Bleeding time in laboratory animals. IV. Effects of prostacyclin, pyrimido-pyrimidine compounds and aspirin in rats

Abstract

It has been suggested that dipyridamole and other inhibitors of phosphodiesterases may impair platelet function by potentiating the inhibitory effect of endogenous circulating prostacyclin (PGI 2), a stimulator of adenylcyclase (1). Treatments combining these drugs with a PGI 2 inhibitor such as aspirin could therefore reduce their inhibitory effect on platelet function (1). We have confirmed that phosphodiesterase inhibitors potentiate the effects of exogenous PGI 2 on human platelets in vitro (2). We report here such a potentiation on bleeding time in rats. However, we were unable to show any reduction in the potency of pyrimido-pyrimidine compounds on bleeding time in rats pretreated with doses of acetylsalicylate (ASA) which totally inhibited endogenous vascular PGI 2 activity (3,4). In contrast, we found that ASA strongly potentiated both exogeneous PGI 2 and dipyridamole-like drugs in this test system. Unanaesthetized male CD rats (230–300 g b.w.) obtained from Charles River, Italy, were used. Bleeding time was measured on the animals' tails (immersed in isotonic saline at 37°C) by a standardized template technique as described (5,6). Prostacyclin, sodium salt (Upjohn, Kalamazoo, Michigan, USA) was dissolved in absolute ethanol (stock solution), kept at −20°C, and diluted with 0.05M Tris buffer, pH 7.4 just before intravenous injection. Lysine acetylsalicylate (Flectadol, Maggioni, Italy) and dipyridamole (Persantin, Boehringer Ingelheim, Italy) were injected intraperitoneally. The pyrimido-pyrimidine compounds SH-869 (2-piperazinyl-4-thiomorpholino-pyrimido (3,2-a) pyrimidine sulphate trihydrate) and VK-774 (4-morpholino-2-piperazinothieno-(3,2-d)pyrimidine-dihydrochloride) from Karl Thomae (West Germany) were dissolved in isotonic saline and injected intravenously. The latter compounds were potent inhibitors of in vivo A.D.P.-induced aggregation of rat platelets (7). Bleeding time measurements were made 5 min after administration of each compound (5 min after the second compound in combined treatments). The effect of PGI 2 was followed up to one hour after administration. Prostacyclin induced a marked, dose-dependent prolongation of bleeding time (Table I). This effect was less but still significant after 30 min, still less decreased after 45 min and has disappeared by 60 min (Table II). The doses of PGI 2 used here were within the range of doses (0.25 – 16 μg/kg, i.v.) reported to produce a vasodepressor effect in anaesthetized male Wistar rats (8). However, the duration of the effect of PGI 2 on bleeding time was apparently longer (half-life about 20 min) than the duration of PGI 2-induced hypotension (2–3 min) (8). Provided the data reported by Armstrong et al. (8) can be extrapolated to the present study, the effect of PGI 2 on bleeding time appears to be dissociated from its cardiovascular effects. As previously reported (6,9), ASA (at doses between 5 and 200 mg/kg b.w.) did not significantly modify bleeding time. However, when rats were pretreated with ASA (50 mg/kg) between 5 min and 24 h before administration of PGI 2 (at a dose which itself did not influence bleeding time) a marked prolongation was observed (Table III). PGI 2 activity also strongly potentiated the prolonging effect of VK 774, when given either 5 min before or 5 min after this pyrimido-pyrimidine compound (Table III).