Reversal of platelet aggregation by prostacyclin

Pharmacological Research Communications, VoL 10, No. 2, 1978

REVERSAL OF PLATELET AGGREGATION BY PROSTACYCLIN Ryszard D. Gryglewski, Department

Ryszard Korbut and Anna Ocetkiewicz

of Pharmacology, Copernicus Academy of Medicine in Cracow, Poland

Receivedin final form 3 February 1978

SUMMARY Platelet thrombi were formed on the surface of blood superfused collag@n strips. Mixed-venous blood from right atrium of heparinized and anaesthetized cats superfused (3 ml/min) a piece of the tendon of Achilles from a rabbit. An increase in weight of the superfused strip was continuously recorded and this gain in weight represented the deposition of platelet clumps. The maximal deposition of the clumps (400 - 600 m 9) occured after 30 - 40 mln of the superfusion. The pretreatment of the collagen strips with prostacyclln (0.5 - 5 ng/ml) inhibited the deposition of the platelet clumps. The treatment of the preformed platelet clumps with prostacyclin (I - 20 ng/ml) resulted in a dispersion of the clumps and a reversal of platelet aggregation. A similar deaggregatory effect of prostacyclln occured after an intra= venous injection of the hormone. IDmm for the in yivo de-aggregatory activity of prostacyclln M~s 7.5 ~g/~g. The in vivo reversal of platelet aggregation by prostac~clin was observed even 3 hours after the platelet clumps were formed. Thus for the first time the de-aggregatory action of prostacyclin in ylvo was demonstrated and quantified.

INTRODUCTION The a n t l - a g g r e g a t . o r y e f f e c t of p r o s t a c y c l i n (PGI~) in vitro (1,2) is closely related to the stimulation of ~ e cyclase In platelets (3,4). We have recently described a new technique for quantification of the anti platelet actlvlty of drugs i~n vivq (5). Presently, using rhls technique we have shown that prostacyclln not only prevents platelets from aggregation, bu~ also it reverses platelet aggregation in heparlnlzed blood. We believe that this demonstration of 0031-6989/78/0010-0185/~01.00/0

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Pharmacological Research Communications, Vol. 10, No. 2, 1978

the de-aggregatory effect of prostacyclin in vivo will have an impact on the clinical consideration of t " ~ " ~ m i n l s t r a t i o n of prostacyclin in thromboembolic diseases.

METHODS Cats body w e i g h t 2 - 3 kg were a n a e s t h e t i z e d w i t h sodium p e n t o b a r b i t o n e (40 mg/kg i . p . and i . v . ) and h e p a r i n i zed ( 2 . 5 0 0 u n i t s / k g i . v . ) . Mixed-venous blood was w i t h d r a w n from tke r i g h t a t r i u m ( c a n n u l a t e d t h r o u g h the r i g h t J u g u l a r v e i n ) by a r o l l e r pump (3 m l / m i n ) . Blood s u p e r f u s e d a c o l l a gen s t r i p of the tendon of A c h i l l e s (30 x 5 mm) and r e t u r n e d by g r a v i t y to the venous system, The w e i g h t of the b l o o d s u p e r f u s e d c o l l a g e n s t r i p was c o n t i n u o u s l y m o n i t o r e d by a HARVARD t r a n s d u c e r type 364 and recorded by a WATANABE p o l y g r a p h . A gain i n w e i g h t of the b l o o d - s u p e r f u s e d c o l l a g e n s t r i p s r e f l e c t e d the i n t e n s i t y of the f o r m a t i o n of p l a t e l e t clumps on the s u r f a c e of s t r i p s ( 5 ) . T h i s gain i n w e i g h t proceeded d u r i n g the f i r s t 30 - 40 min of s u p e r f u s i o n and no f u r t h e r gain i n w e i g h t o c c u r e d . The d e - a g g r e g a t o r y a c t i o n of s y n t h e t i c p r o s t a c y c l i n ( 6 , 7 ) was observed when the hormone was i n f u s e d e i t h e r d i r e c t l y over the b l o o d - s u p e r f u s e d strip or intravenously into the animal after the formation of the platelet clumps was completed, Intensity of the deaggregatory activity of prostacyclln was expressed as the percent of the maximal de-aggregation (Fig. I). The details of our i n y i y o technique for quantification of the antiplatelet potency of drugs were described elsewhere (5).

RESULTS

We have demonstrated t h a t p r o s t a c y c l i n r e v e r s e s p l a t e let aggregation in vivo both when administered directly over the blood-superfused collagen strips or when injected intravenously into the animal (Fig. 1). The de-aggregatory activity of prostacyclin was observed even 3 hour3 after the platelet clumps were formed. As expected prostacyclzn was a weaker de-aggregatory than antiaggregatory agent (Table I). The de-aggregatory potency of prostacyclin in vivo was IO_^ = 7;5 ~g/kg as calculated from ~s~;~l~°~'si~h~ast~gT~0d~-~g~;t~;~ ag c ~ o n Ofwhen administered at further stages of thrombi formation (I - 3 hours duration of blood superfusion) prostacyclin used to have a smaller but a prolonged de-aggregatory action as compared to its de-aggregatory action at the early stage of platelet aggregation (30 60 min duration of blood superfusion). The results presented in Table I and in Fig. 2 refer to the de-aggregatory action of prostacyclin at the early stage of thrombus formation.

Pharmacological Research Communications, Vol. 10, No, 2, 1978

PERCENTOFDE-AGGREGATION:

187

20~n 1

43%

BLOODO

N

56%

- 500.~

~

0

FIG.

1

Oe-aggregatory activity of prostacyclln (PGIo). Platelet thrombl were formed on the surface of collag~n strips which gained in weight (0 - 500 mg) when superfused (3 ml/min) with mixed-venous blood of the heparinlzed, anaesthetized cat. PGI 2 was infused either intravenously into the animal (I.V., 5 ~g/kg during 3 pin) or directly over the bloodsuperfused collagen strip (OIR., 10 ng/ml during 12 min). Percent of loss in weight of the collagen strip which was covered with the platelet thrombl was calculated and used as t h e

expression

for

the de-aggregatory

potency

of

PGI 2.

100-

./

i

°

/

_~.n=3, CU5 I

__ _.

FIG.

, ......

5

2

Quantification of the de-aggregaZory activity of prostacyclin in vivo. Various doses of prostacyclin (PGlo) were infused intravenously into n cats for each dose of PGIA and percent of the de-aggregatory activity was c a l c u l a t e d ~(see Fig.l). Veritical bars represent 2 s.e.m. ID50 for the de-aggregatory activity o f PGI 2 i s 7 . 5 p g / k g .

Pharmacological Research Communications, Vol. 10, No. 2, 1978

188

TABLE I

Comparison of antl-eg~regatory and de-aggregatory activities of prostacyclin (PGIo). PGI o was i n f u s e d a t Concentrations o f 0 ° 5 - 20 n g / ~ l o v e r ~he c o l l a g e n s t r i p s with mixed-venous blood of hepar±nlzed Antl-aggregatory activity was a s s e s s e d

which were superfused and a n a e s t h e t i z e d cats. by an infusion of PGI 2

3 min before blood was allowed to superfuse the strips. Oeaggregatory activity was assessed by an infusion of PGI_ 30 40 min after blood was allowed to superfuse the strips ~Fig.1). 7

i,

±

r

+ :

+

"

Percent of the maxlmnl activity Anti-aggregatory De-aggregatory

Concent ratlon

of PGI 2 (ng/ml) r~_:~

__

T

0.5 :L.0 5.0

:;: =alal~lllll=~lr."

""':"

--

Jlllll

_

.

lllrl

i

i'iii

16 + 3 (n = 5) 37 ~' 5 (n = 4) 65 ~" 2 (n = 4 )

10,0

92;'6

20.0

not"test

(n = 4 ) ed

14+

0 (n = 6 ) 3 (n = 7 )

3B ;" s (n . s) 53 T 5 (n = 6 ) 77 ~' 6 ( n

= 4)

Mean + s.e.m., n = number of experiments

DISCUSSION

Our in vivo technique for studying antl-platelet activity of d r ~ allowed us to quantify the antl-aggregatory potencies of aspirin, indomethacin and nlctindole in the circulating blood of heparinized and anaesthetized cats (5). Presently, we have shown ~hat prostacyclin (1,2) not only shares with cyclo-oxygenase inhibitors and thromboxane A 2 synthetase inhibitors their anti-aggregatory activities in vivo, but also it is capable to deslntegrate the pre-formed platelet clots, even three hours after they were consolidated in circulating blood, This ne~ p r o p e r t y of prostacyclin in vivo is proposed to be named a "de-aggregatory" activity in Contrast to its anti-aggregatory activity.i.e, a potency to prevent plate~ets from aggregation. The demonstration of the de-aggregatory activity of prostacyclin in vlvo gives a rationale for the administration of prostacy-~-li6--in arterial thrombosis,e.g, in fresh myocardial infarctions. However+ + it should be kept in mind that our" experiments were performed in heavily heparinized enimals and thus the extension of these results onto the patients with ~hrombosis might be premature. Mustard and Packham (8) have discussed the chances of a combined anti-coagulant, fibrinolytic and antl-platelet therapy in arterial thrombosis, In the light of+the discovery of the existance of prostacyclin (1,2) ~nd the discovery of its deaggregator V activity in heparinized blood the suggestion of Mustard and Packham became even more interesting. We have recently shown that during the experimental atherosclerosls t h e g e n e r a t i o n of prostacyclin by the c o r o n a ry vascular bed is severely suppressed (9) and platelet aggre

189

Pharmacological Research Communications, VoL 10, No. 2, 1978

g a b i l i t y is Increased ( I 0 ) . Therefore, i t =nay well be that the a d m i n i s t r a t i o n of p r o s t a c y c l i n w i l l o f f e r a s p e c i a l advantage when arterial thrombosis is resulted in by atherosclerosis.

ACKNOWLEDGEMENTS'

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The authors g r a t e f u l l y acknowledge the generous grant for~,equipment from the Trustees of The Wellcome T r u s t , London. U°K.

REFERENCES

I . S. HONCADA, R.O. GRYGLEWSKI, S. BUNTING and D.R..VANE, Nature (London), 263, 663-665 (1976),, 2, "R.-%'~RY~LEWSKI, S. BUNTING,~S. MONCAOA, R.O..FLOWER and 3.R. VANE, Prosta l a n ~ i n s , 1~2, 685-713 (1976). 3. R. ,GORMAN, S. BUNTING-and O.V. MILLER, P r o s t p g l a n d i n s , 13, 377-388" ( 1977 ), 4. 3.E. TATESON, S. MONCADA and O.R. VANE, prostagland,,ins , 13, 3B9-397 (1977). 5. ~'.0. GRYGLEWSKI, R. KORBUT, A . OCETKIEWICZ and T. STACHURA, Naunyn Schmiede,b,,,erq;s Arch. Pharmacol. i n press: 6. R.A. OOHNSON~-~-F.-H'--L~OL'N, D o L , ' ~ M P ~ ~ , NIDY, S.A. MIZSAK and U. AXEN, 3. Am. Chem.,-99o 12-19 (1977) 7. N. WHITTAKER, Tetrahedron--Le~ers,; ~ , ' 2 8 0 5 - 2 8 0 8 (1977): 8. a.P. MUSTARD and-H.A' P A C K ~ ~ F u ~ L E , 9, 19-76 (1975), 9. A, DEMBINSKA-KIE~,. T, GRYGLEWSKA,~. ZMUDA and R.O, GRYGLEWSKIo Prost~glandi'ns, 14, 1025-1034 (1977)~. 10. A. ZHUDA, A. DEHBiN=S~. KiEc, A : C~r~TKOWSKI and ~ R,O, GRYGLEWSKI, P r o s t a g l a n d i n s , ~ , ~ ~-035-1042 (1977) o