PROSTAGLANDINS

EFFECT OF PROSTACYCLIN ON PLATELET-ACTIVATING FACTOR IN=

DUCED RABBIT PLATELET AGGREGATION.

F.Bussolino and G.Camussi

Laboratorio di Immunopatologia.Cattedra di Nefrologia

Ospedale S.G. Battista. 14,C.Polonia

10126 Torino

ABSTRACT

In this paper, the effect of prostacyclin (PGI2) on the

aggregation induced by Platelet-activating factor (PAF), a

phospholipid mediator of anaphylaxis, was studied. Synthe=

tic PGI2 and PGI2 -1ikeactivitygenerated from rabbit aorta

were demonstrated to be effective inhibitors of PAF-in=

duced rabbit platelet aggregation and release of 3 H-sero=

tonin (3H-5HT),

INTRODUCTION

Platelet-activating factor (PAF) is a phospholipid medi=

ator of anaphylaxis brhose structure has been recently iden=

tified as l-O-alkyl-2-acetyl-sn-3-phosphorylcholine(1,2).PAF

is released by leukocytes from various species under immune=

logic stimuli(3-5) and by human and rabbit platelets(6).

PAF causes rabbit and human platelets to change shape , ag=

gregate and release their granule contents(3,5). The activa=

tion of platelets by PAF is independent both from arachido=

nit acid metabolites and released ADP(5,6), thus represen=

ting the mediator of the putative third pathway of platelet

aggregation(6). Prostacyclin (PG12), which is the major cy=

clooxygenase product in blood vessel wails(7), prevents pla=

telet aggregation induced by ADP and arachidonic acid and

has an important role as defensive hormone in the homeosta=

tic interaction between platelets and endothelial cells. In

this report, we present data concerning the inhibitory ef=

feet of PG12 on PAF-induced platelet aggregation and relea=

NOVEMBER 1980VOL.20NO.5 781

PROSTAGLANDINS

Se reaction.

MATERIALS AND METHODS

Isolation and purification of PAF: PAF was obtainedby

previously described procedures(3,4). Briefly , rabbit

buffy coat cells were prepared by differential centrifu=

gation followed by several washes to remove the majority

of contaminant platelets. Removal of erythrocytes was ob=

tained by sedimentation in 2.5% gelatin (Difco)in Tris-

buffered isotonic saline pH 7.4 and subsequent osmotic

shock. PAF bound to bovine serum albumin (BSA) fraction V

(Sigma) was obtained by overnight incubation of buffy co=

at cells in Tris-buffered Tyrode's(Tris-buffered Tyrode's

contained: Tris(hydroxymethyl)amino-methane 1x10 -2M; NaCl

1x10-'M; MgC12 IxIO-~M; CaC12 1x10-3M; KC1 2.6x10m3M; 0.1%

glucose) at pH 9.5 containing 0.25% BSA(8). PAF was extra=

cted by precipitating BSA with three parts of absolute e=

thanol. After concentration under vacuum, the ethanolwater

residue wasextracted with chloroform.The lipid thus obtai=

ned was chromatographed on a silicic acid col.umn developed

with chloroform, chloroform-methanol mixtures(9:1, 7:3, 6:4.

5:5)and pure methanol. The active fraction was eluted from

the column with the pure methanol, chromatographed on high

performance silicic acid column(Varian, Micropak Si5) and

eluted with a mixture of chloroform -methanol-water as

solvent. Evaporated PAP-methanol fraction, was dissolved in

absolute ethanol and kept at -209C. Before each experiment,

0.5 ml aliquots of PAF-ethanol were evaporated under vacuum

and the dry residue was then dissolved in 0.5 ml Tris-buf=

fered Tyrode's containing 0.25% BSA, pH 7.4. PAF activity

was detected by aggregation of rabbit platelets in the pre=

sence of IxIO-~M indomethacin(Sigma)(9). Physicochemical

characterization and treatement with lipases were carried

out on the active fraction in order to characterize them

782 NOVEMBER 1980VOL.20N0.5

PROSTAGLANDINS

as PAF(9-10). Synthetic PAF(Dr.Benveniste's personalgi=

ft)with a specific activity of 6 PAF units(4)/lng was

used as reference substance.

Aggregation and "release reaction" of rabbit plate=

telet by PAF:Suspension of twice washed rabbit platelets

was prepared as previously described(3), and PAF activi=

ty was determined in an aggregometer(Elvi 840) (4). Inso=

me experiments, the platelets were labelled in the first

washing with 2 u Ci. of 3 H-serotonin(3H-5HT, 5+ydroxy-

G-3H tryptamine creatine sulphate, 17.3 Ci. per mmole ,

The Radiochemical Center)for IO min. at 22°C. Five per

107/ml rabbit platelets were stirred at 900 rpm at 37OC

in Tris-buffered Tyrode's containing 0.25% gelatin at

pH 7.3 in the presence of 5 ng PAF or 2x10-4M ADP (Stago).

In some experiments, platelets were preincubated for3 min

at 37'C with several substances: IxIO-~M to 1x10 -I'M PG12

(Upjohn Co.), IxIO-~M Isoproterenol(Sigma), IxIO-~M Theo=

phylline(Sigma), Creatine phosphate(CP, 31.25 pg /IO0 ~1)

/Creatine phosphokinase(CPK,l5 .25~g/lOOvL)enzymatic system

(Sigma), 1x1 OB3M Dibutyryladenosine 3' :5'-cyclic monopho=

spheric acid (Dibutyryl CAMP, Sigma)and 1x10 -5 M Indometha=

tin. All substances were dissolved in Tris-Tyrode's buffer

pH 7.4, except PGI2prepared as 1x10 -3

M stock solution in

0.05 M Tris(hydroxymethyl)amino-methane buffer at pH 9.5at

4'C. Platelet aggregation was expressed in arbitrary units

of aggregation as millimeters of chart distance. 3 H-5HTwas

detected according to Greenberg(l1). Release of 'H-5HTwas

expressed as a % of total labelled 3 H-5HT determined byly=

sis of platelets by Triton X lOO(Sigma).

z2-like activity generation: PG12-like activity gene=

ration from New Zealandrabbitaorta incubated with washed

rabbit platelets was performed with a slight modification

ofHornstra"smethod (12). Aorta was removed , cleared ion=

NOVEMBER 1980VOL.20NO.S 783

PROSTAGLANDINS

gitudinally and kept for at least 90 min at 4O C in Ty=

rode's buffer (NaCl 2.37~1O-~M, KC1 5.36x10-3M,NaH PO

~.SXIO-~M, CaC12 2H20 ~xIO-~M, MgC12 6H20 1.4 x 10 -23;

,

NaHC03 2.3x10e2M, glucose 0.1%) with or without IxI~~M

indomethacin or ~.~xIO-~M tranylcyprom$ne (Sigma), inhi=

bitors of cyclooxygenase and PG12-synthetase respecti=

vely(13,14). A small piece of aorta tissue incubated

with 5x107/ml rabbit platelets under continuousstirring

at 900 rpm for 1 to 10 min at 37O C generated PG12-li=

ke activity (7,13). The amount of PG12 content of the

incubate was evaluated from the difference in ADP-in=

duced aggregation in the presence of aorta-containing

incubates(b) compared with aorta-free incubates(a) and

expressed as % inhibition: Inhibition% = (a-b)/a x 100

according to Hornstra (12). In the experiments performed

in order to evaluate the effect of PG12-like activity

on PAF-induced aggregation, 5 ng of PAF were usedinste=

ad of ADP. As control, the platelets preincubated with

aortic tissue treated with indomethacin or tranylcypro=

mine, which inhibit PG12-like activity generation (7,

?3), were tested in the same conditions as above.

The

ter

ter

RISSULTS

effect of PAF on rabbit platelets starts 2-3 secaf=

addition of the active material into the aggregome=

cuvette. Initially, platelets showed shape change, ?

then aggregation and release of "H-5HT (60.5% at 1 miln)

(fig.la). Both CP/CPK enzymatic system(at a concentsa =

tion able to completely convert 5x10 -5M ALIP to ATP) (5)

and indomethacin, an inhibitor of cyclooxygenase and

then of arachidenic acid-dependent aggregation(l4), had

no effect on PAF-induced rabbit platelet aggregation

(fig.la). Rabbit aorta tissue incubated with washedpla=

telets generated a PGX2 -like activity whLch had anfnhi=

,bitory effect on platelet aggregation induced by ADP

784 NOVEMBER 1980VOL.20NO.S

PROSTAGLANDINS

(Table 1).PG12-,like activity thus generated also inhibi=

ted PAF-induced platelet aggregation.

CP CPK

3H-5HT

f

58x

100 b

1

I f

3H-5HT

s 60X

?

Fig.la: Aggregation and release of 'H-5HT of rabbit pla=

telets induced by PAF. Rabbit platelets were tested in

aggregometry after challenge with 5 ng PAF. In some ex=

periments platelets were preincubated with indomethacin

or CP/CPK. Release of 3

H-5HT was expressed as % of total

platelet 3 H before stimulation. Fig.lb: Time dependent

PG12-likeactivity generation and its rffect on PAF-indg

ted platelet aggregation and 3 H-5HT release.

NOVEMBER 1980VOL.20NO.S 785

PROSTAGLANDINS

Table 1. Comparative Effect of PGI2-Like Activity Produced from

Rabbit Aorta, Isoproterenol,Tbeophylline and Dibutyryl CAMP on

PAF and ADP Induced Platelet Aggregation.

ADP PAF

% Inhibition of Aggregation(a)

Platelet preincubated with rabbit aorta 88.025.4 81.2z2.3

Platelet preincubated with indomethacin tre ated rabbit aorta - 13.8+_.0 4.922.2

Platelet preincubated with tranylcypromine treatedrabbit aorta 2.6+2.0 1.820.6

Isoproterenol(lxl&5M) 48.321.7 48.523.5

Tbeophylline(1x10-4M) 62.653.1 59.525.6

Dibutyry1cAMP(lx10-3M) 67.021.9 60.723.8

(a)Control values for platelet aggregation induced by 2x10m4M ADP

and 5 ng PAF were 95.4+8.6 and 200.0+7.4 aggregation units(mean +

1 S.D. of 10 experiments.)

PGI2-like activity generation was completely suppressed

by cyclooxygenase and PGI2 -synthetase inhibitors(suchas

indomethacin and tranylcypromine respectivelyjthus abo=

lishing the inhibitor effect on PAF-induced plateletag=

gregation(Table 1). As shown in fig. lb the inhibitory

effect on PAF-induced platelet aggregation and release

of 3 H-5HT,obtained preincubating platelets with aorta

tissue/increased proportionally with incubation time.

The PGI2 -like activity generated at 8 min. inhibitedag=

gregation and release of 3 H-5HT and at 10 min. alsosha=

pe change. Synthetic PGI2 was inhibitory at low concen=

tration (10 -10

M) and completely blocked both aggregation

and release of 3 H-SHT at concentration of 10 -8M , but

786 NOVEMBER 1980VOL.20NO.S

PROSTAGLANDINS

was ineffective on shape change.Much h.&ghBr-concenrra =

tion(10W7M) also prevented shape change. However, when

we used a concentrationof PAF(l ng) able to induce only

shape change of platelets, lO+M of PC12 prevented the

shape change(Table 2).

Table 2: Effect of Synthetic PG12 on PAF.Action on RabbitPlatelets

AGGREGATION(a) SHAPE GRANGE 'H-5HT(b)

CONTROL 180,3+ 5.8 YES 60.5+2.5

PGI 2 l~l<~'M 23,6+_ 3.8 YES 18.4t4.0

1x10-'M 5.3+ 0.7 YES 3.120.3

1~10-~M 0 YES 2.220.1

1~10-~M 0 NO 2.220.4

(a)Rabbit platelets vere preincubated with different doses of PG12

for 3 min and stimulated by 5 ng of PAP. Aggregation is expressed

in arbitrary units as nnn chart distance. (b) Release of 3 H-SKI was

expressed as % of total platelet 3H before stimulation.

Isoproterenol,an activator of adenylcyclase(l5), thee =

phylline,an inhibitor of phosphodiesterase(15),that in=

crease the intracellular concentration of c?AMP,as well

as dibutyryl CAMP, had an inhibitory effect on PAF-in=

duced rabbit platelet aggregation(Table 1).

DISCUSSION

PAF-induced platelet aggregation is independent from the

known pathways of platelet aggregation:ADP and thrombo=

xane A 2'

PAF is fully active on platelets in presence of

CP/CPK enzymatic system and indomethacin,at doses that

completely abolished aggregation by ADP and arachidonic

acid respectively. Recently it has been shown thatendo=

NOVEMBER 1980VOL.20NO.5 787

PROSTAGLANDINS

genous PAF from platelets may also partially account for

thrombin-induced platelet aggregation(6). PAF-induced a%

gregation requires extracellular Ca+Tenergy metabolism

and the activation of serine esterase and phospholipase

A2' CAMP levels and platelet contractile elements have

been implicated in platelet response to PAF(5). Release

of PAF from leukocytes (3,4,5)has been implicated in the

onset and amplification of vascular permeability leading

to the deposition of immune complexes in vessels and glo

meruli(5). Release of PAF appears in generalized anaphy=

lactic shock and in intravascular blood coagulation(l6).

The consideration of the crucial physio-pathological rg

le of PAF emphasizes the importance of a physiological@

hibition of PAF-induced platelet aggregation. On thispax

spective,we studied the effect of PG12 on 'PAF-inducedliLa

telet aggregation. PG12 is a potent inhibitor ofthecther

two pathways of platelet aggregation(ADP- and arachiwc

acid-dependent). The PG12 and PG12-like activity fromlab

bit aorta inhibit the action of PAF on rabbit platelets.

PG12inhibits aggregation and release of 3 H-5HT and, at

high concentration, also platelet shape change. The inh&

bitory effect of PG12 on PAF induced platelet aggregation

is probably due to the increase of intracellular cAMP.In

effect PG12 activates platelet adenyl cyclase(l7).Isopro -

terenol,theophylline and dibutyryl cAMP,that increase in -

tracellular cAMP(lS),inhibit PAF-induced plalelet aggre=

gation.It has been reported that CAMP inhibits the mobi!+

zation from storage sites of the cytoplasmic Calcium(l8),

essential for the aggregation and "release reaction"dph_

telets(lg).These data suggest that mobilazation of inter -

nal Ca ++ plays a role also in PAF-induced rabbitpiatelet

aggregation.

788 NOVEMBER 1980VOL.20NO.5

PROSTAGLANDINS

1)

2)

3)

4)

5)

6)

7)

8)

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ACKNOWLEDGEMENTS

The authors wish to thank Upjohn Co. for making the Pro=

stacyclin sodium salt.

Editor: Brendan Whittle Received: 4-30-80 Accepted: 8-20-80

NOVEMBER 1980VOL.20N0.5 791