Manuela Livio

URAEMIC BLEEDING: ROLE OF ANAEMIA AND BENEFICIAL EFFECT OF RED CELL TRANSFUSIONS

Abstract Bleeding time, altered in most uraemic patients, may be influenced by packed cell volume (PCV). Uraemic patients are often anaemic, so the influence of PCV on bleeding time was assessed in several groups of patients with chronic uraemia. The findings were that bleeding times in uraemic patients are profoundly influenced by anaemia, and that the platelet-mediated haemorrhagic tendency in uraemia may be successfully managed by raising PCV values to above 30%.

The Inhibitory Effect of Aspirin on Platelet and Vascular Prostaglandins in Rats cannot be Completely Dissociated

Summary. Blood platelets and vascular endothelium generate prostaglandin derivatives with opposing effects: cyclic endoperoxides and thromboxane A2 (TXA2) aggregate platelets, whereas prostaglandin I2 (PGI2, prostacyclin) inhibits aggregation. Since aspirin inhibits the synthesis and effects of all these metabolites of arachidonic acid, the rationale for clinical use of this drug in thrombosis prevention trials is questionable. Treatment schedules would have to be calibrated to inhibit proaggregating platelet prostaglandins and TXA2 without affecting protective vascular prostaglandins (PGI2). This important clinical information cannot easily be obtained from studies in man.

Salicylate reverses in vitro aspirin inhibition of rat platelet and vascular prostaglandin generation.

Abstract It is generally accepted that aspirin inhibits platelet function by irreversible acetylation of prostaglandin cyclo-oxygenase. The salicylate moiety seems not to be causally involved in the inhibitory effect of aspirin, a concept supported by the virtual inactivity of sodium salicylate. However, prostaglandin synthesis is also inhibited by numerous compounds which have no acetylating properties. Recent evidence indicates that salicylate may prevent the inhibitory effect of aspirin on rabbit platelet cyclo-oxygenase, suggesting that interaction of the salicylate moiety of aspirin with this enzyme is important. This study was aimed at evaluating whether the inhibitory effect of aspirin on platelet prostaglandin generation could be reversed by sodium salicylate. We therefore measured spectrophotometrically malondialdehyde (MDA) generated by arachidonate in rat platelet-rich plasma and evaluated the effect of short-term incubation with either aspirin or salicylate or both. In the experimental conditions used, salicylate not only prevented but also reversed aspirin-inhibition of MDA formation. This interaction was not peculiar for platelets, since salicylate also reversed the in vitro inhibitory effect of aspirin on vascular prostacyclin generation (measured by a bioassay). These findings suggest that irreversible acetylation of cyclo-oxygenase does not account for the early in vitro inhibitory effect of aspirin on prostaglandin synthesis.

Non-steroidal anti-inflammatory drugs react with two sites on platelet cyclo-oxygenase evidence from ‘in vivo’ drug interaction studies in rats

Non-steroidal anti-inflammatory drugs inhibit platelet cyclo-oxygenase activity. This study shows that salicylate, diflunisal, sulphinpyrazone and indomethacin prevention vivo aspirin inhibitory effect on cyclo-oxygenase activity as measured by the formation of malondialdehyde and thromboxane B2, two products of platelet arachidonic acid metabolism. Salicylate also prevents the inhibitory effect of indomethacin. All these drugs therefore appear to interact with the same site on platelet cyclo-oxygenase. Since salicylate is inactive by itself on this platelet enzyme and diflunisal and sulphinpyrazone were used at ineffective doses, it is suggested that their interaction with aspirin (or indomethacin) occurs at the level of a supplementary binding site, rather than directly on the substrate active site. Interaction with this putative supplementary site is necessary but not sufficient for the efficacy of these drugs on cyclo-oxygenase inhibitors. Acetylation by aspirin of the active site appears to be a phenomenon secondary to the binding of salicylate moiety to the supplementary site.

Inhibition of Human Platelet Aggregation by Parathyroid Hormone

Parathyroid hormone (PTH) is a polypeptide which in different in vitro systems raises intracellular cyclic AMP (cAMP) levels via adenyl cyclase activation and stimulates Ca2+ transport across cell membranes. We tested whether, on the basis of this mechanism, PTH would inhibit human platelet aggregation. The latter was tested in vitro by a photometric technique. Platelet aggregation induced by the calcium ionophore A 23187 was inhibited by PTH at concentrations (0.5-3 USP U/ml) similar to those effective in other in vitro systems. Higher concentrations of PTH were required to prevent aggregation initiated by adenosine-5-diphosphate, arachidonic acid, or platelet-aggregating factor. The terminal synthetic fragment 1-34 b PTH was ineffective against all aggregation stimuli. The antiaggregating effect of PTH was potentiated by verapamil and theophylline and was additive to that of PGI2. However, PTH did not appear to increase platelet cAMP levels and was not counteracted by an inhibitor of platelet adenyl cyclase. It is therefore unlikely that PTH inhibits platelet aggregation through an adenyl cyclase stimulated increase of cAMP. Since PTH levels are markedly increased in uremic plasma, it might contribute to the defective platelet function and the bleeding tendency frequently occurring in uremic patients.

Impaired Thromboxane Production by Newly Formed Platelets after Aspirin Administration to Thrombocytopenic Rats

The in vivo inhibitory effect of aspirin on platelet cyclo‐oxygenase is irreversible and lasts for the entire platelet life‐span. Reappearance of cyclo‐oxygenase activity in blood after aspirin has been proposed as a measure of the formation of new platelets and as an indirect indicator of platelet survival. A delay of 24–72 h in recovery, however, has been observed and it has been suggested that aspirin might also inhibit megakaryocyte cyclo‐oxygenase. To test this possibility, aspirin (100 mg/kg) or saline were administered i.p. to rats made thrombocytopenic 2 h later (platelet count less than 5% of basal value) by a specific antiplatelet antiserum. Malondialdehyde (MDA) and thromboxane B2 (T×B2) production by platelets was measured by spectrophotometry and radioimmunoassay respectively, during the period of platelet count restoration. By 24 h after thrombocytopenia was induced, platelet count was about 15% of basal values in control and aspirin‐treated rats. However, while in controls MDA and T×B2 production was restored to about 20% of basal values, in aspirin‐treated rats less than 5% return of activity was detected. A marked difference between the two groups was still found 96 h after induction of thrombocytopenia, when platelet count restoration was similar. Since aspirin disappeared very rapidly from the circulation, the delay in recovery of cyclo‐oxygenase activity supports the hypothesis of a megakaryocyte effect of this drug.

Salicylate fails to prevent the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on human platelet cyclo-oxygenase and lipoxygenase activities

Sodium salicylate is inactive both on cyclo-oxygenase and lipoxygenase prepared from human platelets. It prevents the inhibition of cyclo-oxygenase induced by aspirin, but does not counteract the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on both enzymes. It also fails to interfere with the inhibitory activity of nordihydroguaiaretic acid on lipoxygenase. These data indicate that, unlike eicosatetraynoic acid, non-steroidal anti-inflammatory drugs interact with a site on cyclo-oxygenase distinct from the catalytic site, although related to it. Such a supplementary binding site is lacking on lipoxygenase.

Inhibition of human platelet cyclo-oxygenase activity by sulfinpyrazone and three of its metabolites

Sulfinpyrazone and three of its major metabolites were compared in vitro for their inhibitory effect on human platelet cyclo-oxygenase activity. Sulfinpyrazone appeared to be about 15-20 times less potent than its sulfide metabolite (G25671) and 6-7 times less potent than the other two compounds, the sulfone metabolite (G31442) and p-hydroxysulfide (G33378). All four compounds were apparently competitive inhibitors of platelet cyclo-oxygenase activity. Comparison of the potency of sulfinpyrazone and its metabolites, as determined in the present study and the plasma levels previously measured in man, indicates that sulfinpyrazone and G33378 were not potent enough to be effective in man. G31442 showed inhibitory potency slightly lower than its corresponding plasma levels, whereas G25671 was effective at concentrations well below those found in human plasma. This study supports the hypothesis that sulfinpyrazone metabolites (in particular the sulfide) rather than the drug itself affect platelet function when administered therapeutically.

Long-lasting inhibition of platelet prostaglandin but normal vascular prostacyclin generation following sulphinpyrazone administration to rats

Buchanan et al (1978) have reported that the inhibitory effect of sulphinpyrazone on collagen-induced platelet aggregation in vivo persisted for up to 18 h after drug administration. Since unchanged sulphinpyrazone could not be detected in plasma of animals within 4 h of administration, these authors suggested that the inhibitory effect might be ascribed to some metabolite(s). Similar findings have been presented by Butler et a1 (1979) who found the inhibitory effect on arachidonic acid-induced platelet aggregation in guinea-pigs was greater 7 h than 1 h after oral administration of sulphinpyrazone. Kirstein Pedersen & Jacobsen (1979) have recently identified two active metabolites of sulphin-pyrazone after intravenous administration of this drug to rabbits. We report here a dose-related inhibition of platelet prostaglandin generation in rats given sulphinpyrazone orally. This effect was maximal between 3 and 18 h after drug administration and disappeared within 36 h. These results extend to another animal species the concept that the metabolism of sulphinpyrazone may play a major role in the drug’s platelet inhibitory activity. The observation that vascular prostacyclin activity was not inhibited at any time after sulphinpyrazone could also be relevant for the antithrombotic activity of this compound. Male CD-COBS rats (Charles-River, Italy), 25(r 350 g, were given a single oral dose of either sulphinpyrazone (Ciba-Geigy, Italy) or its suspending vehicle (0.5% carboxymethylcellulose). Animals were killed from 90 min to 36 h thereafter by ether anaesthesia. Blood was collected and the abdominal aorta and inferior vena cava removed and processed as previously described (Villa et a1 1979). Malondialdehyde (MDA) formation was measured by a modification (Villa et al 1979) of the spectrophotometric assay described by

Sulphinpyrazone prevents in vivo the inhibitory effect of aspirin on rat platelet cyclo-oxygenase activity

Abstract Sulphinpyrazone reportedly inhibits in vitro platelet cyclo-oxygenase activity. This study shows that Sulphinpyrazone administration (200 mg/kg) to rats was followed by long lasting inhibition of platelet cyclo-oxygenase, as measured by malondialdehyde generation by sodium arachidonate. The inhibition was apparently competitive and could in fact be ascribed to supposed metabolites of the drug. When given 30min–6 hours before aspirin (5–25mg/kg), Sulphinpyrazone and to a considerable extent its metabolites significantly prevented permanent inhibition of platelet cyclo-oxygenase activity normally produced in vivo by aspirin. Sulphinpyrazone at 100 mg/kg was unable by itself to modify platelet malondialdehyde or thromboxane B2 generation, yet if effectively interfered with aspirin activity. This suggests that Sulphinpyrazone and its metabolites may interact with a binding site on cyclo-oxygenase not directly involved with the enzyme activity. Interaction of aspirin with this binding site would be a prerequisite for its inhibitory effect on the enzyme active site. The clinical implications of this study include a reappraisal of the pharmacological basis for the association of Sulphinpyrazone and aspirin in thrombosis prevention trials.