PROSTAGLANDINS

FORMATION 3Y PHOSPHOLIPASE A OF PROSTAGLANDINS AND THROMBOXANEA LIKE ACTlLlTY IN THE PLATELE?S OF NORMAL AND ESSENTIAL FATTY ACID2 DEFICIENT RATS. COMPARISON WITH EFFECT ON HUMAN AND R~BB IT PLATELETS. J.E.

Vincent

and

F.J.

Zijlstra

Department of Pharmacology, Erasmus 1738, Rotterdam, The Netherlands

University

Rotterdam,

P.O.Box

Abstract When rat platelets are incubated with phospholipase A2, thromboxane A2-like activity and prostaglandins are formed. The amounts are approximately similar, whether aggregation is induced after the incubation or not. No aggregation is observed when the platelets are incubated with phospholipase A2. In the platelets of essential fatty acid deficient rats, only small amounts of thromboxane A2-like activity and prostaglandins are formed. No formation of these substances occurs when human and rabbit platelets are incubated with phospholipase A2. The results indicate that formation of thromboxane AZ-like activity enhances aggregation in rat platelets, but that aggregation is not induced . Introduction During the aggregation of blood platelets, arachidonic acid (AA) is partially transformed into the prostaglandin endoperoxides PGG and PGH2 and into thromboxane A (TxA2) (1). The initial step in z his * reaction is the release of &A from the 2-position of the phospholipids by phospholipase A (PLA ). This enzyme is activated by several aggregation-inducing aggnts ($,3,4). The specificity of the action of PLA2 was recently demonstrated (5). When rat platelets are incubated with PLA a biphasic effect upon collagen-induced aggregation is observed. T?I: latter is enhanced after shorter incubation periods with the enzyme, whereas longer incubation periods lead to an inhibition (6). The first phase is suppressed after the addition of indomethacin. In the experiments described here, the effect of PLA2 on formation of PG-endoperoxides, thromboxane AZ-like activity and prostaglandins (PG’s) in platelets has been determined. These effects were measured in the platelets of both normal and essential fatty acid (EFA] deficient rats. A comparison was made with the action of PLA2 upon human and rabbit platelets. Methods

and materials

The PG’s, PG endoperoxides and TxA2 were determined by bioassay using the cascade method. The organs were superfused with Krebs buffer and

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PROSTAGLANDINS

a mixture af antagonists and indomethacin In the cascade, the (71. following organs were present: - Rabbit aorta, with which the sum of PG endoperoxides and TxA2-like activity is measured (1); - Rabbit coeliac and mesenteric arteries. It has been shown that TxA2 induces contractions in these organs (8); PG activity is determined as PGE2. - Rat stomach. The measurement of platelet aggregation and the Induction of EFA deficiency have been described before (9). Thin

layer

chromatography

14

C) arachidonicicid was added to a suspension of 500 ul 0.1 u Ci (lrat platelets and incubated for 3 min. This was followed by the addition of 100 ug PLA2 and incubation for 3 min. The suspension was centrifuged and the supernatant acidified with formic acid and extracted with 5 ml ethylacetate. The ethylacetate was evaporated and the residu dissolved in chloroform and applied to a TLC plate (SiliTwo dimensional chromatography was performed according to cagel). Bailey etal(lO).The following solvents were used: chloroform, methanol, acetic acid, water (90:8:1:0,8) (first direction) and diethylether, methanol, acetic acid (gO:l:Z) (second direction). A Berthold thin-layer scanner with dot printer-was used in the scanning of the chromatograms. In order to determine the position of the non-labelled compounds, the plates were sprayed with a solution of 5% phosphomolybdic acid in ethanol and heated for 5 min at IOO’C. Blue spots indicate the presense of thromboxane B2 and the prostaglandins. Materials ‘,:;;;“,yl from the ous gift Research,

A2 of porcine pancreas Phigin, was obtai;td from BoehC) arachidonic acid, (lC) PGE2 and (lC) PGF2, Radiochemical Centre, Amersham, England. PGD was a generof Upjohn and thromboxane B2 of Dr. D.H. Nug ! eren, Unilever Vlaardingen, Holland.

f;:%

Results During platelet aggregation induced by collagen, a substance with rabbit aorta contracting (RCS) activity is present after 20 sec. After 1 min this activity reaches a maximum (Fig. l,a,b). No activity for 1.5 min with is seen when platelet poor plasma (PPP) is incubated When platelet rich plasma (PRP) is incub40 ug/ml PLA2 (Fig. 1,~). ated with 40 ug/ml PLA2 during 1.5 min, a considerable increase in the amount of RCS formed occurs (Fig. 1 ,d). Similar amounts are form ed when PRP is incubated with PLA during 1.5 min, followed by aggregation induced by collagen (Fig. 3,e).

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DECEMBER 1977 VOL. 14 NO. 6

PROSTAGLANDJNS ‘JORMAL 2LATELETS

Aggregation

v d

b 23

1

merenteric

k . b

c . 0

ng All

rabbit aorta IO

h

\ ng

All

\

:I

A

2

.

b

0

d

f

rat stomach

ng PGE2

\

. 0

1

\

. b

PRPcollagen 20”

collagen

PLA2

A d

PRP-

PRP-PLA2

PRP-lmidozole

PLA2

collagen

PLA2

I’

Legend to Fig. 1: Determination of RCS, PG and mesenteric artery contractina activities Aggregation was induced in 200 1.1~PRP with of PRP of normal rats. 40 ug/ml collagen. PLA2 40 ug/ml was incubated during 1.5 min with PRP. 100 ul PRP was added to a cascade consisting of a strip of rabbit aorta, rat stomach and rabbit mesenteric artery. of PRP with PLA2; d: incubation of a,b: aggregation; c: incubation followed by aggregation of PRP with PLA PRP with PLA2; e: incubation 0.5 min, followed f: incubation of PRP with 3.10m3M imidazole 3 uring by incubation with PLA2 and aggregation. Reference substances were teric artery and PGE2 for

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angiotensin rat stomach.

1977 VOL. 14 NO. 6

II

for

rabbit

aorta

and

mesen-

1045

PROSTAGLANDINS

These data indicate that formation of PG endoperoxides and TxA2like activity occurs during the aggregation of rat platelets. Unexpected is the formation of this activity when the platelets are incubated with PLA2. A comparatively high amount of RCS activity is formed which does not lead to aggregation. When aggregation is induced after incubation the amount of RCS formed is not increased. The effect of the TxA -like activity, formed in these experiments on human platelets was a 3 so investigated. When human platelets are incubated with 100 I-g/ml PLA , no aggregation occurs. The addition of 50 ul of rat PRP incubate 3 for 1.5 min with 100 pg/ml PLA to 200 ~1 human PRP induced aggregation. As the addition of rat PR6 to human PRP leads to a slow aggregation after prolonged stirring, the experiment was repeated with the filtrate of rat PRP treated with PLA2. This also induced aggregation. It has been observed, that incubation with PLA2 during shorter periods leads to an increase in collagen-induced aggregation (6). These data can be explained in several ways: First, the assumption can be made that in rat platelets the formation of TxA2-like activity can enhance, but not induce aggregation. that formation of PG endoperoxides and Second, it could be possible PG’s occurs on the outer membrane of the platelet, but that TxA2like activity is not formed. The RCS-activity is considered to be a mixture of the PG-endoperoxides and TxA2 (1). the above menti:oned experiments In order to investigate this point, and the activity of the samples determined 6y measurwere repeated, ing the contractions of strips of coeliac and mesenteric arteries of a male rabbit. In these organs, contractions are induced by TxA2, butnotby PG endoperoxides (81. The results indicate that formation of TxA -like activity occurs when platelets are incubated with PLA (Fig. !,e). RCS- coeliac and mesenteric artery contracting act?vities show a parallel course It has been reported recently, that imidazole in these experiments. inhibits the thromboxane-synthetase system in platelet microsomes (111. When this s&stance was added to the system used here, RCScoeliac and mesenteri,c artery contracting activi~ties were reduced, whereas an increase in PG formation occurred (Fig. :,f)l. This is an indication that TxA 2 is formed when rat platelets are incubated with PLA2.

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PROSTAGLANDINS

The products formed from AA were also determined by thin-layer,4 A rat platelet suspension was incubated with (lC) chromatography. AA and after incubation with PLA2 extracted and the products separatThe positions of PGD2 and thromboxed by thin-layer chromatography. ane B2 (TxB2) were determined by chromatographing these substances and spraying the plates with a phosphomolybdic acid solution in alThe peaks corresponding to 12L-hydroxy-5,8,10_heptadecatriencohol . oic acid (HHT) and 12L-hydroxy-5,8,10,14-eicosatretranoic acid (HETE) are tentatively identified, using the Rf values reported (10). The products formed after incubation with PLA2 are shown in Fig. 2. The first p’eak,appearing after the origin consists mainly of TxB2 and PGE2. When imidazole is added, the amount of PGE2 increases and PGF and PGD2 appear (Fig. 3). P”he same experiments have been performed with the platelets of EFA deficient rats. Only small amounts of RCS and rat stomach strip contracting activities are found, indicating the absence of formation of prostaglandins and TxA -like activity (Fig. 4). In essential fatty acid deficiency, AA (2&4 ~6) is replaced to a large extent by another fatty acid, 20:3 a9 which is normally present in small amounts. This substance is not a precursor of the PG’s. Platelet aggregation by collagen is nearly normal (12) and enhanced by PLA (62. The explanation could be, that a substance is formed from t t!e 2D:3 w8 acid which enhances platelet aggregation and has no RCS activity. The results of these two experiments are represented in Fig. 5. A dose-response curve for the formation of RCS activity by PLA2 is shown in Fig. 6A. When platelets are incubated with PLA2 in increasing amounts before aggregation, PGE2 formation increases (Fig. 6B). Different effects are observed when PLA is incubated with human and rabbit platelets. In these cases, no 3 ormation of prostaglandins and TxA2-like activity is observed. Discussion The data reported here indicate that the following events most probably occur when the platelets are incubated with PLA2: First, AA is released from the phospholipids. Second, this substance is transformed by enzyme systems on the out7 side membrane into PG-endoperoxides, a TxA2-like substance and PG’s. Unexplained remains why no aggregation takes place when TxA2-like activity is present. This effect can most probably not be attributed to an impaired reactivity towards aggregating stimuli due to the action of enzyme or to the formation of an inhibitor as collagen-i,nduced aggregation is enhanced after incubation with PLA2 during shorter peri,ods. It seems ltkely that TxA2 or the equivalent substance formed

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1977 VOL. 14 NO. 6

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PROSTAGLANDINS

2nd.,;

/

’

/I ,

E2 F2c! -

B ” I+ sz

Fig.

1048

2.

Thin Layer incubation and PLA2.

chromatogram of of a rat platelet

the products, suspension

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after obtaine 14 C) AA with (l-

1977 VOL. 14 NO. 6

PROSTAGLANDINS

AA’ PLA2

Fig.

3.

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Thi,n layer chromatogram of the products.ohtabed,iffter the incubation of a rat platelet suspension with (lC)AA, imidazole and PLA . 14 After incubatik with (lC)AA the suspension was incubated with imidazole 3.10-3 M for 3 min followed by PLAZ.

1977 VOL. 14 NO. 6

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PROSTAGLANDINS

V

v

r

r-

C

EFAD platdets i

rabbit

aorta

@T*> a

b

C

d

20 rat stomach PGE2 “9 > a PRP collagen

Fig.

1050

4.

-F+b PPPPLA;!

> C

PRP PLA;

d

PRP-PLA2 collagen

Determination of RCS and PG activities of PRP of EFAdeficient rats. Induction of aggregation and incubation with PLA2 the same as in fig. 1. a: Aggregation. Sample taken after 1 min. b: Incubation of PPP with PLA2 c: Incubation of PRP with PLA followed by aggregation. d: Incubation of PRP with PLAZ,

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1977 VOL. 14 NO. 6

PROSTAGLANDINS

Normal

platelets

PRP-PLA2

PPP-PLA2

PRPcollagen

PRP-PLA2

PRP-lmidazole

collagen

PLA2

EFAD platelets

0

aggregation

a

mesenteric art.

q rabbit I

aorta

rat stomach

b

Fig.

PRP-

PPP-

collagen

PLA2

5.

PRPpL%

PRP-PLA2 collagen

Sumnary of effects of incubation of PRP of normal and EFA deficient rats with PLA . The height of the bars corresponds to the values in fig. l’and 2. The increase in PG content in the PRP-lmidazole-PLA group is statistically significant compared to the PRP-P?A2 collagen group. (P