Platelet Prostaglandin Biosynthesis Research Articles

Cycle Network Model of Prostaglandin H Synthase-1.

The kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was developed to investigate its complex network kinetics and non-steroidal anti-inflammatory drugs (NSAIDs) efficacy in different in vitro and in vivo conditions. To correctly describe the complex mechanism of PGHS-1 catalysis, we developed a microscopic approach to modelling of intricate network dynamics of 35 intraenzyme reactions among 24 intermediate states of the enzyme. The developed model quantitatively describes interconnection between cyclooxygenase and peroxidase enzyme activities; substrate (arachidonic acid, AA) and reducing cosubstrate competitive consumption; enzyme self-inactivation; autocatalytic role of AA; enzyme activation threshold; and synthesis of intermediate prostaglandin G2 (PGG2) and final prostaglandin H2 (PGH2) products under wide experimental conditions. In the paper, we provide a detailed description of the enzyme catalytic cycle, model calibration based on a series of in vitro kinetic data, and model validation using experimental data on the regulatory properties of PGHS-1. The validated model of PGHS-1 with a unified set of kinetic parameters is applicable for in silico screening and prediction of the inhibition effects of NSAIDs and their combination on the balance of pro-thrombotic (thromboxane) and anti-thrombotic (prostacyclin) prostaglandin biosynthesis in platelets and endothelial cells expressing PGHS-1.

Effects of dietary fish oil supplementation on the fatty acid composition of the human platelet membrane: demonstration of selectivity in the incorporation of eicosapentaenoic acid into membrane phospholipid pools.

The fatty acid composition of membrane phospholipids of stimulated and unstimulated platelets was studied in six normal volunteers given a daily dietary supplement of a fish oil rich in eicosapentaenoic acid (EPA) for 4 weeks. The supplement was equivalent to 1.8 g of EPA daily. Thromboxane synthesis and platelet aggregation responses to sodium arachidonate, thrombin and the ionophore A23187 were also investigated. A marked increase in the relative EPA content of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was noted after 2 and 4 weeks fish oil supplementation. However, there was no incorporation into phosphatidylinositol (PI) or phosphatidylserine (PS). The relative arachidonic acid (AA) content of PC and PE was significantly reduced at 2 and 4 weeks but that of PI and PS remained unchanged. Significant reductions in the relative linoleic acid content of total phospholipids, PC and PE were also noted. Stimulation of platelets obtained after 4 weeks fish oil supplementation by thrombin and A23187 was associated with a marked reduction in the AA content of PI and a minor reduction in that of PE. There was no change in the relative proportions of EPA in PI, PS, PC or PE after stimulation. Throughout the study there were no significant changes in platelet aggregation responses or in platelet thromboxane production. Our results indicate that the incorporation of EPA into the platelet membrane phospholipids is selective and that if PI is the major source of AA for platelet prostaglandin biosynthesis then the reported beneficial effects of EPA on haemostasis cannot be explained on the basis of its incorporation into and mobilization from the platelet membrane phospholipid pool.

Platelet-vessel wall interactions in hemostasis: implication of 5-hydroxytryptamine.

5-Hydroxytryptamine (5-HT) is implicated in the platelet-vessel wall interactions during hemostasis. Indeed, the tail bleeding time and the initial blood loss in rats are significantly increased by platelet amine depletion with reserpine/parachlorophenylalanine, by the specific 5-HT2 receptor antagonists ketanserin, R 55 667 and R 56 433 and by the ergot derivatives metergoline and methysergide. In the dose range affecting hemostasis, the serotonergic receptor antagonists also inhibit the 5-HT-amplified aggregation of ADP-sensitized platelets in rat whole blood. Bleeding times are also prolonged by alpha 1-adrenergic receptor antagonism with prazosin or phentolamine, by specific thromboxane A2-synthetase inhibition with R 59 655 or dazoxiben, and by anticoagulant treatment. The latter three mechanisms of action are not explanatory for the effect of the tested serotonergic antagonists, since bleeding times are prolonged by specific 5-HT2 receptor antagonists without an effect on alpha-adrenergic receptors, on platelet prostaglandin biosynthesis or on coagulation and fibrinolysis. The present study shows the interplay between the platelet-derived mediators 5-HT and TXA2 at the level of the platelet or the blood vessel to be of major importance in the platelet-vessel wall interactions during hemostasis.

Near-normal glycaemic control does not correct abnormal platelet reactivity in diabetes mellitus.

The effect of improved glycaemic control, effected by 16 weeks continuous subcutaneous insulin infusion (CSII), on platelet aggregation and platelet prostaglandin biosynthesis has been assessed in a group of 11 diabetic patients with painful peripheral neuropathy. Before CSII and compared with results obtained on samples from age- and sex-matched control subjects, there was enhanced reactivity of the platelets from diabetic patients to ADP, collagen and sodium arachidonate (NaAA). There was also increased thromboxane B2 (TXB2) production after platelet stimulation by NaAA. In contrast, collagen-induced thromboxane production by platelets from diabetic patients was significantly less than that of platelets from controls. Treatment by CSII resulted in a statistically significant improvement in glycaemic control and this was maintained for the 16 week period of the study. At 16 weeks and in the presence of near-normal glycaemic control, the enhanced platelet reactivity in response to collagen and NaAA persisted and that to ADP was further increased. Collagen-induced thromboxane production was, however, corrected by CSII.

Biosynthesis of prostanoids, tissue fatty acid composition and thrombotic parameters in rats fed diets enriched with docosahexaenoic (22:6n3) or eicosapentaenoic (20:5n3) acids

The objective of this experiment was to elucidate the effect(s) of eicosapentaenoic (20:5n3) vs docosahexaenoic (22:6n3) acid on prostaglandin biosynthesis and related thrombotic parameters. Diets were formulated to contain oils absent in (control) or enriched with either 20:5n3 (EPA) or 22:6n3 (DHA). The diets were fed to rats for three weeks and the following evaluated: 1) bleeding time; 2) blood viscosity; 3) platelet aggregation; 4) tissue fatty acids; 5) serum thromboxane (TXB 2), aortic prostacylin (6-keto) and 6) arachidonic acid conversion to eicosanoids by lung microsomes. There were no significant differences between treatments for bleeding time, red blood cell viscosity or platelet aggregation. In EPA fed rats 20:5n3 increased significantly in platelet and aorta phospholipids. In platelets and aorta 20:4n6 was slightly decreased in EPA and DHA animals. Platelet 22:6n3 levels were not altered by treatment, but 22:6n3 increased in the aorta of EPA and DHA fed rats. Similar changes were noted in lung and liver fatty acid composition. Serum TXB 2levels were significantly decreased only in the EPA vs control group. No differences were noted for aortic 6-keto levels or in the amount of hydroxy fatty acids, PGE, TXB 2or PGF 2 produced by lung microsomes. While fish oils have been shown to alter hematologic parameters in humans this study suggests that the rat is not similarly affected. Furthermore, it is evident that in the rat, 20:5n3 and not 22:6n3 is responsible for the alterations in platelet prostaglandin biosynthesis; however, these observations may not be directly applicable to other species.

Alteration of monocrotaline pyrrole‐induced cardiopulmonary effects in rats by hydralazine, dexamethasone or sulphinpyrazone

The effects of intraperitoneal hydrallazine, dexamethasone, or sulphinpyrazone on the toxicity of monocrotaline pyrrole (MCTP) were examined in rats 14 days after injection of MCTP (5 mg kg-1, i.v.). MCTP alone caused increases in lung weight, and of both lactate dehydrogenase activity and protein concentration in bronchopulmonary lavage fluid. Right ventricular hypertrophy also occurred. Hydrallazine (3 mg kg-1, daily), a vasodilator and platelet prostaglandin synthesis inhibitor, reduced the degree of right ventricular hypertrophy and the elevation in the concentration of protein in lavage fluid. Dexamethasone (27 micrograms kg-1, daily), an anti-inflammatory agent and inhibitor of phospholipase, also reduced the right ventricular hypertrophy and the increased protein concentration in lavage fluid caused by MCTP. Sulphinpyrazone (100 mg kg-1, twice daily), an inhibitor of platelet prostaglandin biosynthesis, prevented right ventricular hypertrophy in the MCTP treated rats without affecting any of the indices of lung injury. These results provide further support for the hypothesis that platelets and vasoconstrictor agents play a role in the development of MCTP-induced pulmonary hypertension.

Activation of platelet prostaglandin biosynthesis pathway during neoplastic cell-induced platelet aggregation.

In a previous study we found a correlation between metastatic potential and platelet aggregating activity in sublines of a benzopyrene-induced murine fibrosarcoma ( mFS6 ); the purpose of the present work was to elucidate the role of thromboxane biosynthesis by platelets and/or by neoplastic cells in the activation of platelets in this system. The cells of the more malignant subline induced higher aggregation and TxB2 production than those of the non metastasizing one. The supernatants of aggregating cell suspensions contained very few TxB2; furthermore, preincubation of platelets with ASA or Apyrase resulted in inhibition of aggregation and TxB2 production, while preincubation of the cells was ineffective; these results suggest the platelet origin of the measured TxB2 and indicate that platelet-derived ADP plays an important role in their activation, while the production of ADP by the cells does not seem to be relevant in this model. The involvement of platelet prostaglandin biosynthesis pathway in neoplastic cell induced platelet activation could play an important role in the development of platelet-dependent tumour metastasis.

Aspirin, prostaglandins, and peripheral gangrene

The effect of aspirin on platelet thromboxane and venous prostaglandin production was determined in a group of 15 healthy adults. Ten received a single 300 mg dose of aspirin and one a single 150 mg dose. The remaining four persons received 40 mg aspirin four times daily. Platelet thromboxane production was measured by radioimmunoassay for thromboxane B 2; vein biopsy was performed before and at a predetermined interval after aspirin ingestion; prostacyclin production was assessed by radioimmunoassay for 6-keto prostaglandin F 1α-. One hundred percent inhibition of thromboxane B 2 production and 61 to 100 (mean 83) percent inhibition of 6-keto prostaglandin F 1α were observed in the five persons in whom repeated biopsy was performed within 12 hours of receiving a single dose of aspirin. This compares with 75 to 100 (mean 90) percent thromboxane B 2 inhibition and 63 to 100 (mean 77) percent 6-keto prostaglandin F 1α inhibition in six persons in whom repeated biopsy was performed within 24 to 72 hours after aspirin ingestion. In the four persons receiving 40 mg aspirin four times daily, there was a statistically significant (p < 0.02) reduction in venous 6-keto prostaglandin F 1α production compared with pre-aspirin values. In this smaller group, the mean percentage 6-keto prostaglandin F 1α inhibition was 87 percent, compared with 98 percent inhibition of thromboxane B 2 production. These results indicate that, in healthy adults, there is little difference in the inhibitory effect of aspirin on prostaglandin biosynthesis by platelets and venous tissue. The clinical response to aspirin in a group of patients with essential thrombocythemia complicated by painful toes associated with pregangrene or gangrene and with or without transient cerebral ischemic attacks is described. Fourteen patients had painful toes with evidence of peripheral ischemia. Three of these also had amaurosis fugax. Two further patients complained solely of transient blurring of vision. All patients received a myelosuppressive agent ( 32p or busulphan) and aspirin. Eleven patients were given 300 to 600 mg of aspirin daily; five received 40 mg of aspirin daily. Only one patient failed to respond to aspirin. In 12 patients, there was complete loss of pain and reversal of the pregangrenous changes of the toes. Following aspirin ingestion, there were no recurrences of the transient ischemic attacks. Pain relief occurred more promptly in persons receiving the larger dose of aspirin. These results support the view that certain complications of essential thrombocythemia, notably painful peripheral ischemia and amaurosis fugax, are platelet-derived and can be controlled by the inhibition of platelet prostaglandin biosynthesis. Aspirin has an inhibitory effect on this hemostatic mechanism.

Effect of alpha-tocopherol on sodium arachidonate-induced pulmonary embolism with platelet aggregates.

The effect of vitamin E (VE) on arachidonate-induced respiratory distress and sudden death in rats was examined. Fourteen VE deficient rats (VEDR), 11 normal control rats (NCR) and 12 VE supplemented rats (VESR) were injected with sodium arachidonate into the central vein, VEDR developed various arrhythmias, respiratory distress, apnea, hypotension, and 11 out of 14 VEDR (79%) died shortly after the injection. Many of these rats demonstrated obstruction of the small pulmonary vasculature with platelet aggregates. On the other hand, NCR and VESR developed much attenuated respiratory insufficiency and 5 out of 11 (45%) and 2 out of 12 (17%) died respectively. Twenty-six VEDR, 16 NCR and 25 VESR were used for an in vitro study of the platelet and vessel wall. VEDR demonstrated increases in platelet counts and aggregation as compared with NCR and VESR. Lipid peroxide in platelets, plasma and the aorta was significantly increased in VEDR. Prostacyclin-like activity in the aorta was not different among VEDR, NCR and VESR. The above results indicate that the pronounced respiratory distress, high mortality rate and pulmonary microembolism with platelet aggregates in VEDR may be attributed to the increase in platelet number and aggregability. The increased platelet aggregability was suggested to be the result of increased prostaglandin biosynthesis in platelets. The increased lipid peroxidation in vessel wall and plasma might also contribute to the enhanced respiratory distress. The respiratory distress and mortality as well as in vitro laboratory results were not different between NCR and VESR. This study indicates that vitamin E supplementation does not protect against the development of respiratory distress and sudden death induced by arachidonic acid over the normal control rats.

The effect of isolated leukaemic blasts on platelet aggregation.

The effect of blastic cells isolated by Böyum's method from the blood of 20 nontreated patients with acute myelogenous leukaemia on normal platelet aggregation has been studied. The isolated blasts in concentration of 2,000/microliter and higher, inhibit normal platelet aggregation induced by ADP or collagen, but have no influence on aggregation initiated by epinephrine. This effect is unaffected by the presence of metabolic poisons or by the disruption of the blastic cell membrane. Our results seem to exclude interference of blastic cells with platelet prostaglandin biosynthesis. In patients with acute leukaemia and an increased leukocyte count the preparation of platelet suspension free of blastic cells is not possible, even by the albumin gradient method or by gel filtration. The leukaemic blast content in platelet-rich suspensions may account for the variations in the aggregation behaviour which are found in these patients. The influence of blastic cells may be an important factor in the bleeding tendency in myelogenous leukaemia

Comparative Studies on the Activities of Prostaglandin Biosynthesis in Platelets and Aortas of Normotensive and Spontaneously Hypertensive Rats

Comparative Studies on the Activities of Prostaglandin Biosynthesis in Platelets and Aortas of Normotensive and Spontaneously Hypertensive Rats

Endotoxin-induced platelet aggregation and secretion I. morphological changes and pharmacological effects

Endotoxin lipopolysaccharide (LPS) from Acinetobacter 199A induced aggregation of blood platelets from immune adherence-positive species (rat, rabbit) but not from immune adherence-negative species such as pig and man. Aggregation occurred in 2 phases: the first was not accompanied by secretion of platelet constituents, was apparently a consequence of C3 activation, and was selectively inhibited by EGTA. The second phase of aggregation was associated with secretion of platelet granule contents, and with a lesser amount of cytoplasmic leakage. Secondary aggregation was abolished by the sulphydryl alkylating agent N-ethylmaleimide, and by agents which increased the level of cyclic AMP in platelets, such as prostaglandin E1 (a stimulator of adenylate cyclase) and methyl xanthines (inhibitors of phosphodiesterase). Secondary aggregation was partly inhibited by agents which block platelet prostaglandin biosynthesis (e.g. aspirin, indomethacin). Primary aggregation was unaffected by these inhibitors at concentrations which blocked secondary aggregation.

Dihomo-γ-linolenate suppresses platelet aggregation when administered in vitro or in vivo

Dihomo-γ-linolenate effectively prevented the irreversible aggregation of human platelets induced by collagen or epinephrine. Also, platelets from rats which received daily oral doses of dihomo-γ-linolenate showed significant reductions in platelet aggregatory responses to collagen and ADP which were attributable to increases in plasma and platelet ratios of dihomo-γ-linolenate to arachidonate. Similar results were obtained in rabbits. This data, together with that of enzymatic studies supports a hypothesis that oral ingestion of dihomo-γ-linolenate may effectively prevent arterial thrombosis in man by causing a redirection of platelet prostaglandin biosynthesis. Thus PGE 1 and its non-aggregatory endoperoxide intermediate (PGR 1) are formed at the expense of PGE 2 and LASS endoperoxide which together may induce platelet thrombus formation.