Platelet Prostaglandin Synthesis Research Articles

Low dose aspirin, platelet function and prostaglandin synthesis: Influence of epinephrine and alpha adrenergic blockade

The present investigation has evaluated the effects of low doses of oral aspirin on platelet prostaglandin synthesis and function. Whole (80 mg) or half (40 mg) tablets of baby aspirin given to adults had no effect on the response of their platelets to thrombin, ADP and epinephrine, but selectively inhibited aggregation induced by threshold concentrations of arachidonate 16–20 hours after ingestion. Larger amounts of arachidonate overcame the inhibition imposed by low dose aspirin, but not by adult aspirin tablets (600 mg). Epinephrine, in concentrations too low to cause aggregation, restored the sensitivity of aspirin-treated platelets to arachidonate. Studies with a-adrenergic agonists, antagonists and calcium channel blockers demonstrated that the corrective effect of epinephrine was mediated by an a-adrenergic receptor influence on calcium modulation of the platelet membrane.

Effect of Ergotamine on Platelet Prostaglandin Synthesis—in vitro and in vivo Experiments

We studied the effect of ergotamine on the formation of metabolites of [1 - 14C] arachidonic acid by human blood platelets incubated with ergotamine (10(-5) or 5.10(-5)M) in vitro or after 0.5 mg i.m. administration in vivo. The in vitro experiments confirmed our earlier observation that ergotamine inhibits prostaglandin synthesis. This effect, however, seemed to be restricted to the toxic dose range of the drug as in vivo it occurred only in those subjects who became nauseated or vomited after administration of the drug.

Effect of gliclazide on prostaglandin and thromboxane synthesis in guinea-pig platelets

The inhibitory effect of gliclazide, a hypoglycemic sulfonylurea, on prostaglandin and thromboxane synthesis in guinea-pig platelets was studied. Gliclazide inhibited the thrombin-induced release of radioactive arachidonic acid from [ 14C] arachidonate-labeled platelets, and the concentration of 50% inhibition (IC 50) was 680 μg/ml. Gliclazide also inhibited in vitro the platelet malondialdehyde (MDA) formation induced by collagen but not by arachidonic acid, and IC 50 was 333 μg/ml. Gliclazide did not block the conversion of [ 14C] arachidonic acid to radioactive thromboxane A 2 (measured as a stable form, thromboxane B 2) in platelets. In ex vivo experiments, the platelet MDA formation induced by collagen but not by arachidonic acid was significantly inhibited by a single administration of gliclazide at 300 mg/kg, and this inhibition was enhanced by the repeated administrations for 7 days: namely, the significant inhibition was observed at 30 mg/kg/day. Thus gliclazide seems to inhibit in vitro and ex vivo arachidonate release from platelet phospholipids.

Vitamin E and platelet aggregation.

In healthy persons, a daily dose of 2,000 IU of vitamin E given for 10 days does not prolong the bleeding time and has no influence on collagen- and ADP-induced platelet aggregation nor on platelet prostaglandin synthesis. Therefore, even high doses of vitamin E have no place in platelet function suppressing therapy.

Role of platelet-prostaglandin synthesis in shear-induced platelet alterations

Role of platelet-prostaglandin synthesis in shear-induced platelet alterations

Role of Platelet-Prostaglandin Synthesis in Shear-Induced Platelet Alterations

Exposure of citrated human platelet-rich plasma (C-PRP) to mechanical shear stress has been shown to induce platelet aggregation (PAG), release from platelets of dense granule contents, and platelet lysis. The mechanisms involved in these shear-induced platelet alterations have not been defined. To examine the possible role of prostaglandin (PG) synthesis in shear-induced platelet reactions, C-PRP was sheared in a concentric cylinder viscometer for 5 min at stresses ranging from 50 to 460 dyn/sq cm in the presence or absence of acetylsalicylic acid (ASA), a potent inhibitor of platelet thromboxane-A2 synthesis. With increasing shear stress, a progressive loss of platelets from the sheared C-PRP was observed due to PAG and adherence of the aggregates to the viscometer surfaces. PAG was associated with a progressive release from platelets of serotonin and β-thromboglobulin (β-TG) and formation of malondialdehyde (MDA) a stable end-product of platelet cyclooxygenase-mediated arachidonate metabolism reflecting thromboxane-A2 synthesis. At stresses above 150 dyn/sq cm, a progressive loss of lactic dehydrogenase (LDH) was observed, indicating platelet damage. ASA (0.1 mM) completely abolished shear-induced MDA formation and caused a 50% reduction of shear-induced release of serotonin and β-TG. However, ASA did not cause an appreciable reduction of shear-induced PAG and did not alter the extent of or threshold for shear-induced platelet lysis. The findings indicate that (1) shear-induced release from platelets of dense and α-granule contents is associated with and potentiated by stimulation of thromboxane-A2 synthesis and (2) thromboxane-A2 synthesis appears to play no appreciable role in shear-induced PAG or platelet lysis in this system.

Effects of Aspirin and Related Drugs in Vitamin E-Deficient Rats

Evidence from the literature indicates that in vitamin E-deficient animals prostaglandin (PG) synthesis in platelets is enhanced while it is decreased in the muscle and testis. In the present study the effects of aspirin, a known inhibitor of PG biosynthesis, on vitamin E deficiency signs in the rat were investigated. Administration of aspirin to vitamin E-deficient rats had no protective effect on fetal mortality, incisor depigmentation, body weight gain or red blood cell peroxidative hemolysis. Aspirin prevented the anemia and thrombocythemia observed in vitamin E-deficient rats. Aspirin, salicylic acid and a carbazole prostaglandin inhibitor exacerbated testis degeneration in vitamin E-deficient animals. Addition of aspirin to the diet more than doubled the vitamin E requirement for reversal of necrotizing myopathy.

The differential inhibition of prostaglandin synthesis in platelets and vascular tissue in response to aspirin

Since aspirin inhibits the platelet as well as vascular prostaglandin synthesis it may therefore, paradoxically induce a thrombotic tendency when used as an antithrombotic agent. The in vivo effect of therapeutic doses of aspirin on the prostaglandin synthetic capacity of the rat platelets and vascular tissue was therefore studied to determine the significance of this paradoxical aspirin effect. A single aspirin dose of 5 mg/kg or greater was found to significantly decrease the synthesis of prostaglandin E by rat platelets. Even the normal augmentation of PGE synthesis by N-ethyl maleimide was significantly reduced by this single aspirin dose. In contrast, doses as high as 20 mg/kg of aspirin failed to reduce the production of 6-keto PGFla by aortic slices from rats pretreated with aspirin. These results indicate that the cyclo-oxygenase enzyme (CO) system in the prostaglandin biosynthetic pathway within blood vessel walls has the capacity to recover from this inhibitory effect of aspirin or it is less susceptible to inhibition than the CO in platelets. This relative resistance of CO to the inhibitory effects of aspirin may serve to protect the organism from excessive thrombotic tendencies brought about by the effect of aspirin on the vascular prostaglandin generating system. These results indicate that an aspirin dose of 5–10 mg/kg would be optimal if aspirin were to be used as an antithrombotic agent.

Effects of 2,2′-dipyridyl and related compounds on platelet prostaglandin synthesis and platelet function

2,2′-dipyridyl, a chelator of ferrous iron and inhibitor of platelet aggregation, was studied together with several similar compounds to determine the mechanism of their effects on platelets. All of these compounds were more potent inhibitors of arachidonic-acid-mediated aggregation (IC 50, 0.17–1.8 mM) than of ADP-mediated aggregation (IC 50, 7.6–19.7 mM). At low concentrations required to inhibit arachidonic-acid-mediated aggregation, 2,2′-dipyridyl, 4,4′-dipyridyl and 2-chloropyridine specifically inhibited the platelet cyclo-oxygenase. The mechanism of inhibition of ADP-induced aggregation was investigated, but was not explained. At concentrations needed to inhibit ADP-induced aggregation, 2,2′-dipyridyl did not alter cell ultrastructure, serotonin or nucleotide content or interfere with release of [ 14C]arachidonic acid or calcium movements. Therefore, our results indicate that 2,2′-dipyridyl and related compounds have two effects on platelets, both due to the unprotonated form. The inhibition of cyclo-oxygenase by low concentrations of these compounds is not due to bidentate iron chelation, since 4,4′-dipyridyl was almost as effective as 2,2′-dipyridyl, but is compatible with binding of these inhibitors to the iron in the heme of the cyclo-oxygenase.

Effect of heterologous antibody on human platelet prostaglandin synthesis.

Rabbit anti-human platelet antiserum (RPA) has been shown to mediate human platelet aggregation and 14C-serotonin release. This effect is not inhibited by aspirin. However, it is not known whether antibody induces platelet prostaglandin synthesis. We, therefore, investigated the effect of RPA on human platelets by measuring malondialdehyde (MDA) production as a reflection of prostaglandin synthesis and comparing it with 14C-serotonin release. Independently, ATP release was also measured as a further measure of platelet secretion. Normal rabbit serum did produce a small amount of MDA (0.54 +/- 0.09 SEM nmole/10(9) platelets). However, RPA produced a much larger amount of MDA (1.43 +/- 0.18 nmole/10(9) platelets) and pre-incubation with aspirin significantly decreased MDA production (0.16 +/- 0.07 nmole/10(9) platelets) (p less than .001). Aspirin did not significantly impair the capacity of RPA to release 14C-serotonin or ATP. These results demonstrate that prostaglandin synthesis occurs when heterologous antibody is added to human platelets. However, prostaglandin formation must be a secondary event in this process since the inhibition of prostaglandin synthesis by aspirin had little effect on the ability of RPA to induce 14C-serotonin release or ATP release.

Is tartrazine-induced asthma related to inhibition of prostaglandin biosynthesis?

Since it has been suggested that aspirin-induced asthma is due to inhibition of prostaglandin (PG) biosynthesis, the present investigation was undertaken to determine whether tartrazine would display a similar profile. Use was made of the exquisite sensitivity of blood platelets, both in humans and in rats, to aggregating substances generated from arachidonic acid during PG biosynthesis and of the ability of aspirin to inhibit aggregation and generation of those substances. Failure to affect aggregation by arachidonic acid as well as the accompanying formation of thromboxane A2, demonstrates that neither tartrazine nor its metabolite sulfanilic acid inhibit platelet PG synthesis. It seems unlikely that tartrazine-induced asthma results from inhibition of PG biosynthesis.

Platelet dysfunction in homozygous beta-thalassemia.

This report details the results of studies performed in nine patients with homozygous beta-thalassemia evaluating platelet function and prostaglandin formation. Platelet malonyldialdehyde (MDA) formation in the presence of N-ethyl maleimide (NEM 1 mM) or thrombin (0.5 u/ml) was used as an indicator of platelet prostaglandin synthesis. The data on the nine patients revealed two distinct subgroups of patients. Six of nine thalassemics, demonstrated platelet abnormalities. Their mean bleeding time was 7.5 +/- 2.5 min (1 SD), significantly prolonged (P less than 0.005) when compared to a value of 3.5 +/- 1.0 min in normal controls. MDA formation in the presence of NEM was significantly decreased (P less than 0.005) to 2.41 +/- 0.49 (1 SD) when compared to a control value of 3.24 +/- 0.33 nmoles MDA/10(9) platelets. Similarly, the mean value for thrombin induced MDA was 0.98 +/- 0.18 nmoles which was decreased (P less than 0.02) when compared to a value of 1.26 +/- 0.2 in the controls. Platelet aggregations with adenosine diphosphate (ADP), epinephrine, and collagen were abnormal in all six patients. However, when platelets from these patients were mixed with platelets from donors who had ingested aspirin 2-8 hr before donation mutual correction and secondary irreversible aggregation of the mixture resulted. No mutual correction was observed when the thalassemic platelets were preincubated with aspirin in vitro before mixing with platelets from donors who had recently ingested aspirin. Although the total amount of platelet malonyldialdehyde formed by the thalassemic platelets in response to NEM and thrombin was decreased when compared to normal controls, this reduction was not the cause of the platelet aggregation abnormalities. This appears to be so because the amount of MDA, and, thus, prostaglandin endoperoxides synthesized by these platelets in response to external stimuli was sufficient to cause irreversible aggregation of platelets from donors who had recently ingested aspirin, and were, therefore, unable to synthesize their own endogenous platelet endoperoxides. In the remaining three patients, bleeding times, platelet aggregation, and MDA formation was normal. No correlation was observed between the platelet abnormalities noted and the magnitude of iron overload, presence of fibrin degradation products, liver function abnormalities, or the use of iron chelators in the individual patient. Family studies were normal. Although the platelet dysfunction does not appear to be of major significance in the usual patient with thalassemia major under normal circumstances, antiplatelet aggregating agents should be used with caution. Aspirin inhibits platelet endoperoxide and prostaglandin formation and this effect may potentiate the platelet dysfunction present in some patients with thalassemia major.

Antiplatelet Drugs

Because platelets are so important in thrombus formation, drugs which inhibit platelet function (the 'antiplatelet drugs') have considerable potential as antithrombotic agents. Among the antiplatelet drugs, only aspirin, sulphinpyrazone, dipyridamole, hydroxychloroquine, and clofibrate have had wide clinical trial. Their effects on platelet metabolism differ. Aspirin prevents platelet prostaglandin synthesis by acetylating and irreversibly inactivating platelet prostaglandin synthetase, while sulphinpyrazone is a reversible inhibitor of the same enzyme. Both aspirin and sulphinpyrazone impair the platelet release reaction and reduce platelet aggregation, but neither prevents platelet adhesion to the subendothelium or to foreign surfaces. On the other hand, dipyridamole reduces platelet adhesion as well as aggregation, probably by inhibiting phosphodiesterase and so raising platelet cyclic AMP levels. The effects of hydroxychloroquine and clofibrate have been less well defined. As the antiplatelet drugs form a diverse group of substances with differing effects on platelet function, it is hardly surprising that every potential clinical application of each antiplatelet drug or drug combination has had to be tested separately, and that these drugs have not proved to be equally effective. One or more antiplatelet drugs have now been evaluated in each of the following situations.

Biochemical studies in Hermansky-Pudlak syndrome.

In order to find the basic defect in the Hermansky-Pudlak Syndrome (HPS), biochemical studies of platelets and leucocytes were undertaken. Glutathione levels of platelets were normal and regeneration of GSH similar to controls occurred after incubation with diamide (a specific agent for GSH oxidation). Phospholipid and fatty acid composition of HPS platelets was normal. The amount of peroxides found in platelet membranes was not elevated. A subnormal aggregation with arachidonic acid could be obtained in PRP using a high concentration of arachidonic acid (2 mM), but normal malondialdehyde levels were measured, suggesting a normal prostaglandin synthesis in HPS platelets. Glutathion peroxidase and p-phenylenediamide-mediated peroxidase (PPD-peroxidase) were normal in leucocytes of 1 HPS patient. Lysosomal enzymes as far as investigated were normal.

Platelet Malonyldialdehyde Formation: An Indicator of Platelet Hyperfunction

SummaryIn 25 normal controls (Group I) and in 23 patients (Groups II and III) platelet malonyldialdehyde formation in the presence of N-ethyl maleimide (NEM) or thrombin was evaluated as an indicator of enhanced platelet function. 12 patients (Group III) with diabetes mellitus, nephrotic syndrome, or acute lymphatic leukemia in remission either exhibited spontaneous platelet aggregation (6/12), or enhanced platelet aggregation to minimal concentrations of ADP and Epinephrine (6/12). In these same Group III patients platelet malonyldialdehyde formation was markedly increased (p <0.0005) when compared to the values in the Group I normal controls. Studies in the 12 abnormal subjects later revealed normal aggregation patterns in 6/12 on follow-up evaluation. Concomitant estimation of platelet malonyldialdehyde at this time revealed a decrease into the normal range for platelet malonyldialdehyde values in the presence of thrombin in these 6 patients. Although significantly decreased from initial levels, (p <0.0005) malonyldialdehyde estimation in the presence of NEM was still slightly above the normal range. In 11 patients (Group II) both platelet aggregation tracings and malonyldialdehyde formation was normal and similar to values in the control population. Platelet malonyldialdehyde is a byproduct of the platelet endoperoxides which are intermediaries in the sequence of platelet prostaglandin synthesis. These results, therefore, suggest that part of the phenomenon of platelet hyperaggregability is intrinsic to the platelet and may involve platelet prostaglandin synthesis. The estimation of platelet malonyldialdehyde is a simple technique which appears to detect platelet hyperfunction.

Ethanol-inhibited platelet prostaglandin synthesis in vitro.

Inhibition of human platelet malonyldialdehyde (an indicator of platelet prostaglandin synthesis) in platelet-rich plasma, not found after 100 mg of ethanol per 100 ml in vitro, was dose-related at 200, 400 and 750 mg per 100 ml.

Metabolism of arachidonic acid by platelets: utilization of arachidonic acid by human platelets in presence of linoleic and dihomo-gamma-linolenic acids.

In vitro human platelet prostaglandin synthesis has been studied from added radioactive arachidonic acid (i) as function of substrate concentration, (ii) as function of platelet concentration and (iii) as function of pH. Platelets, as in platelet rich plasma when labelled with arachidonic acid, washed and treated with thrombin, released radioactivity mainly from phosphatidylcholine and phosphatidylinositol. The released radioactivity was mostly accounted for by the formation of the previously identified oxygenation products of arachidonic acid. Platelet utilization or arachidonic acid was also studied in presence of linoleic and dihomo-gamma-linolenic acids, the two essential fatty acids known for antithrombotic effect. At its high concentrations linoleic acid decreased platelet cyclo-oxygenase activity as seen by a decreased formation of endoperoxides from arachidonic acid. Dihomo-gamma-linolenic acid was found to be a mutually competitive substrate with arachidonic acid for the platelet prostaglandin synthetase thus causing reduced utilization of arachidonic acid as shown by measuring the various oxygenation products of arachidonic acid. These two acids were utilized differently by platelet prostaglandin synthetase.

Sex differences in the antithrombotic effects of aspirin

Aspirin inhibits platelet function by acetylating platelet cyclooxygenase. Recent clinical trials indicate that aspirin is a promising antithrombotic agent against both venous and arterial thrombosis, but somewhat surprisingly this protective effect appears to be limited to males. To examine the potential sex-related differences in response to aspirin, we developed an animal model for quantitating fibrin accretion into an injury-induced thrombus and used it to study the effects of aspirin on thrombus size in male and female rabbits. Platelet prostaglandin synthesis was estimated by assay of platelet malondialdehyde and was significantly decreased in both male and female rabbits following treatment with 10 mg/kg aspirin (p less than 0.001). This inhibitory effect was not different for platelets from male and female rabbits. Thrombus size was significantly decreased in aspirin- treated male rabbits when compared to controls (p less than 0.05), but this aspirin effect was not apparent in female rabbits or rabbits of either sex treated with 10 mg/kg sodium salicylate. These findings support the results of clinical trials that were obtained by retrospective subgroup analysis. The reason for the sex difference is not known, but the findings raise an important issue in relationship to this mechanism of the antithrombotic effect of aspirin.

Role of Prostaglandin Synthesis in Rabbit Platelet Activation Induced by Basophil-Derived Platelet-Activating Factor

Abstract Rabbit platelet-activating factor (PAF) is a lipid released from IgE-sensitized basophils and mast cells during challenge with antigen. PAF induces shape change, aggregation, and secretion of granule-associated 3[H]-serotonin in rabbit platelets. This study investigated the capacity of PAF to initiate rabbit platelet prostaglandin synthesis and the requirement for generated prostaglandin endoperoxides and thromboxane A2 in PAF-induced platelet aggregation and secretion. PAF in submaximal concentrations induced synthesis in rabbit platelets of PGE2, PGF2α, and prostaglandin endoperoxides in amounts comparable to that initiated by collagen, but greater than that caused by thrombin. PAF stimulation of platelets preloaded with 14[C]-arachidonic acid caused platelet production of 14[C]-thromboxane B2, the stable derivative of 14[C]-thromboxane A2. When compared kinetically to secretion, PAF induced platelet endoperoxide production peaked just before maximal release of granular 3[H]-serotonin content. However, the cyclooxygenase inhibitors, aspirin and indomethacin, failed to alter PAF-induced platelet shape change or aggregation. Similarly, secretion was unaffected by indomethacin except for a slight attenuation seen at the lowest stimulus concentrations examined. Platelet production of endoperoxides occurred throughout the range of PAF concentrations examined, even those causing barely detectable levels of secretion. PAF thus initiates platelet synthesis of prostaglandin endoperoxides and thromboxane A2, but aggregation and secretion induced by PAF occur independent of these products of the cyclooxygenase pathway.

Oral contraceptive use alters the balance of platelet prostaglandin and thromboxane synthesis

The ability of platelet microsomes to generate platelet aggregating activity on addition of arachidonic acid was evaluated in women taking oral contraceptives and in controls taking no medication but matched for age, sex, and family history. Oral contraceptive users generated significantly more platelet aggregating activity per 100 ug of platelet microsomal protein than controls. Variation in generation of platelet aggregating activity during the menstrual cycle was also observed with highest activity during the third week. These studies show an altered balance of platelet prostaglandin and thromboxane synthesis in oral contraceptive users which may contribute to their increased incidence of thromboembolic phenomena.