iFiB.OSIBOSTSRESEARCH Printed in Great Brirain

Vol.

pp. ;01-TO!&, Pergamon Press,

11,

13;: Ltd.

NORWLPROSIXCYCLIX-LIKE ACTIVITY IN VASCULJRTISSUES FRCN THR@IROCk-TOPENIC RATS Silvia

Villa,

Antonella

Callioni

and Giovanni de Gaetano

Laboratories of Cardiovascular Clinical Pharmacology and of the Autonomic Nervous System Istituto di Ricerche Farmacologiche “4lario Negri” Via Eritrea, 61 - 20157 Milan, Italy (Received

5.8.1977; Accepted

in by

revised

Editor

L.

form Roka)

2.9.1977.

ABSTRACT Arterial and venous tissues from normal rats generate a potent inhibitor of platelet aggregation with the characteristics of prostacyclin Vascular tissues from rats made moderately (PGI,). 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 wssibility that chronic thrombocytopenia might affect prostacyclin generation by inducing endothelial damage, cannot be excludel. I OX I XTRODUCT

Vascular tissues from humans and from several laboratory animals, including the rat, generate prostacyclin (PGI2), a potent inhibitor of platelet aggregation (1,2). Prostacyclin derives from the enzymatic conversion of arachidonic acid through the formation of labile prostaglandinendoperoxides (PGG2, PGH2) (1). The observation that human vessels generate much more prostacyclin from endoperoxides than from arachidonic acid, led Xoncada et al. (1) to suggest that blood platelets could be an important source of endoperoxides as a substrate for vascular prostacyclin synthetase. The purpose of this study was to evaluate whether basal release of prostacyclin was affected in rats made thrombocytopenic (by heterologous platelet antiserum). MATERIALS ANDMETHODS Prostacyclin activity was assessed as platelet aggregation inhibitory Antiserum against rat platelets Xis activity as previously described (2).

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702

PG12 ACTIVITY

IS R&T VASCCZAXRE

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raised in rabbits according to Ashida and Abiko (3). It was administered to rats either i.v. or subcutaneously or both, following different treatment schedules, in order to obtain various degrees and duration of thrombocytopenia. Thrombocytopenic rats did not show any overt sign of haemorrhage. Acetylsalicyclic acid was given to both normal and thrombocytopenic animals in a single i-v. dose of 200 mg/kg, in the form of its soluble lysine salt (Flectadol-Maggioni, Milan, Italy) as described (4). RESULTS Different degrees of thrombocytopenia were obtained, ranging from 500,oO The platelet count in all control rats was over to 10,ooO platelets&l. The animals were killed from one to 72 hours after antiserum l,oco,ooo/~l. administration. Groups of three animals received one i.v. and two subcutaneous injections of antiplatelet antiserum with 24 hours between each. During the 72 hour observation period their platelet count ranged between 170,CCC and 9O,CEO/~l. Prostacyclin-like activity released from abdominal aorta and inferior vena cava of thrombocytopenic rats was consistently comparable to that released from vessel walls of control rats (Figure 1).

vein

control

. 1 min

. FIG. 1

Inhibition of platelet aggregation by PGI2 generated from rings of rat vascular tissue. Cut rings (6 mg) were incubated in 200 ul of tris buffer (0.05 mol/l, pH 7.4) for 4 min at room temperature. Samples of 50 ul of the supernatant from the incubation mixtures, incubated for 1 min in PRP produced a marked inhibition of platelet aggregation induced by ADP (1 umol/l). Results were very similar with rings from normal or thrombocytopenic rats.

x-01.11,x0.6

PGI 1 .?,CTIl-ITYIS RAT

, lmin

VASU”iXTlJRE

,

control

FIG. 2 Effect of i.v. injection of lysine acetylsalycilate (200 mg/kg b.w.) on the antiaggregatory activity of rat vascular tissues removed 5 min after drug injection. Results were very similar with rings from normal or thrombocytopenic rats.

Figure 2 shows that i.v. administration of acetylsalicylate (200 mg/kg b.w.) to control rats or to rats made thrombocytopenic (4 20,ocO platelets/ul) by an i.v. injection of antiserum one hour earlier, produced the same degree of inhibition of prostacyclin-like activity from vascular tissues. DISCUSSION This study shows that vascular tissue from normal or thrombocytopenic rats releases comparable amounts of prostacyclin-like (antiaggregatory) activity. Neither the degree nor the duration of the antiserum-induced thrombocytopenia affected the rat vascular tissues ability to generate this activity. We suggest that the basal release of prostacyclin from rat vessels does not depend on the number of circulating platelets at the moment of removal This would imply that of vascular tissues or during the three days before. rat vessels possess both the substrate and the enzymatic activities required The observation that acetylsalicyclic acid for prostacyclin production. administration to both normal and thrcxnbocytopenic rats is followed by almost immediate inhibition of prostacyclin-like activity supports this view (4). However, the possibility that chronic thrombocytopenia, by inducing

PGI

2

ACTIVITY

IX RAT VXSCCLXTl_-RE

endothelial damage, might reduce the ability prostacyclin, cannot at present be excluded.

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of vessel walls to generate

Judith Baggott, Anna Mancini, Paola Bonifacino helped in the preparation of this manuscript.

and Vincenzo De Ceglie

REFERENCES Humanarterial and venous tissues 1. PKNNN, s., HI&S, EA., and VA!! J.R. of platelet generate prostacyclin (prostaglandin X), a potent inhibitor aggregation. Lancet 1 , 18-21, 1977. 2. VILLA, s., WSLIWIEC, M. , and de CAETANO,G. Prostacyclin Lancet 1 , 1216-1217, 1977. atherosclerosis in rats.

and

effect of pantethine on experi3. ASHIDA, S.I., and ABIKO, Y. Protective Thromb. Diath. Haemorrh. 33, 528-539, mental thrombocytopenia in the rat. 1975. activity in rat vascular 4. VILLA, S.) and de GA!ZIWiO,G. Prostacyclin-like tissues. Fast, long-lasting inhibition by treatment with lysine acetylProstaglandins, submitted. salycilate.

Normal prostacyclin-like activity in vascular tissues from thrombocytopenic rats.

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