Unsaturated platelet-activating factor: Influence on aggregation, serotonin release and thromboxane synthesis of human thrombocytes

THROMBOSIS RESEARCH 41; 699-706, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.

UNSATURATED PLATELET-ACTIVATING FACTOR:INFLUENCE ON AGGREGATION, SEROTONIN RELEASE AND THROMBOXANE SYNTHESIS OF HUMAN THROMBOCYTES

1

R.Korth' , H.Riess2 , G.Brehm2 and E.Hiller2 INSERM U 200, Universite

Paris Sud, 32 rue des Carnets, 92140 Clamart,

France

'Medizinische Klinik III Klinikum Grosshadern, Universitat MUnchen, Marchioninistr. 15, 8006 MUnchen 70, Federal Republic of Germany (Received 12.6.1984; Accepted in revised form 8.10.1985 by Editor R. Gollwitzer) (Received in final form by the Executive Editorial Office 14.12.1985) ABSTRACT Unsaturated platelet-activating factor (paf-acether) aggregated thrombocytes of healthy male volunteers like saturated pafacether. Unsaturated paf-acether released serotonin in the presence of imipramine. Within one minute the release increased depending on the concentrations of unsaturated paf-acether up to 45% of the serotonin. Human thrombocytes synthesized only a small amount of thromboxane B (TXB ) after aggregation induced by unsaturated paf-acethe6. Unsgturated paf-acether prevented the binding of radiolabelled saturated paf-acether to intact washed thrombocytes in the same extent as saturated pafacether.

INTRODUCTION The platelet activating factor is a biological active phospholipid with a chemical structure of I-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (AcGEPC, paf-acether, PAT)(l-3). Monocytes,macrophages and neutrophils as well as cultured human endothelial cells and thrombocytes themselves release the factor (4-6). Preparations of paf-acether containing saturated and/or unsaturated alkyl moieties (7) aggregate human thrombocytes, induce the release of serotonin (8-12) and modulate the migration of human leukocytes (13). The effects of saturated or unsaturated PAF-acether seem to be independent of the cyclooxygenase (10-12). Intact washed thrombocytes or platelet membranes exhibit specific binding sites for satu ated paf-acether which can be measured in the presence of albumin and Ca !! +(l4-18). Key words: Unsaturated plateles-activating factor, aggregation, lease, thromboxane synthesis, H-paf-acether-binding. 699

serotonin re-

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The purpose of the present study was to compare unsaturated paf-acether (from ratfish-Hydrolagus colliei-liver oil) with synthetic saturated paf-acether on aggregation of human thrombocytes suspended either in plasma (PRP) or in buffer (washed thrombocytes). Serotonin release and thromboxane B synthesis were also investigated used the unsaturated compound. Finally we g nalysed the ability of unsaturated paf-acether to desplace saturated radiolabelled pafacether bound to intact washed thrombocytes.

MATERIAL AND METHODS __Preparations of paf-acether containing 20% saturated and 80% monounsaturated alkyl moieties (16,7% hexadecyl and 78,2% octadecyl moieties) were prepared from ratfish liver oil by T.Muramatsu (Tokyo Medical and Dental University, Tokyo, Japan) as described previously (7). Saturated synthetic paf-acether was obtained from Bachem (Bubendorf, Switzerland). The preparations of pafacether were dissolved as described previously for washed (19) or unwashed (9,ll) human thrombocytes. Thin layer chromatography was carried out as previously described (20). The hemisynthetic and synthetic preparations of pafacether used proved to be pure with regard to lipid class composition. Venous blood of male volunteers was taken from the cubital vein in 0.1 volume acid-citrate dextrose (ACD) and centrifuged (18Oxg:15 min) to obtain PRP (platelet rich plasma). Thrombocytes were washed as previously described (19) and aggregation was performed in an aggregometer (Fresenius KG, D-6380 Bad Homburg) with PRP or washed thrombocytes in the presence of fibrinogen (0,16 mg/ml, Kabi, Stockholm, Sweden). The aggregation was induced by various concentrations of saturated or unsaturated paf-acether. TXB2 was measured after aggregation of thrombocytes in PRP using 90 nmol/l unsaturated paf-acether (n=6). Reaction was stopped after three or six minutes with an EDTA-Indomethacin (Sigma Chemicals GmbH, D-8028 Taufkirchen) solution and cooled immediately to 0°C. The suspension of thrombocytes was frozen at -40°C. After thawing the lysed thrombocytes were sedimented by centrifugation. A modified radioimmunoassay of TXB wa carried out ;4s described (21). 2. 3H-serotonin ( H-5HT, 28.2 Ci/mol, 38.9 Incubation of thrombocytes in PRP with nmol/l, New England Nuclear, D-6723 Eichenhain) was performed as described (9,ll). The release of serotonin was induced in the presence of imipramine (25 umolll, Sigma Chemicals GmbH) using different concentrations of unsaturated paf-acether for 1 minute. Lytic effects of unsaturated paf-acether were excluded since no increase of lactate dehydrogenase (LDH) could be measured in the platelet supernatant after three minutes aggregation using 1 nmol/l to 10 umol/l unsaturated paf-acether (n=2)(22). In some experiments the thrombocytes were preincubated with verapamile (0.1 nmol/l, Knoll AG, D-6700 Ludwigshafen) and clonidine (10 umol/l, Boehringer KG, D-6507, Ingelheim)(n=6). For the bigding studies thrombocytes were kept in a Tyrode's-Hepes buffer without Ca +(pH 6.4) containing 0.25% fatty acid-free bovine serum albumin (BSA, Sigma Chemicals GmbH). Thrombocytes were then r5Tuspended in the same buffer brought to pH 7.4 and containing 1.3 mmol/l Ca . Unlabelled saturated or unsaturated paf-ace her was added to 0.5 ml of t e platelet suspension (I(;"," ;l;telets x ml -1 ) with 26.1 nCi(0,65 nmol/l 3 H paf-acether, Amersham, . n=3). Following incubation at 20°C without stirring the thrombocytes'were'4eparated after different time intervals from the suspension by vacuum filtration through Whatman GF/c filters (45210 Feriere, France) with a Millipore vacuum system (67120 Molsheim). Filters were washed with 10 ml cold Tyrode's buffer. The radioactivity linked to the filter was countedbystandard procedure. The mean duplicates were substracted by the corresponding zerovalues without thrombocytes.

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701

PAF-ACETHER

a b C

0 = 6. ._ 3 ‘E’4.

g $2. E

00

d . 0

2

0

b

2

min

FIG.1 Aggregation of washed thrombocytes using acether(a:70,b:1.4,c:0.7,c:O.O7nmol/l.One

(A) saturated or (B) unsaturated paf. experiment representative of three.

loo_

5

.90ii E

=60_

I

0

I

I

1o-8

1o-g

I

1o-7

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10m6 mol/l unsaturated PAF

FIG.2 Maximal aggregation of thrombocytes in ACD-PRP by unsaturated paf-acether (n=lZ). Percent of maximum (o reversible and ??irreversible aggregation).

UNSATURATED

JO2

8

80

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-

4 P60.-s c

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0

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E~tumted FIG.3

Release of platelet serotonin by unsaturated in PRP in the presence of imipramine (n=6).

paf-acether

after one minute

RESULTS Aggregation of washed thrombocytes started immediately after addition of unsaturated or saturated paf-acether and reached its maximum after two or three minutes (Figure 1). Unsaturated hemisynthetic paf-acether aggregated washed aspirinated human thrombocytes as well as saturated synthetic paf-acether did. At concentrations of saturated paf-acether lower than 1 nmol/l aggregation of washed thrombocytes was reversible. At concentrations higher than 1 nmol/l aggregation became irreversible. In ACD-PRP maximal aggregation induced by unsaturated paf-acether increased between 1 nmol/l and 500 nmol/l to reach a plateau at 500 nmol/l (Figure 2). In the presence of imipramine unsaturated paf-acether releasedserotonindosedependently within one minute up to an average of 45.13219% of the total platelet serotonin content (Figure 3). The synthesis of TXB2 induced by unsaturated paf-acether increased from 3.322.57 ng/ml after three minutes to 8.66fJ.6ng/ml after six minutes aggregation induced by 90 nmol/l unsaturated paf-acether. In the absence of the agonist TXB2 rose from 0.2420.3 at three minutes to 0.420.38 ng/ml at six minutes. Unsaturated paf-acether (50 nmol/l) prevented the binding of tritium labelled saturated paf-acether (0.65 nmol/l) to intact washed thrombocytes as well as unlabelled saturated paf-acether (50 nmol/l)(Figure 4). Clonidine did not inhibit unsaturated paf-acether induced aggregation ofthrombocytes in ACD-PRP (66.4?15.5,n=6). Verapamile inhibited aggregation induced by unsaturated paf-acether (13.1?10.6%,n=6).

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s

1 2

5

15

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PAF-ACETHER

30

I

.

60 min

FIG.4 Effect of unlabelled unsaturated (+) or saturated (A) paf-acether (50 nmol/l) on the binding of tritium-labelled saturated paf-acether (0.65 nmol/l)(2? ) to intact washed thrombocytes in the presence of BSA (0.25%) and Ca (1.3 mmol/l). One experiment representative of three.

DISCUSSION The unsaturated paf-acether aggregated washed and unwashed human thrombocytes as well as saturated paf-acether. In concentrations which induced an irreversible aggregation unsaturated paf-acether released serotonin. Compared with the results described previously on unsaturated paf-acether the release of serotonin was more reproducible in the presence of imipramine (11). Unsaturated paf-acether synthesized less than 10% of the TXB2 synthesized by human thrombocytes after an aggregation using collagen (lug/ml synthesized 40 ng/ml TXB2 at three minutes, own unpublished datas). Aggregation of washed aspirinated thrombocytes remained irreversible as described with aggregation and serotonin release induced by unsaturated paf-acether in the presence of indomethacin (11). Unsaturated paf-acether activates human thrombocytes independently from cyclooxygenase-dependent metabolities of arachidonate (10-12). Unsaturated paf-acether prevented the binding of tritiumlabelled saturated paf-acether as well as saturated unlabelled paf-acether did. This result suggests that unsaturated paf-acether uses the same platelet binding sites as saturated paf-acether (14-18). An activation of a-receptors is not important for the aggregation induced by unsaturated paf-acether, because clonidine showed no inhibitory effect. The calcium channel blocker verapamil inhibited aggregation induced by unsaturated paf-acether. The same results were published using saturated paf-acether (23). Preparations of paf-acether containing unsaturated alkyl moities activate human thrombocytes in a comparable manner as saturated synthetic paf-acether (9,11,24). The bulk of these results indicate that modification of the alepha-

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tic chain at the position 1 do not notably influence paf-acether activity. This was already described with variations of alkyl ether chain length of saturated paf-acether (25).

ACKNOWLEDGEMENT We gratefully acknowledge support by the Deutsche Forschungsgemeinschaft. The binding studies were performed in the laboratory INSERM U 200 and we thank Dr. Benveniste for the helpful discussions and the permission to publish the results in this article. We thank Prof. Mangold and Prof. Muramatsu (Federal Center for Lipid Research, Institute for Biochemistry and Technology H.P.Kaufmann-Institute, Piusallee 68, 4400 Miinster, RFA)for the friendly gift of unsaturated paf-acether and B. Reinhard for expert assistance.

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