Degree and duration of prostaglandin E1-induced inhibition of platelet aggregation in the rat

EUROPEAN JOURNAL OF PHARMACOLOGY 15 (1971) 343-349. NORTH-HOLLAND PUBLISHING COMPANY

DEGREE AND DURATION

OF PROSTAGLANDIN

INHIBITION OF PLATELET

AGGREGATION

El-INDUCED IN THE RAT

G. HORNSTRA Unilever Research, Vlaardingen, The Netherlands

Received 1 February 1971

Accepted 19 April 1971

G. HORNSTRA, Degree and duration of prostaglandin El-induced inhibition of platelet aggregation in the rat, European J. Pharmacol. 15 (1971) 343-349. The inhibiting action of prostaglandin El (PGE1) on platelet aggregation in circulating rat blood was studied quantitatively. PGE1, given intravascularly, produced very strong but short-lasting inhibition of ADP-induced platelet aggregation. When PGE1 was given in a mixture with ADP, the relation between log dose PGE1 (0.16-5.0 X 10-~ ug/30 sec) and aggregation inhibition was positive and rectilinear; the inhibiting action of all doses tested lasted less than 10 min. However, after subcutaneous, intramuscular or intraperitoneal (i.p.) injection of 50 ~g PGE1, inhibition lasted 10-20, 30--40 and over 90min, respectively. When 3.125, 6.25, 12.5 or 25 #g PGE1 was administered i.p., duration of the aggregation inhibiting action (AIA) was about 10, 10, 60 or 70 min, respectively. Intraperitoneal administration of 12.5-50/~g PGE1 caused a biphasic AIA, possibly as a result of a PGEl-induced serotonin release. The results indicate that PGE1 is suitable for the prevention of intravascular thrombocyte aggregation and may be useful clinically.

Prostaglandin E 1 Thrombocyte aggregation inhibition

1. INTRODUCTION Since Kloeze (1967) observed the inhibiting action of prostaglandin El (PGE1) on platelet aggregation and adhesion in vitro, numerous investigators have confirmed and extended his findings. The possible application of PGEI as an anti-thrombotic agent is worthy of investigation since thrombocyte aggregation may play an important role in thrombogenesis. To this end, several techniques have already been applied and it has been shown very clearly that PGE1 inhibits the formation of platelet aggregates and platelet thrombi in vivo very effectively (Chandrasekhar, 1967; Emmons et al., 1967; Itoh et al., 1967; Kloeze, 1970a,b). However, the techniques used were not suitable for obtaining quantitative information about the degree and duration of the PGEl-induced aggregation inhibiting action (AIA). We therefore

ADP-induced platelet aggregation Serotonin release

investigated the AIA of PGEI in rats by means of the filter-loop technique, which proved suitable for measuring platelet aggregation in circulating rat blood (Hornstra, 1970).

2. MATERIALS AND METHODS The degree of platelet aggregation in circulating rat blood was determined by continuous measurement of the blood pressure in front of and behind a microfilter connected to the extracorporeally extended arterial circulation of rats. Aggregation was induced by infusing 0.06 gg ADP/30 sec (adenosine diphosphate di-sodium salt, Boehringer) in front of the filter. The aggregates obstruct the filter, resulting in changes of pressure across the filter. From these changes, the degree of aggregation, the aggregation-

G.Hornstra, PGE l-induced inhibition of platelet aggregation

344 mmHg 100

a

_

EP

the first aggregation index indicated whether or not the AIA lasted longer than 10 min.

1

90 80 70 60 50

,EP 2

40

120 110 100 90

60 sec i

80

i

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r--'l

ADP

Fig. 1. Recording o f an aggregation measurement: (a) blood pressure behind the filter; (b) blood pressure in f r o n t o f the filter; A = 100(t - EP2/RP2 X RPI/EP;).

index A, can be calculated (see fig. 1). The time interval between two consecutive determinations in the same animal was standardized at 10 min. The influence of PGE1 on the blood pressure was measured by continuous recording of the reference pressure (systolic blood pressure in front of the fdter). PGE~ was administrated intra-arterially (mixed with ADP), intravenously, subcutaneously, intramuscularly or intraperitoneally; it was dissolved in 0.9% NaCl-solution (pH adjusted at 7.42 with 0.01 N NaOH, 38°C, 100/ag PGEI/ml saline). The controls received corresponding amounts of saline without PGE1. The experiments were divided into 3 series.

2.1. In tra-arterial administration of PGE ~ mixed with ADP In 7 groups, each consisting of 8 male Wistar rats (body weight 2 7 5 - 3 0 0 g), 3 consecutive aggregation measurements per animal were carried out. In the first and third determinations only ADP was administered (control). In the second measurement the ADP was mixed with PGE1 to give doses varying from 0.0-0.5/2g PGEI/30 sec. Each group received a different PGEl-dOse. The difference between the first and second aggregation index indicates the degree of the PGE1-induced AIA. Comparison of the third with

2.2. Intravenous, subcutaneous, intramuscular and intraperitoneal administration of 50 lag PGEI In 8 groups (1 control and 1 experimental group for each route of administration), each consisting of 6 young-adult male Wistar rats (mean body weight 275 g), 6 consecutive aggregation measurements per animal were performed. 1 min before the second measurement 0.5 ml saline (control) or 50~ug PGEI in 0.5 ml saline (experimental) was administered intravenously, subcutaneously, intramuscularly or intraperitoneally (i.p.). Degree and duration of the PGE~induced AIA were catculated from the differences in A within the experimental groups with respect to the first measured value, with the corresponding differences in the control groups. 2.3. Intraperitoneal administration of different doses of PGE1 In groups of 6 - 1 2 young adult male Wistar rats, body weight 275-300 g, 18 consecutive aggregation measurements were performed. PGE~, 50, 25, 12.5, 6.25 or 3.125#g in saline (experimental) or the corresponding amounts of saline without PGE1 (control) were administered i.p. Degree and duration of the AIA were calculated by comparing the differences in A within the experimental groups with respect to the first measured value, with the corresponding differences in the control groups.

3. RESULTS

3.1. Intra-arterial administration of different doses of PGE1 mixed with ADP The results of the aggregation measurements and the aggregation inhibiting actions of various doses of PGE1 are given in table 1. There were no significant differences between the mean A 1-values (aggregation index at the first measurement) in the 7 groups nor was the difference between the aggregation indices at the first and the second measurement (A1 and A~) in the control groups significant. Therefore the AIA of PGE1 could be expressed as the difference between A1 andA2 (z~l).

G.Hornstna, PGE l-induced inhibition of platelet aggregation Table 1 Aggregation inhibiting action (AIA) of PGE l when mixed with ADP (n = 8). A 1: f'trst aggregation-index, induced by ADP only; A 2 : second aggregation-index, induced by ADP + PGE1 ; A 3: third aggregation-index, induced by ADP only. PGE 1 0zg X 10-2/30 sec)

A:

A2

A3

AIA ± SE (AA)

0.0 0.15625 0.3125 0.625 1.25 2.5 5.0

78.9 76.1 68.5 63.0 66.0 69.0 61.7

77.4 69.6 57.8 42.9 43.0 26.9 18.1

68.9 75.4 68.4 59.8 57.3 64.9 55.6

6.5 ± 3.6 10.7 ± 3.7 20.1 ± 5.0 23.0 _+5.6 42.1 ± 5.4 43.6 ± 2.1

As illustrated in fig. 2, there was a significant positive rectilinear relationship between log dose PGE1 and its AIA, represented by y = 71.4 + 26.8 x, in which y = the aggregation inhibiting action (AA) and x = log dose PGEI. In no group was A3 significantly lower than A I ; thus it was concluded that the AIA of PGE~ in intra-arterial doses of up to 0.05/ag/30 sec lasted less than 10 min. P G E : , 1.25 X 10-2 #g/30sec or less, did not influence systolic blood pressure; 2.5 or 5.0 × 10-2 /ag PGE1/30 sec produced a small, short-lasting fall in pressure of 3.7 and 7.9%, respectively.

345

3.2. Intravenous, subcutaneous, intramuscular and intraperitoneal administration of 50 Izg PGE~ The mean aggregation indices (A) of the four control and experimental groups are given in table 2. Within each administration route, there were no significant differences between the first measured aggregation indices (A 1) of control and experimental groups. Therefore the influence of PGEI on ADP-induced aggregation could be calculated by correcting the course of A with respect to A ~ in the experimental group by that in the control group; the results of these calculations are given in fig. 3. The results show that the AIA after 50 #g PGE~ persisted for 10 min after intravenous administration, 20 rain after subcutaneous administration, and more than 3 0 m i n after intramuscular administration. The AIA of 50/ag PGE~ was longest after i.p. injection. However, the experimental time was too short (41 rain) to determine the exact period of action. Therefore, the dose-response relation of i.p. administered PGE~ was investigated in experiments lasting 3 hr (§ 3.3.). PGE1 administration resulted in a slight, rapidly occurring decrease of blood pressure which always lasted less than 10 min. This period is much shorter than the duration of AIA after subcutaneous, intramuscular and intraperitoneal administration.

50

Table 2 Mean aggregation-index on six-fold ADP-administration (A 1-A 6) in rats injected with 50 #g PGE 1 or 0.5 ml saline (control) via four different routes (n = 6).

30

,O,o

Route

Group

A1

A2

A3

A4

As

A6

Intravenously

P G E 1 56.0 8.5 4t.9 48.3 48.8 27.9 control 69.1 53.2 51.4 45.0 41.0 33.5

Subcutaneously PGE1 67.0 41.7 34.5 38.2 44.7 44.9 control 53.5 55.6 51.7 47.0 42.1 38.6 0.15625

0.3125

0.625

1,25

2.5

50

!.o9 dose PGE 1 x 10 -2 jJg/30 sec

Fig. 2. Relationship between log dose PGE1 and aggregation inhibiting action (AA) ± SE of PGE1 ; PGE1 was administered intra-arteriaUy, mixed with ADP. y = 71.4 + 26.8 x.

Intramuscularly PGE: 60.3 35.7 32.5 31.0 25.3 32.7 control 55.0 58.0 53.2 48.9 43.2 40.1 PGE 1 69.7 43.0 45.0 42.4 32.8 29.5 Intraperitoneally control 62.4 61.2 64.1 60.8 54.6 53.3

G.Hornstra, PGE l-induced inhibition o f platelet aggregation

346

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3.3. Intraperitoneal administration o f different doses

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In fig. 4, the course of the aggregation index A is given after i.p. administration of 0 (4 control groups see § 2.3), 3.125, 6.25, 12.5, 25 and 50/ag PGE1. No significant differences existed between the mean A 1-values of the 9 groups. Moreover, the courses of the 4 control groups did not differ significantly from each other. Therefore the AIA values of the 5 different doses of PGE1 were calculated with a common, mean control group. The course of the AIA of PGE1 is represented in fig. 5.

First phase ~ A _+SE

Time (min)

- 1 8 . 6 _+6.4 -26.026.1 - 2 9 . 7 -+ 3.8 -22.8+8.8 - 2 5 . 2 _+4.7

1 1 1 11 11

'T _ 50

Fig. 3. Course of the aggregation inhibiting action (AA) _+SE of 5 0 ~ g PGE1, administered via different routes. On the abscissa the time after PGE1 administration is given. Probability legend: - , P l > 0.1; ?, 0.075 < P l < 0.1;+, 0.05 < PI < 0.075; *, 0.01 < P1 < 0.05; **, 0.001 < P1 < 0.01; ***, P1 < 0.001.

3.125 6.25 12.5 25 50

Second phase AA +_SE - 3 0 . 8 + 6.0 -34.4 +9.8 -38.7 +6.8

Time (min) 41 51 51

Table 4 Change o f the systolic blood pressure after intraperitoneal administration of PGE1. Control groups

Experimental groups PGE ] (ug)

3.125 6.25 12.5 25 50

n

6 6 6 6 11

P1 a ( m m Hg)

99.8 96.3 114.0 109.6 123.6

a For explanation see text.

Depressor frequency

4 3 5 5 11

Max. pressure change Time t (sec)

&P (% of P1)a

230 164 186 131 140

-0.9 -4.8 -3.6 -10.2 -9.9

n

P1 a ( m m Hg)

&P at time t

Probability

6 6 6 6 9

100.5 100.5 115.3 124.2 116.4

+2.3 0.0 +4.0 +1.1 -0.8

P2 > 0.10 P2 > 0.10 0.05 > P 2 > 0.01 0.075 > P~ > 0.05 P2 < 0.001

G.Hornstra, PGEl.induced inhibition of platelet aggregation

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After 12.5, 25 and 50~g PGE1, the AIA-curves showed a biphasic course. The maximal AIA's of both inhibitory phases are stated in table 3. In all animals, blood pressure responses (AP) after PGE1 administration were measured with respect to the systolic blood pressure just before injection of PGE1 (Pl). PGE~ did not decrease blood pressure in all rats (sometimes even an increase occurred). In those rats in which PGE~ reduced blood pressure, the time that elapsed (min) between injection of PGE1 and the

On

maximal reaction was determined and the average time of maximal pressure change within each group was calculated (time t). The pressure changes at time t in the remaining animals of each PGE1 group (i.e. those rats in which PGEI did not lower blood pressure) and the corresponding control group were then measured and the mean maximal pressure change per group was calculated. In table 4 the results of these calculations are given; in all cases the slight depressor effect of PGEI disappeared within 10 min of its administration.

G.Hornstra, PGE 1-induced inhibition o f platelet aggregation

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4. DISCUSSION PGEx strongly inhibited thrombocyte aggregation in circulating rat blood. The method used permitted

the exact degree and duration of the AIA of PGE] to be determined. When administered intravascularly, either intraarterially or intravenously, the AIA was of short duration. This can be explained by rapid disappearance of PGE1 from the circulation. Ferreira and Vane (1967) demonstrated that on infusing 0.5-1.O/zg PGEx/min, 80-90% was inactivated after one single passage through lungs or liver of cats, rabbits and dogs. In exploratory investigations with isolated rat livers, we found that at a concentration of 0.3-1.0/lg PGE1/ml perfusate and a flow rate of 10 ml/min, about 80% of the PGE~ was metabolized in one passage through the liver. The duration of AIA of PGE~ was greater after subcutaneous, intramuscular or intraperitoneal administration, probably because of slower absorption. When administered i.p., 12.5-50/ag PGE~ caused a biphasic aggregation inhibiting action; inhibition was monophasic after 3.125 and 6.25/lg PGE]. The maximal AIA-values of the first inhibitory phase produced by different doses of PGE1 were not significantly different from each other; this also applied to the second inhibitory phase if it was present. It is very unlikely that the second inhibitory phase is due to PGE1 alone. If so, then the PGE1concentration in the blood 4 0 - 5 0 min after its administration must be equal to or higher than its concentration after 1-10 min, since PGE~-induced AIA is dose-dependent (see fig. 2). However, 51 min after intraperitoneal administration of 25/ag PGE~, the concentration in blood was far less than at 11 min; the concentration was determined by a newly developed method of Jouvenaz et al. (1970). Therefore, the second inhibitory phase, seen after i.p. administration of PGE~, is in part due to an aggregation inhibiting PGE]-metabolite or to another aggregation inhibiting substance that is released into the circulation following administration of PGE 1 • Several investigators have demonstrated that PGE~, administered intravascularly or i.p., releases catecholamines in dogs (Kayaalp and Tiirker, 1967) and rats (May et al., 1969). However, it is doubtful whether these amines are responsible for the second phase of aggregation inhibition, since, in general, they stimulate aggregation (Ardlie et al., 1966; Sinakos and Caen, 1966). Moreover, biphasic aggregation inhibition was absent after intravascular infusion of

G.Hornstra, PGE l-induced inhibition of platelet aggregation PGEI for 10 min (Hornstra, to be published). Thus, if a substance released by PGE1, is responsible for the second inhibitory phase seen after intraperitoneal injection, then this release takes place in the peritoneum before and/or during absorption. In the peritoneum of rats a large number of mast cells are found, which contain serotonin (Benditt et al., 1955). It has been shown that if the amount of serotonin in the body is increased~ then the tendency of the blood to form thrombi is decreased (Shimamoto, 1960; Baumgartner et al., 1964). Baumgartner and Born (1968) demonstrated that, under certain conditions, serotonin inhibits ADP-induced thrombocyte aggregation in vitro. Conflicting data are given in the literature, concerning possible serotonin-release by PGE1 (yon Euler, 1968; Thompson and Angulo, 1969). Since exploratory investigations have shown that the second phase of aggregation inhibition after i.p. PGEl-administration is decreased considerably after treatment with Compound 48/80, PGE~ may release serotonin and this may, at least in part, be responsible for this second phase. This hypothesis is now under investigation. In our experiments, 12.5-50/2g PGEr given intraperitoneally, caused a slight drop in blood pressure. Vergroesen et al. (unpublished results) did not observe a depressor effect in untreated rats after intraperitoneal administration of 6 - 2 0 0 #g P G E t . Therefore, the hypotensive effect may be due to extension of the arterial circulation by the extracorporeal filter system.

ACKNOWLEDGEMENT The author wishes to thank Miss A. v.d. Windt, Miss S.Y. Gielen and Miss L.J.M. Kurvers for their technical assistance and Mr. R.N. Lussenburg and Mr. R. v. Splunter for the statistical evaluation of the results.

REFERENCES Ardlie, N.G., G. Glew, C.J. Schwarz, 1966, Influence of catecholamines on nucleotide-induced platelet aggregation, Nature 212, 415. Baumgartner, H.R., A. Studer, K. Reber, 1964, Influence of 5-hydroxytryptamine, 5-hydroxytryptophan, dopamine,

349

norepinephrine and reserpine on thrombotic deposits in the rabbit, Thromb. Diath. Haemorrh. 12, 169. Baumgartner, H.R., G.V.R. Born, 1968, Effects of 5-hydroxytryptamine on platelet aggregation, Nature 218, 137. Benditt, E.P., R.L. Wong, M. Arase, 1955, 5-Hydroxytryptamine in mast cells, Proc. Soc. Exptl. Biol. Med. 90, 303. Chandrasekhar, N., 1967, Inhibition of platelet aggregation by prostaglandins, Blood 30 (4), 554. Emmons, P.R., J.R. Hampton, M.J.G. Harrison, A.J. Honour, J.R.A. Mitchell, 1967, Effect of prostaglandin E, on platelet behaviour in vitro and in vivo, Brit. Med. J. 2, 468. Euler, U.S. von, 1968, Prostaglandins, Clin. Pharmacol. Therap. 9 (2), 228. Ferreira, S.H., J.R. Vane, 1967, Prostaglandins: their disappearance from and release into the circulation, Nature 216,873. Hornstra, G., 1970, Method to determine the degree of ADP-induced platelet aggregation in circulating rat blood ("filter loop technique"), Brit. J. Haematol. 19, 321. Itoh, T., N. Kobayashi, Y. Suzuki, M. Nakamura, 1969, The effect of prostaglandin E1 (PGE1) on blood platelet agglutination, Proc. Symp. ONO Pharm. Comp. Kyoto, pp. 97-108. Jouvenaz, G.H., D.H. Nugteren, R.K. Beerthuis, D.A. van Dorp, 1970, A sensitive method for the determination of prostaglandins by gas-chromatography with electron capture detection, Biochim. Biophys. Acta 202 (1), 231. Kayaalp, S.O., R.K. TfJrker, 1967, Release of catecholamines from the adrenal medulla by prostaglandin El, Eur. J. Pharmacol. 2, 175. Kloeze, J., 1967, Influence of prostaglandins on platelet adhesiveness and platelet aggregation, in: Prostaglandins, eds. S. Bergstr~Sm and B. Samuelsson (Almqvist and Wiksell, Stockholm) pp. 241-252. Kloeze, J., 1970a, Prostaglandins and platelet aggregation in vivo, I. Influence of PGEI and to-homo-PGE1 on transient thrombocytopenia and of PGEI on the LD 50 of ADP, Thromb. Diath. Haemorrhag. 33 (2), 286. Kloeze, J., 1970b, Prostaglandins and platelet aggregation in vivo. II. Influence of PGE1 and PGFla on platelet thrombus formation induced by an electric stimulus in veins of the rat brain surface, Thromb. Diath. Haemorrhag. 33 (2), 293. May, B., K. Jeuck, D. Helstead, 1969, Primate und sekund/ire Prostaglandineffekte auf den Fett- und Kohlenhydratstoffwechsel, Med. Ern~ihrung10 (8), 172. Shimamoto, T., 1960, Anti-thrombolic effects of monoamino-oxydase inhibitor, Circ. 22, 687. Sinakos, Z., J. Caen, 1966, Le comportement des plaquettes chez le rat, Thromb. Diath. Haemorrh. 16,163. Thompson, J.H., M. Angulo, 1969, Prostaglandin-induced serotonin release, Experientia 25 (7), 721. Vergroesen, A.J., H.M.v.d. Graaf, Unpublished observation.