF1 alpha), and thromboxane (TXB2) using the MRL-lpr and NZB X NZW F1 hybrid mouse strains with predictably progressive forms of renal disease that mimic the human illness. Mice were evaluated for renal disease by measuring urinary protein excretion and renal immunopathological conditions and these features were related to renal eicosanoid production. These studies show that: (a) intrarenal synthesis of TXB2 increased incrementally in MRL-lpr and NZB X NZW F1 hybrid mice as renal function deteriorated and renal pathologic events progressed; (b) there were no consistent increases in the levels of two other cyclooxygenase metabolites, PGE2 or 6 keto PGF1 alpha; (c) increased TXB2 production occurred in the renal medulla, cortex, and within enriched preparations of cortical glomeruli; (d) when renal disease was prevented by pharmacologic doses of PGE2, intrarenal TXB2 did not increase; (e) administration of a dose of ibuprofen (9 mg/kg), a cyclooxygenase inhibitor capable of reducing 90% of platelet TXB2 without affecting intrarenal levels, did not retard the progression of renal damage. Taken together, these data indicate that the intrarenal level of TXB2 rises in relation to the severity of murine lupus nephritis. Furthermore, because of the potential deleterious effects of TXA2, enhanced production of this eicosanoid may be an important mediator of renal injury.

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro , to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection. The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE 2 , PGD 2 , PGF 2α , TXB 2 and 6-keto-PGF 1α . In all types studied PGE 2 and TXB 2 were the major products formed. The identification of PGE 2 and TXB 2 was confirmed by GC/MS with multiple ion monitoring. The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.

Previous studies have indicated that the regional distribution of the arachidonic acid metabolites around a focal ischemic lesion may be important in the pathogenesis of cerebral ischemia. To determine the functional significance of this regionalization, we examined the effect of imidazole (a thromboxane synthetase inhibitor) on the distribution of the vasoconstrictor thromboxane and the vasodilators prostacyclin and prostaglandin E2 (PGE2) and on the distribution of cerebral blood flow (CBF) around a focal ischemic lesion, middle cerebral artery (MCA) occlusion in the cat. The study was conducted in two phases. The first phase examined regional distribution of tissue arachidonic acid metabolites and the effect of imidazole treatment on that distribution. The second phase examined the effect of imidazole treatment on the distribution of blood flow about the focal ischemic lesion as well as on electrocortical function and edema production. MCA occlusion resulted in increased thromboxane, prostacyclin, and PGE2 levels in the ipsilateral hemisphere. These increases were greatest in the region of marginal ischemia and were present both 3 and 6 hours after occlusion. Imidazole pretreatment (50 mg/kg i.p.) significantly inhibited thromboxane production, but augmented production of prostacyclin and PGE2. In the blood flow studies, imidazole was without effect on regions of dense cerebral ischemia (CBF less than 20 ml/minute/100 g for more than 12 of 24 postocclusion hours). In regions of marginal ischemia (20 less than CBF less than 30 ml/minute/100 g for more than 12 of 24 postocclusion hours), imidazole pretreatment significantly increased blood flow in both gray and white matter compared with saline-treated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryA group of 186 patients with Transient Ischaemic Attacks (TIA) or cerebral Infarction (Cl) was found to demonstrate in vitro Spontaneous Platelet Aggregation (SPA) in 39% of those studied. Of the 176 normal subjects studied the incidence of in vitro SPA was found to be 5%. Further investigation of the phenomenon of SPA revealed that:1. it is associated with ADP-hyperaggregability, i. e. the threshold concentration to induce second wave aggregation is decreased;2. it is dependant on the increase in pH which occurs in platelet-rich plasma stirring in an aggregometer while concurrent ADP-hyperaggregability is independant of this change in pH;3. it is associated with malondialdehyde production and the release of endogenous 5- hydroxytryptamine; and that4. in addition Km and Vmax values for [14C]-5HT incorporation are normal; and that5. no gross abnormalities of the platelet membrane glycoproteins were apparent although occasionally glycoprotein III was found to be increased.This study demonstrates abnormal platelet behaviour in patients with TIA and Cl where the enzyme system involved in thromboxane production is sufficiently stimulated, by stirring alone, to induce aggregation of platelets and the release reaction.Acetylsalicylic acid abolishes SPA and prolongs the bleeding time with similar characteristics as has been described for normal individuals. Plasma (3-thromboglobulin levels are significantly increased in the patients studied. However, no correlation was established with the incidence of in vitro SPA.

A group of 186 patients with Transient Ischaemic Attacks (TIA) or cerebral Infarction (Cl) was found to demonstrate in vitro Spontaneous Platelet Aggregation (SPA) in 39% of those studied. Of the 176 normal subjects studied the incidence of in vitro SPA was found to be 5%. Further investigation of the phenomenon of SPA revealed that: 1. it is associated with ADP-hyperaggregability, i. e. the threshold concentration to induce second wave aggregation is decreased; 2. it is dependant on the increase in pH which occurs in platelet-rich plasma stirring in an aggregometer while concurrent ADP-hyperaggregability is independant of this change in pH; 3. it is associated with malondialdehyde production and the release of endogenous 5- hydroxytryptamine; and that 4. in addition Km and Vmax values for [14C]-5HT incorporation are normal; and that 5. no gross abnormalities of the platelet membrane glycoproteins were apparent although occasionally glycoprotein III was found to be increased. This study demonstrates abnormal platelet behaviour in patients with TIA and Cl where the enzyme system involved in thromboxane production is sufficiently stimulated, by stirring alone, to induce aggregation of platelets and the release reaction. Acetylsalicylic acid abolishes SPA and prolongs the bleeding time with similar characteristics as has been described for normal individuals. Plasma (3-thromboglobulin levels are significantly increased in the patients studied. However, no correlation was established with the incidence of in vitro SPA.

Objectives. We assessed the production of eicosanoids and the effects of very low dose aspirin in patients with stable angina under basal conditions and during rapid atrial pacing.Background. Platelet activation occurs in acute ischemic syndromes but is still controversial in stable angina. Very low dose aspirin is known to be platelet selective and can be used to test the hypothesis of the platelet origin of increased thromboxane production in stable angina.Methods. Urinary excretion of eicosanoids was measured in 42 patients, including 24 patients with and 18 patients without coronary artery disease. The effects of 50 mg/day of aspirin were measured at rest and during pacing-induced ischemia in 10 patients with stable angina and were compared with a similar group of patients not treated by aspirin.Results. Excretion of 11-dehydro-thromboxane B2was 2.6 times higher in patients with stable angina than in healthy subjects (mean [±SEM] 74.8 ± 13.0 [24 patients] vs. 29.0 ± 5.4 [18 patients] ng/mmol of creatinine, p < 0.01). Urinary prostacyclin metabolite levels did not differ between the two groups. Treatment for 8 days with 50 mg/day of aspirin inhibited platelet cyclooxygenase, as reflected by the 97% reduction of in vitro serum thromboxane production. This aspirin regimen normalized the level of urinary thromboxane metabolites in patients with angina (17.3 ± 3.4 ng/mmol of creatinine [10 patients], p < 0.001 from baseline level before treatment) and did not change prostacyclin metabolite levels. Atrial pacing in patients with angina not treated with aspirin caused lactate and thromboxane release into the coronary sinus. In patients with very low dose aspirin therapy, pacing did not cause thromboxane release despite inducing myocardial ischemia. However, fractional lactate extraction decreased less sharply in patients with than without aspirin therapy.Conclusions. Thromboxane production is greatly increased in patients with stable angina. Very low dose aspirin administered to these patients reduces thromboxane synthesis to normal levels, preserves prostacyclin biosynthesis and prevents acute thromboxane release into the coronary circulation during pacing-induced ischemia. Our data suggest that platelets (not monocytes/ macrophages) are activated in stable angina to produce thromboxane.

Reduced glomerular thromboxane receptor sites and vasoconstrictor responses in diabetic rats. Glomerular thromboxane production and urinary thromboxane excretion are increased in early diabetes, but in spite of this renal blood flow and glomerular filtration rate are significantly higher than in control animals. To study the possibility of a defect in thromboxane actions in the kidney, we have measured glomerular thromboxane receptors and the renal hemodynamic response to the administration of a stable thromboxane analog in diabetic rats. Glomerular thromboxane receptors were studied in hyperglycemic diabetic rats 7 to 10 days after injection of streptozotocin (65 mg/kg, i.v.) and in normal controls. Scatchard analysis of equilibrium binding using the thromboxane antagonist, [3H]-SQ29548, demonstrated one class of high affinity thromboxane receptor sites in control (Kd =19.9 ± 2.6nM, N = 16) and diabetic rats (Kd = 19.8 ± 2.1nM, N = 8, P = NS). The number of thromboxane receptors was reduced by 44% in diabetic rats (control, 374 ± 20 vs. diabetic, 210 ± 21 fmol/mg, P < 0.01). Thromboxane binding in diabetic rats was not restored to normal levels by thromboxane synthetase inhibition with OKY046. Diabetic rats had higher renal blood flow (diabetic, 7.03 ± 0.18 vs. control, 6.33 ± 0.13 ml/min, P < 0.05) and glomerular filtration rate (2.42 ± 0.10 vs. 1.96 ± 0.07 ml/min, P < 0.05). Infusion of the stable thromboxane agonist, U46619 (0.1 μg/kg/min), reduced renal blood flow and glomerular filtration rate in all animals, but the constrictor responses were blunted by 50% in hyperglycemic diabetic rats compared with normal controls or euglycemic diabetic rats (P < 0.05). Control of blood glucose with insulin normalized the number of glomerular thromboxane receptor sites, reversed hyperfiltration and restored glomerular responses to thromboxane agonist. The abnormalities of glomerular thromboxane receptors are similar to changes in angiotensin II receptors, and suggest a generalized defect in vasoconstrictor receptors in the diabetic kidney.

Plasma exudation has been suggested to be an important component of the inflammatory response in asthma. Bradykinin elicits many of the features of asthma, including bronchoconstriction, cough, plasma exudation and mucus secretion. In an attempt to quantify local plasma exudation, we have employed a novel low-trauma technique with the aim of challenging and lavaging a central part of the bronchial tree, by selecting a medium sized bronchus.A fibreoptic bronchoscopy was performed in non-smoking healthy volunteers. The instrument was placed proximally in the right upper lobe bronchus. A plastic catheter, equipped with an inflatable latex balloon, was inflated with air (2–4 cmH2O). A solution (100 μ l of either two different concentrations of bradykinin: 0·09 and 0·9 mg ml−1; or normal saline) was instilled through the catheter and distal to the balloon. Eight minutes later a lavage procedure with 10 ml of saline was performed through the catheter. The procedure was then repeated twice, with the other solutions, but from the lingular and middle lobe bronchi. All solutions were given in a blinded fashion, and two different studies were performed. Lavage concentrations of albumin and IgG were quantified as measurements of plasma exudation.In our first study we found that bradykinin challenge significantly increased concentrations of albumin and IgG. In study two, there was no numeric increase in plasma proteins after local bradykinin challenge, but the concentration of thromboxane was significantly increased in lavages from bradykinin-challenged bronchi.Thus, local bronchial administration of bradykinin has the capacity to induce exudation of large plasma macromolecules into the bronchial lumen, as well as local thromboxane production.

Circulating thromboxane (TX) is elevated following endotoxin, and platelets become hyperaggregable. Thromboxane synthesis was therefore studied in platelets during endotoxemia. Rabbit blood and platelets were taken at 0, 60 and 120 min after start of E. coli endotoxin infusion (1.10 microgram/kg min, i.v.). Blood incubation with arachidonic acid (AA, 10(-4) M) generated TXA2, which was measured using a superfused rabbit aorta bioassay. Washed platelets were stimulated with 1-14C AA (0.1 microCi) to generate radiolabeled TXB2, which was isolated by TLC and quantitated by scintillation spectrometry. Control (0 time) platelet count was 488 +/- 10(3)/mm3. In the test group, platelet counts fell to 65% of control at 60 min and to 52% at 120 min, while TXA2 generation was 95% (60 min) and 94% (120 min) of control. In contrast a serial dilution of untreated platelets yielded a progressive decline in thromboxane generation. In endotoxemic platelets, the conversion of 1-14C AA to TXB2 (percent/10(9) platelets) was increased at 120 min (0 min, 34.7; 120 min, 40.0: P less than 0.05). Endotoxemic platelets generated greater amounts of thromboxane than did normal platelets, and this condition may account for platelet hyperaggregability in shock.

Acetylsalicyclic acid (aspirin) inhibits prostanoid synthesis1,2 by irreversible acetylation of fatty acid cyclooxygenase (EC 1.14.99.1)3. It thereby inhibits synthesis of pro-aggregatory thromboxane A2 (TXA2) by platelets2,4 and is widely used in the treatment and prophylaxis of vascular disease. Its efficacy, however, may be reduced since it also inhibits formation of prostacyclin (PGI2)5,6 which is a vasodilator and anti-aggregatory agent7,8. There is uncertainty over the optimum dose regimen for aspirin since although it inhibits platelet thromboxane production for many days4, the magnitude and duration of its effect on PGI2 production by vascular endothelium in vivo is unknown. Resting plasma concentrations of PGI2 (measured as the stable hydrolysis product 6-oxo-PGF1α) are at or below the limit of sensitivity of the most sensitive assays9 and cannot therefore be used to demonstrate a reduction in production. Bradykinin stimulates PGI2 synthesis by cultured human vascular endothelial cells10 and we have shown that it stimulates PGI2 production by man in vivo11. We report here that an oral dose of aspirin (600 mg) causes rapid and substantial inhibition of bradykinin-stimulated PGI2 production, but recovery occurs within 6 hours; this implies that endothelial PGI2 synthesis would be spared most of the time during dosing once daily with even this relatively large dose of aspirin.

To study the effect of the cardiopulmonary bypass (CPB) on the productions of antiaggregatory prostacyclin (PGI2) and proaggregatory thromboxane A2 (TxA2), we collected serial plasma samples from seven patients before, during, and after CPB and assayed them for 6-keto-PGF 1 alpha and thromboxane B2, the main metabolites of PGI2 and TxA2, respectively. The PGI2 production rose significantly (p less than 0.05) following cannulation of the large vessels and remained elevated during the CPB. After discontinuation of CPB, the PGI2 decreased progressively. The TxB2 production also rose during CPB, but later than the increase in PGI2. There was a significant correlation between 6-keto-PGF 1 alpha and TxB2 levels (r = 0.429, p less than 0.001, n = 77). Thus the deficient PGI2 production, or an imbalance between PGI2 and TxA2, does not seem to be responsible for the platelet loss during CPB. By contrast, the human body appears to be protected from platelet aggregation by a surge in endogenous PGI2 during CPB.

SummaryPlatelet aggregation, secretion of 5-hydroxy tryptamine and production of thromboxane B2 were monitored simultaneously in human platelet suspensions in the absence and presence of cyclooxygenase or thromboxane synthetase inhibitors. Aggregation, secretion and thromboxane B2 formation in response to either sodium arachidonate or epinephrine were blocked by aspirin or by 1-N-butyl imidazole suggesting that thromboxane biosynthesis was an essential requirement for platelet activation by these agents. In contrast, thrombin and collagen could apparently induce aggregation and secretion via two pathways: at low doses involving thromboxane production, but at higher doses by a direct mechanism independent of thromboxane biosynthesis. In the case of ADP, inhibition of thromboxane production blocked secretion but had little effect on aggregation, indicating that secretion was probably dependent on thromboxane biosynthesis which probably occurred as a result of aggregation. Thus it appears that although the processes of thromboxane production, release of dense granule constituents and aggregation may often be intimately linked, each process can occur independently of the other, depending upon the stimulus used.

Human peripheral blood polymorphonuclear leukocytes (PMNs) and murine peritoneal macrophages (M phi) were stimulated to generate thromboxane upon treatment with highly purified human interleukin 1/leukocytic pyrogen (IL1/LP) at various concentrations. Thromboxane B2 was measured by radioimmunoassay in the cell-free supernatants of cell suspensions after 1 hour incubation at 37 degrees C. Thromboxane B2 amounts increased in a way which depended on the dose of IL1 added to the cell cultures.

In six awake sheep the control heparin-protamine reaction was associated with a 150-fold rise in arterial plasma thromboxane B2 (TxB2) levels, a 4.5-fold increase in pulmonary vascular resistance, a 20% decrease in cardiac output, a 30% decrease in arterial PO2, and a 30% reduction in arterial white blood cell concentrations. Depletion of 99% of circulating platelets by antibodies did not prevent either acute and severe pulmonary hypertension or increased plasma TxB2 levels induced by heparin-protamine administration. We produced sheep platelet aggregation in vitro with bovine thrombin and measured marked TxB2 release (36.3 +/- 16.3 ng/10(9) platelets). In contrast, neither heparin, protamine, nor heparin-protamine complexes over a 10,000-fold range of concentrations induced platelet aggregation and release of thromboxane in vitro. Therefore sheep platelets are not the source of thromboxane production associated with acute pulmonary hypertension during the heparin-protamine reaction, and other cells must produce the thromboxane.

1. The aspirin concentrations previously reported to inhibit platelet aggregation in vitro (40–500 μmol/l) are much greater than those required in vivo in man (5 μmol/l). 2. Human platelet-rich plasma was incubated with buffer or various aspirin concentrations at 37°C for up to 4.5 h. Platelet aggregation and thromboxane generation were measured in response to collagen (0.4–6.3 μg/ml) and adenosine 5′-pyrophosphate (0.5–4 μmol/l). 3. The concentration of aspirin needed to inhibit platelet aggregation in response to a critical concentration of aggregating agent (lowest concentration to cause greater than 50% aggregation) was lower than that required for higher concentrations of aggregating agent. 4. With more prolonged incubation times with aspirin, lower concentrations of aspirin inhibited platelet aggregation. 5. Inhibition of platelet aggregation and thromboxane formation by 10 μmol/l aspirin was maximal by 90 min. There was progressive inhibition by 3 μmol/l aspirin during incubation for 270 min. By the end of this time there was also significant inhibition by 1 μmol/l aspirin. 6. The apparent discrepancy between inhibitory aspirin concentrations in vivo and those observed in vitro in previous studies appears to have been resolved by extending the incubation time of platelets with low aspirin concentrations, thus mimicking the conditions in vivo.

A reproducible electrical injury to the hind limb was produced in rats, an injury characterized by progressive tissue necrosis. Serial histochemical examinations of cross sections with a peroxidase-antiperoxidase method revealed increased production of arachidonic acid metabolites, especially thromboxane, at distal sites near the entrance wound and in periosseous tissues more proximally. Levels of these vasoactive substances remained elevated during the time of progressive necrosis and demarcation of seemingly normal, uninjured tissue. Treatment with agents capable of blocking thromboxane production allowed tissue salvage, as evidenced by a decreased autoamputation rate and an increased total surviving length. From this study it appears that an electrical injury is thermal trauma, producing elevated levels of arachidonic acid metabolites in areas of greatest heat production. Prolonged thromboxane excess, with resultant vasoconstriction and thrombosis in the microcirculation, is seen to play a key role in the progressive tissue loss characteristic of the injury. The use of antithromboxane agents may be of benefit in halting this progression and salvaging tissue in these devastating injuries.

3-Hydroperoxy-3-methylphthalide (3-HMP), a structural analog of acetylsalicylic acid (ASA), was found to have some antiinflammatory properties which are distinct from those of ASA. 3-HMP inhibits human platelet aggregation and ATP release in response to low concentrations of collagen but is less effective than ASA. 3-HMP inhibits prostaglandin and thromboxane production from exogenous [14C]arachidonic acid by human platelet lysates in vitro and does so at lower concentrations than ASA (3-HMP IC50 = 10 microM; ASA IC50 = 50 microM). 3-HMP is also more effective than ASA as an inhibitor of prostacyclin-like activity production by rings of rabbit aorta. Human polymorphonuclear (PMN) leukocyte [14C]arachidonic acid metabolism is inhibited by 3-HMP but not ASA. In urethane-anesthetized rats, 3-HMP (10 mg/kg intravenously) is effective in inhibiting PMN leukocyte accumulation in response to intrapleural carrageenan administration whereas ASA is ineffective (100 mg/kg intravenously). This hydroperoxy analog of ASA has antiinflammatory activity which may result from a combination of the ASA-like and hydroperoxide-related pharmacological properties.

Tissue contents of prostaglandin E2 (PGE2), thromboxane B2 (TxB2, a metabolite of proaggregatory thromboxane A2) and 6-keto-PGF1 alpha (a metabolite of antiaggregatory prostacyclin, PGI2) were measured from ovarian cancer (n = 13), borderline malignant, benign ovarian tumor and normal ovary of postmenopausal women. TxB2 contents were significantly (p less than 0.05) increased in metastatic ovarian cancer tissue, compared to local ovarian cancer, borderline malignant, benign tumor or normal ovarian tissues, while in 6-keto-PGF1 alpha production there was no difference between these groups. PGE2 contents were also increased in metastatic ovarian cancer tissue. These data suggest the association between local PG production and advanced ovarian cancer tissue.

This study examines the question of whether the aspirin-induced delay in the recovery of platelet cyclooxygenase pathway activity, as measured by RIA of thromboxane B2, results from a direct effect on megakaryocyte cyclooxygenase. From our measurement of recovery of TXB2 and information on megakaryocyte transit time in rats, we propose that thromboxane synthesis may represent a relatively late step in the differentiation of megakaryocytes. Megakaryocyte thromboxane production was depressed by 70% and that of platelets by 85% at two hr after 20 mg/kg oral aspirin dissolved in DMSO. Full megakaryocyte thromboxane recovery occurred by 72 hr and preceded complete platelet thromboxane recovery by 24 hr. Whereas megakaryocyte thromboxane synthesis showed substantial recovery by 36 hr after aspirin, platelet recovery did not begin for 24 hr and achieved a maximal recovery rate over the following 12 hr. This finding is consistent with predictions based upon human data for both megakaryocyte labeling studies and post-aspirin platelet recovery. We conclude from our data and from estimates of megakaryocyte maturation times in marrow, that thromboxane synthesis develops in rat megakaryocytes after approximately 48 hr of cytoplasmic differentiation toward platelet shedding. This metabolic capacity therefore serves as a marker of megakaryocyte differentiation.

The effect of atrial pacing on intracoronary thromboxane production was investigated in 35 patients with stable (n = 19) or unstable (n = 16) angina. Arterial and coronary sinus thromboxane B2, the stable metabolite of thromboxane A2, myocardial lactate extraction and thermodilution coronary sinus flow were measured before, during and immediately after atrial pacing until the onset of angina. Pacing did not significantly increase coronary sinus thromboxane B2 (rest, 233 +/- 107 pg/ml; pacing, 249 +/- 154 pg/ml; postpacing, 330 +/- 309 pg/ml) (mean +/- standard deviation) despite a moderate increase in arterial thromboxane B2 (rest, 270 +/- 170 pg/ml; pacing, 387 +/- 364 pg/ml; postpacing, 446 +/- 420 pg/ml) (all changes probability [p] less than 0.05). A positive transmyocardial thromboxane B2 gradient, suggesting intracoronary thromboxane A2 production, occurred in only five patients at rest (gradient = 60 +/- 35 pg/ml). During pacing, a transmyocardial thromboxane B2 gradient was not observed despite myocardial lactate production in 18 patients. A postpacing gradient was observed in eight patients (gradient = 284 +/- 349 pg/ml). These gradients were significantly more frequent in patients who produced lactate during pacing (7 of 18) than in patients without lactate production (1 of 17) (p less than 0.05). In patients with and without a postpacing gradient, coronary vascular resistance decreased with pacing and returned to rest levels immediately after pacing, suggesting that a postpacing thromboxane gradient does not significantly alter coronary tone. These data suggest that: 1) pacing-induced angina is usually not associated with substantial intracoronary thromboxane A2 production; 2) in a minority of patients who develop intracoronary thromboxane A2 production, the amount is small and does not produce significant coronary vasoconstriction.

Publisher Summary This chapter illustrates the biochemical mechanism of glucocorticoid and phenytoin-induced cleft palate. The production of cleft palate by glucocorticoids and phenytoin is a complicated interference in a complex developmental program involving many genetic and biochemical processes. The H-2 histocompatibility region includes genes that affect (1) susceptibility to glucocorticoid- and phenytoin-induced cleft palate, (2) glucocorticoid receptor level in a variety of tissues including maternal and embryonic palates, adult thymuses, and lungs, and (3) the degree of inhibition of prostaglandin and thromboxane production by glucocorticoids and phenytoin in thymocytes. A gene linked to a minor histocompatibility locus on the second chromosome also influences susceptibility to glucocorticoid- and phenytoin-induced cleft palate. Phenytoin is an alternate ligand for the glucocorticoid receptor affecting prostaglandin and/or thromboxane production. The capacity of glucocorticoids to induce cleft palate is correlated with their anti-inflammatory potency. Some of the anti-inflammatory effects of glucocorticoids can be explained by the inhibition of prostaglandin and/or thromboxane release, which in turn could be caused by the inhibition of arachidonic acid release from phospholipids.

The eicosanoids constitute a large and still growing family of oxygenated and biologically active compounds, which are derived from certain polyunsaturated, essential fatty acids. The best known of these are the prostaglandins (PGs) and thromboxanes (TXs) of the 2-series, i. e. bisenoic compounds originating in arachidonic acid.

Effects of a new imidazole derivative, sodium 4-[alpha-hydroxy-5-(imidazolyl)-2-methylbenzyl]-3,5-dimethyl benzoate dihydrate (Y-20811), on thromboxane (TX) production and platelet aggregation were investigated. Y-20811 inhibited TX synthetase (IC50 = 2.2 X 10(-8) M) and platelet aggregation induced by arachidonic acid (AA) in human, guinea pig and rabbit platelets in vitro. Administered orally to rabbits, Y-20811 at a dose of 1 mg/kg decreased serum TXB2 concomitant with increasing 6-keto PGF1 alpha and at a dose of 3 mg/kg inhibited AA-induced platelet aggregation, in both cases for at least 48 hours. Y-20811 (0.3 mg/kg/day) administered to rabbits for 7 days decreased serum TXB2 levels by 50-90% during the medication, and these levels were restored to initial values 3 days after withdrawal of the drug. At a dose of 1 mg/kg Y-20811 protected rabbits against death induced by AA (2 mg/kg). These results indicate that Y-20811 is a selective and long-lasting TX synthetase inhibitor and an anti-aggregating agent useful in preventing thrombotic disorders.

The metabolism of thromboxane B2 was studied in the rabbit. The aim of the study was to identify metabolites in blood and urine that might serve as parameters for monitoring thromboxane production in vivo. [5,6,8,9,11,12,14,15-3H8]-Thromboxane B2 was administered by i.v. injection to rabbits, and blood samples and urine were collected with brief intervals. The metabolic profiles were visualized by two-dimensional thin layer chromatography and autoradiography, and the structures of five major metabolites were determined using chromatographic and mass spectrometric methods. In urine the major metabolites were identified as 11-dehydro-TXB2 and 2,3,4,5-tetranor-TXB1, and other prominent products were 11-dehydro-2,3,4,5-tetranor-TXB1, 2,3-dinor-TXB1 and 2,3-dinor-TXB2. In the circulation, TXB2 was found to disappear rapidly. The first major metabolite to appear was 11-dehydro-TXB2, which also remained a prominent product in blood for the remainder of the experiment (90 min). With time, the profile of circulating products became closely similar to that in urine. TXB2 was not converted into 11-dehydro-TXB2 by blood cells or plasma. The dehydrogenase catalyzing its formation was tissue bound and was found to have a widespread occurrence: the highest conversion was found in lung, kidney, stomach and liver. The results of the present study suggest that 11-dehydro-TXB2 may be a suitable parameter for monitoring thromboxane production in vivo in the rabbit in blood as well as urinary samples, and possibly also several tissues. This was also demonstrated in comparative studies using radioimmunoassays for TXB2 and 11-dehydro-TXB2.

We studied the effect of prostaglandin E1 (PGE1) and prostacyclin (PGI2) infusion on thromboxane A2 (TxA2) production in endotoxin-induced lung injury by using a chronic lung lymph fistula model in sheep. Seven sheep received alternately Escherichia coli endotoxin and endotoxin plus PGE1 (1 μg/kg/min). Six sheep received PGI2 (0.2 μg/kg/min) instead of PGE1. The infusion of both PGE1 and PGI2: (1) decreased TxA2 production (as measured by RIA of its metabolite TxB2); (2) decreased pulmonary hypertension; and (3) decreased interstitial edema. The effect of PGE1 and PGI2 infusion on TxA2 production may be independent of effects on the reaction of platelets and leukocytes.

In studies aiming at establishing a role for thromboxanes in various physiological or pathological conditions, it is often desirable to measure the endogenous thromboxane production. Since TXA2 is difficult to measure as such, a common approach in such studies is to monitor its stable hydrolysis product, TXB2, instead, and to compare obtained TXB2 levels in, for example, plasma samples in patient and control groups. Numerous such studies have been published to date; however, even the so called “basal” TXB2 levels reported therein vary widely. Not infrequently, “basal” TXB2 levels were found to be around 100–200 pg/ml plasma or even higher, however, others claim to find values below 15, 10 or even 2 pg/ml (e.g., refs. 1–6). It is difficult to explain such differences between studies where identical assay methods have been used (for example a commercially available TXB2 radioimmunoassay); that is, if the obtained data really reflect the endogenous situation.

Placentas obtained from women with preeclampsia produce more thromboxane and less prostacyclin than normal. They also produce more progesterone than normal. This study tested whether a progesterone concentration equivalent to that in the medium after in vitro incubation of placentas from women with preeclampsia (greater than or equal to 1.5 x 10(-5) mol/L) could cause an imbalance of increased thromboxane and decreased prostacyclin production by normal placentas. Fresh term placental tissues were incubated for 48 hours in the absence or presence of various concentrations of progesterone and/or estradiol. Prostacyclin and thromboxane were determined by radioimmunoassay of their stable metabolites, 6-keto-prostaglandin F1 alpha and thromboxane B2. A progesterone concentration of 1.5 x 10(-5) mol/L significantly (p less than 0.001) inhibited prostacyclin production, compared with control, but lower concentrations did not. Estradiol at concentrations present in the medium after incubation of either normal or preeclamptic placental tissue did not significantly affect prostacyclin production, nor did it prevent progesterone at 1.5 x 10(-5) mol/L from inhibiting prostacyclin. Thromboxane production was not affected by either progesterone or estradiol.In vitro addition of progesterone at a concentration equivalent to that in the medium after incubation of preeclamptic placentas inhibited prostacyclin production by normal placentas to a rate characteristic of preeclampsia. However, it did not increase thromboxane.Increased trophoblast progesterone in preeclampsia may act by a paracrine mechanism to inhibit prostacyclin synthesis in placental vasculature. Progesterone's selective inhibition of prostacyclin without affecting thromboxane may be due to the compartmentalization of thromboxane production to trophoblast and stroma and prostacyclin production to placental vasculature.

1. Exposure of platelets to exogenous arachidonic acid results in aggregation and secretion, which are inhibited at high arachidonate concentrations. The mechanisms for this have not been elucidated fully. In our studies in platelet suspensions, peak aggregation and secretion occurred at 2-5 microM-sodium arachidonate, with complete inhibition around 25 microM. 2. In platelets loaded with quin2 or fura-2, the cytoplasmic Ca2+ concentration, [Ca2+]i, rose in the presence of 1 mM-CaCl2 from 60-80 nM to 300-500 nM at 2-5 microM-arachidonate, followed by inhibition to basal values at 25-50 microM. Thromboxane production was not inhibited at 25 microM-arachidonate. Cyclic AMP increased in the presence of theophylline, from 3.5 pmol/10(8) platelets in unexposed platelets to 8 pmol/10(8) platelets at 50 microM-arachidonate; all platelet responses were inhibited with doubling of cyclic AMP contents. 3. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine attenuated the inhibitory effect of arachidonate, suggesting that it is mediated by increased platelet cyclic AMP and that it is unlikely to be due to irreversible damage to platelets. 4. Aspirin or the combined lipoxygenase/cyclo-oxygenase inhibitor BW 755C did not prevent the inhibition by arachidonate of either [Ca2+]i signals or aggregation induced by U46619. 5. Thus high arachidonate concentrations inhibit Ca2+ mobilization in platelets, and this is mediated by stimulation of adenylate cyclase. High arachidonate concentrations influence platelet responses by modulating intracellular concentrations of two key messenger molecules, cyclic AMP and Ca2+.

The susceptibility of diabetic patients to pathophysiological complications and abnormalities in their platelet function prompted this investigation. A group of 180 insulin-dependent subjects were randomly selected: 38.9% with no other clinical complications; 16.7% with retinopathy; 15.0% - neuropathy; 3.9% - nephropathy; 4.4% - peripheral vascular disease; 21.1% - coronary arterial disease. Twenty seven subjects with no diabetic disease were used as controls. Thromboxane B 2 [stable metabolite of thromboxane A 2 (TXA 2 )], generated by exogenous arachidonic acid and quantitated by electron-capture gas chromatography (EC-GC) was significantly higher in platelets of diabetics with clinical complications than in controls. Platelets in platelet rich plasma (PRP) processed from citrated whole blood of diabetic subjects were more sensitive to ADP- or collagen-induced aggregation than those of non-diabetics. Platelets similarly obtained from diabetics, but separated on agarose gel column rather than obtained in PRP, aggregated with greater sensitivity to arachidonic acid than those of controls. Plasma total cholesterol and triglyceride concentrations were similar in diabetic and non-diabetic subjects. Platelet total phospholipid concentration, percent composition of molecular species and fatty acid distribution were similar in both groups of subjects indicating a similar substrate concentration for prostaglandin biosynthesis. It is postulated that in the platelets of diabetic subjects: a) the observed increase in TXA 2 synthesis is due to the increased activity of the thromboxane synthetase system at one or more sites; b) this increased enzyme activity is the mechanism of greater platelet sensitivity to aggregation.

We have examined the influence of an allogeneic stimulus on T lymphocyte prostanoid synthesis. PGE2 and TXB2 (the stable product of TXA2) were determined by radioimmunoassay. When T cells were derived from alloimmunized animals, the production of PGE2 and TXA2 was significantly higher than that of non-immunized cells. Moreover, T immune lymphocytes in the presence of the immunized alloantigen showed an increment in prostanoid production. We propose that the allogeneic stimulus provides a signal to the T lymphocytes for an increase in prostanoid synthesis.

To study the production of the vasodilatory prostacyclin (PGI2) and vasoconstrictory thromboxane A2 (TxA2) by the human umbilical vessels, vascular segments collected from the cords of healthy newborns (n = 15) after normal pregnancies were superfused with Eagle's medium at 37 degrees C under continuous oxygenation. The stable metabolites of PGI2 and TxA2, 6-keto-prostaglandin F1 alpha and thromboxane B2 (TxB2) respectively, were measured from the superfusates with radioimmunoassays. All vascular segments studied produced 6-keto-PGF1 alpha and TxB2 without any significant difference between the arteries and veins. 6-keto-PGF1 alpha was produced some 10-15 times more than TxB2. 17-beta-estradiol at concentrations of 10, 100 and 1000 ng/ml dose-dependently stimulated the 6-keto-PGF1 alpha formation, but had no effect on TxB2 generation. Progesterone 50, 500 and 5000 ng/ml caused no changes in 6-keto-PGF1 alpha or TxB2 generations but even the smallest concentration of progesterone abolished the stimulating effect of estradiol. These results thus suggest that the high circulating levels of 17-beta-estradiol and progesterone in the umbilical vessels may be important in the regulation of PGI2 synthesis in the umbilical vessels in vivo.

The discovery of the imbalance of increased thromboxane and decreased prostacyclin production in preeclamptic women has explained the cause of the major clinical symptoms of this disorder and has formed the basis and rationale for clinical studies with low-dose aspirin to treat preeclampsia. Low doses of aspirin (60 to 81 mg/day) have a remarkable ability to inhibit thromboxane production selectively without significantly inhibiting prostacyclin production. Therefore the actions of thromboxane to increase vasoconstriction, stimulate platelet aggregation, increase uterine contractility, and decrease uteroplacental blood flow are attenuated, and the ratio of thromboxane to prostacyclin is altered in favor of prostacyclin. Prostacyclin promotes vasodilation, inhibits platelet aggregation, decreases uterine contractility, and increases uteroplacental blood flow. The initial clinical studies with low doses of aspirin are very encouraging with respect to the treatment and prevention of preeclampsia. Substantial evidence already indicates that low-dose aspirin therapy decreases the incidence of preeclampsia; it decreases the maternal systemic arterial pressor response to angiotensin II; and it does not seem to be harmful to the fetus. Treatment of preeclampsia with prostacyclin appears to be contraindicated because prostacyclin is a potent systemic vasodilator and the clinical outcome of preeclamptic women infused with prostacyclin has been poor. The mechanism whereby low-dose aspirin preferentially inhibits thromboxane synthesis is not known.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study examines extracorporeal prostaglandin production during routine and simulated haemodialysis in healthy volunteers. The roles of dialyser membranes and alcohol washing procedures were investigated. The source of extracorporeal prostaglandin E2 was estimated by a specific platelet cyclo-oxygenase antagonist. Extracorporeal thromboxane production, with and without antagonist, was compared in an attempt to substantiate the role of the cyclo-oxygenase pathway by sources other than platelets. Clinical investigations show that prostaglandin liberation in the extracorporeal bloodstream is detectable. Additionally, laboratory results suggest an association between the type of dialyser membrane and extracorporeal prostaglandin release. The amount of prostaglandin E2 was reduced when dialysers were pre-washed with alcohol. Furthermore, it was experimentally possible to determine that a large part of extracorporeal prostaglandin E2 is released by sources other than platelets, suggesting a possible role of monocytes in extracorporeal prostaglandin production.

Leukotriene D4 (LTD4) administered intravenously to anesthetized, spontaneously breathing guinea pigs elicited decreases in dynamic lung compliance (Cdyn) and airway conductance (GAW) with a maximal response achieved at 0.5 min. Simultaneously, plasma levels of the thromboxane metabolite, TxB2, and the prostacyclin metabolite, 6-keto-PGF1 alpha, increased 10-fold over pre-LTD4 levels. Pretreatment of the guinea pigs with meclofenamic acid delayed the onset of the LTD4-induced bronchoconstriction, antagonized the magnitude of the decreases in Cdyn and GAW, and blocked the increase in plasma TxB2 and 6-keto-PGF1 alpha levels. The thromboxane synthetase inhibitor, UK 37,248, suppressed the LTD4-induced bronchoconstriction, while it completely blocked TxB2 production without significantly affecting 6-keto-PGF1 alpha. The SRS-A end organ antagonist, FPL 55712, blocked both the LTD4-induced bronchoconstriction and the production of the arachidonic acid metabolites. These results suggest that thromboxane A2 plays an important role in mediating part of the bronchoconstriction elicited by intravenously administered LTD4 in the guinea pig.

Previous work has shown enhanced aggregation and thromboxane synthesis by platelets from diabetic subjects. We have compared thromboxane synthesis by platelets from normal subjects with that of platelets from two groups of insulin-dependent diabetic patients: one group receiving conventional depot insulin therapy and the other continuous subcutaneous insulin infusions. Thromboxane synthesis was significantly higher with platelets from the conventionally-treated diabetic patients than that observed for control subjects. Patients on continuous insulin infusions were similar to control subjects. This group of patients also had better control of glycemia. The effect on thromboxane production might be related to normalization of plasma lipids which occurs with continuous infusion insulin therapy.

Prostacyclin and thromboxane production was measured in human umbilical cord arteries bathed in clotting human blood and compared with arteries bathed in Krebs buffer or clotting blood without vessels. In comparison with the combined system, vessels in buffer generated only minute amounts of immunoreactive thromboxane B2 while blood alone generated only minute amounts of immunoreactive 6-oxo-PGF1 alpha. Incubation of vessels in blood was associated with an enhanced 6-oxo-PGF1 alpha formation at 1-2 h of incubation, demonstrating an active prostacyclin synthetase and a transfer of the platelet endoperoxide precursor to this enzyme. This new combined system was used to reevaluate the selectivity of cyclooxygenase inhibitors for vascular and platelet derived eicosanoid formation. With respect to 6-oxo-PGF1 alpha accumulation, the IC50 value [mumoles/l] for tiaprofenic acid (8.5 +/- 3.0) was significantly higher than that for diclofenac (0.14 +/- 0.03) (P less than 0.05) while acetylsalicylic acid (18.0 +/- 7.0) was less potent than diclofenac and indomethacin (2.4 +/- 1.0) (P less than 0.05). With respect to thromboxane B2 formation, the IC50 values for diclofenac (0.26 +/- 0.04), indomethacin (IC50 0.30 +/- 0.05) and tiaprofenic acid (IC50 0.71 +/- 0.08) were not significantly different from each other. Acetylsalicylic acid (7.7 +/- 1.8) was less potent than either of the other compounds (P less than 0.01). While these IC50 values might suggest different potencies for inhibition of vascular and platelet cyclooxygenases by tiaprofenic acid and, possibly, indomethacin, statistical analysis was not possible because of different slopes of the dose-response curves.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the relationship between glucose levels and platelet thromboxane production or aortic prostacyclin production, using radioimmunoassay (RIA) to measure thromboxane B2 and 6-keto-PGF1 alpha. We found a direct relationship (p less than .05) between plasma glucose levels and thromboxane A2 production by arachidonate stimulated platelets in platelet rich plasma of normal mice. However, when mice were deprived of food overnight, the glucose level fell but the TxB2 production rose significantly. Moreover, mice with streptozotocin diabetes had significantly elevated glucose levels, but normal TxB2 production, which also rose significantly after fasting. Thus in our laboratory both fasting and diabetes nullify or reverse the direct relationship between glucose levels and TxB2 production seen in normal fed mice. This makes it difficult to ascribe the correlation between glucose and TxB2 levels in normal fed animals to cause and effect. RIA revealed an inverse correlation between glucose levels and 6-keto-PGF1 alpha production which was highly significant in aortas taken from fasted mice and stimulated for 10 minutes with 0.1 mM arachidonate. This inverse correlation was present with either normal or diabetic aortas. Moreover, fasting increased the production of 6-keto-PGF1 alpha. However there was a significant elevation of 6-keto production by aortas of mice with diabetes of 5-6 weeks duration, compared to aortas of normal mice. Therefore either diabetes in these mice reversed a normal inhibitory effect of glucose on 6-keto production, or else the inverse correlation between glucose levels and 6-keto production does not represent a cause and effect relationship between the two variables.

Megakaryocytes isolated in high purity from guinea pigs produced thromboxane B2 in response to exogenously provided arachidonic acid. This production was inhibited by in vitro treatment with acetylsalicylic acid with a concentration response relationship similar to that seen in platelets. During in vitro culture, the aspirin-treated megakaryocytes recovered thromboxane synthetic ability. Following a lag of 12 hours, recovery of megakaryocyte thromboxane production resumed at a rate of 16% of control per day. This recovery was inhibited by the addition of cycloheximide to the culture medium.

We have compared the production of prostaglandins in fibroblast-like cells and endothelial cells in culture. Of the fibroblasts studied 10T1/2, SHE, BP6T and KD produce significant amounts of PGI2, PGE2 and PGF2F2 under optimal culture conditions, but only 3T3 and BHK produce TXA2 in addition to PGI2. The adult bovine aortic endothelial cells (ABAE) and fetal bovine heart endothelium (FBHE) synthesise PGI2 but not TXA2, either from endogenous or exogenous substrates. Both cultured endothelial cells and fibroblasts apparently lack 15-hydroxyprostaglandin dehydrogenase pathway and the ability to convert 6-Keto PGF1 alpha into 6-Keto PGE1. PGI2 production by ABAE was 3-5 times that of FBHE, about twice that of SHE cells and 6-8 times that of 10T1/2 or BP6T cells. Supernatants or media obtained from these cells inhibited aggregation of human platelet-rich plasma, a known biological effect of PGI2. This effect was abolished when cell monolayers were preincubated with indomethacin or tranylcypromine. RIA and chromatographic data of 6-Keto PGF1 alpha from these experiments confirmed that the inhibition of platelet aggregation was due to the formation of PGI2. The production of all prostanoids by endothelial cells or fibroflasts was significantly higher during the exponential phase of growth as compared to confluent monolayers. We propose that fibroblasts 10T1/2 or SHE can serve as useful experimental models for the study of metabolism and transport of PGI2 and/or TXA2 in cells of nonendothelial nature.