Silvia Villa

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.

Prostacyclin-like activity in rat vascular tissues. fast, long-lasting inhibition by treatment with lysine acetylsalicylate

Both arterial and venous tissues obtained from normal rats released prostacyclin (PGI2)-like activity, as marked by its potent inhibitory effect on platelet aggregation. Intraperitoneal or intravenous administration of a single dose of a soluble lysine salt of acetylsalicyclic acid (L-ASA, 1-400 mg/kg) resulted in abolition or substantial reduction of prostacyclin-like activity released from rat vasculature. The inhibitory effect of L-ASA was evident one minute after its i.v. administration to the animals, persisted for at least 24 hours and was still detectable (in venous tissues only) 168 hours later. Venous tissues were inhibited by doses of L-ASA as low as 1 mg/kg, whereas arterial tissues were not inhibited by doses of LA-ASA lower than 10 mg/kg. This difference may possibly be related to the lower prostacyclin-like activity shown by rat venous tissues compared to arterial ones. It is suggested that L-ASA or part of its molecule may bind to and inhibit cyclo-oxygenase in the blood vessel wall in a manner similar to the acetylation of platelt cyclo-oxygenase.

Platelet Aggregation and Antithrombin III Levels in Diabetic Children

We studied platelet function and antithrombrin III levels in 30 insulin-dependent diabetic children with no clinically evident vascular complications. 9 were in-patients and 21 were out-patients. The disease had been discovered within the previous 10 years. 25 control subjects of comparable age and body weight were studied simultaneously. Template bleeding time, threshold concentrations of ADP or adrenaline required to induce irreversible platelet aggregation and plasma antiherparin activity (platelet factor 4) did not differ significantly in control and patient groups. In contrast, the immunological levels of plasma antithrombine III were significantly higher in the diabetic group. These results suggest that diabetic children, with no clinical signs of microanigopathy, show no laboratory changes suggesting increased platelet function. The unxpected increase in the antithrombin III level could reflect a very early defense mechanism against activation of the blood clotting system.

Arachidonic acid and leukotriene B4 induce aggregation of human peripheral blood mononuclear leucocytes in vitro

Peripheral human blood mononuclear leucocytes (MNL) aggregated in response to arachidonic acid (AA) in vitro. This phenomenon was similar to that already described for polymorphonuclear cells (PMN). The effect of AA was concentration‐dependent and was shared only by the structurally related di‐homo‐y‐linolenic acid among the other fatty acids tested. A number of agents able to induce platelet aggregation such as ADP, collagen, serotonin and a stable prostaglandin analogue all failed to stimulate MNL or PMN aggregation. Inhibitors of cyclo‐oxy‐genase activity such as acetylsalicylic acid and indomethacin not only did not prevent AA‐induced aggregation, but even potentiated it. In contrast, both nordihydroguaiaretic acid and BW 755C, two inhibitors of cyclo‐oxygenase and lipoxygenase, strongly prevented MNL aggregation. Thus AA seems to aggregate MNL through the mediation of lipoxygenase products. This is supported by the observation that leukotriene B4 (LTB4) also induced MNL aggregation. When highly purified lymphocyte and monocyte preparations were assessed separately, the latter responded to AA similarly to mixed MNL whereas lymphocyte aggregation was inconsistent, small and reversible even at high concentrations of AA. Although the pathophysiological significance of the MNL aggregation described here is still obscure, assembly of these cells‐particularly monocytes‐at the site of injury might be a crucial event.

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

Normal prostacyclin-like activity in vascular tissues from thrombocytopenic rats

Abstract Arterial and venous tissues from normal rats generate a potent inhibitor of platelet aggregation with the characteristics of prostacyclin (PGI2). Vascular tissues from rats made moderately or severely thrombocytopenic (by administration of heterologous antiplatelet antiserum) release prostacyclin-activity in the same way as the corresponding vessels from normal rats. This activity was inhibited by administration of acetylsalicylate to both normal and thrombocytopenic rats. It is suggested that the basal release of prostacyclin from rat vasculature is independent of the number of circulating platelets and that rat vessels possess both the substrates and the enzymes required for prostacyclin generation. The possibility that chronic thrombocytopenia might affect prostacyclin generation by inducing endothelial damage, cannot be excluded.

Vitamin E and vitamin C inhibit arachidonate-induced aggregation of human peripheral blood leukocytes in vitro

SummaryArachidonate induces aggregation of human polymorphonuclear (PMN) and mononuclear (MNL) blood leukocytes. This is mediated by the lipoxygenase pathway, as it is prevented by lipoxygenase inhibitors and can also be induced by leukotriene B4 (LTB4). Vitamin E and vitamin C have profound effects on the functional state of leukocytes, some of which may involve the lipoxygenase pathway. This study shows that both vitamins inhibit arachidonate-induced aggregation of PMN and MNL, in a concentration-dependent way. BW-755, previously shown to inhibit arachidonate-induced PMN and NML aggregation, was found to potentiate the inhibitory activity of both vitamins. When LTB4 was used as an aggregating agent, vitamin E markedly inhibited PMN and MNL aggregation, whereas vitamin C was ineffective.The prevention of PMN and MNL aggregation by vitamin E might account, at least partially, for the reported beneficial effects of vitamin E supplementation in some experimental syndromes characterized by leukocyte activation.

Decreased plasminogen activator but normal prostacyclin activity in rat veins during development of experimental thrombosis

Abstract The course of two vascular activities (plasminogen activator and prostacyclin) was measured on venous specimens taken from rats before and during development of experimental venous thrombosis (induced by venous stasis). Vascular plasminogen activator activity (measured by a histochemical technique) increased immediately after ligature of the inferior vena cava, then progressively declined up till the appearance of a visible red thrombus; it virtually disappeared in concomitance with the formation and growth of the thrombus. Blood fibrinolytic activity closely followed the course of vascular plasminogen activator activity. In contrast, vascular prostacyclin (platelet aggregation inhibitory) activity remained within the normal range throughout the observation period (20 minutes - 48 hours). These results indicate that the course of two potential antithrombotic defence activities may be dissociated during the development of experimental venous thrombosis. The model used may provide some hints as to the relative roles of fibrinolytic and platelet inhibitory activities released from vascular endothelium in response to thrombogenic stimuli.

Increased vascular prostacyclin activity in rats after endotoxin administration.

Endotoxin did not interact in vitro with prostacyclin activity but stimulated its release from vascular tissues when administered in single doses to rats 30 min before testing.

Platelet Aggregation: Methodology and Physiopathology

The physiopathological role of platelet aggregation in some thromboembolic and atherosclerotic complications is strongly suggested on the basis of many indirect findings. The qualitative methodological approach to this problem generally used until recently is rapidly giving way to a quantitative, biochemical approach. Platelet aggregation, however, even if expressed in terms of nanomoles of a product obtained in a sophisticated reaction system, will continue to deceive investigators and clinicians who fail to view it in the adequate (although still uncertain) context of rheological and vascular interactions.