THROMBOSIS RESEARCH 43; 425-433, 1986 $3.00 t .OO Printed in the USA.

0049-3848/86 Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

MECHANISM

OF PAF-INDUCED

PLATELET

AGGREGATION

IN MAN

L.J. Kiister, J. Filep and 3.C. Frolich Department of Clinical Pharmacology, Hannover Medical School, P.O.Box 61 01 80, 3000 Hannover 61, Germany (FRG)

(Received 19.3.1986; Accepted in original form 23.5.1986 by Editor H. Schrijer)

ABSTRACT The present study was designed to investigate the mechanisms involved in aggregation induced by platelet-activating factor (PAF) in human plateletrich plasma (PRP). PAF induced dose-dependent aggregation over the range of 50 nM to 14 NM, with a threshold dose of about 100 nM. BN 52021, a recently described PAF antagonist, completely abolished the effect of PAF at a ten-fold higher concentration. None of the concentrations of PAF used significantly increased TXB release. In plasma obtained from volunteers who had taken 500 mg ace 3ylsalicylic acid over five days, no change of PAF-induced aggregation could be observed in comparison to the control state. The lipoxygenase inhibitors nordihydroguaiaretic acid and BW 755 C also failed to significantly modify the PAF-induced platelet response. Pretreatment of PRP with the calcium channel blockers verapamil and nifedipin and the calmodulin antagonist trifluoperazine inhibited platelet aggregation by PAF over the entire range tested. These data indicate that PAF may utilize a specific membrane receptor, which can be blocked by BN 52021. Its aggregatory effect is probably mediated via the calcium-calmodulin system. Moreover, derivatives of arachidonic acid do not appear to be primarily involved in PAF-induced aggregation.

Key words: PAF

thromboxane

calcium

platelets

verapamil

BN 52021

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INTRODUCTION Since its discovery in 1972 (I), platelet-activating factor (PAF, I-O-Alkyl-ZOacetyl-sn-glycero-3-phosphorylcholine), has been found to be involved in numerous inflammatory and allergy-related processes (for review see 2). More recently, it has also been suggested that PAF might be an important mediator in shock, causing systemic hypotension with acute circulatory collapse (3, 41, pulmonary hypertension and increased vascular permeability (5) when injected into experimental animals. Furthermore, PAF is one of the most potent inducers of platelet aggregation known in man (6) as well as in most animal species (7). Irreversible platelet aggregation is associated with the release reaction with rapid granule depletion. Thus, PAF has been reported to stimulate the secretion of ADP and serotonin (8) and to increase thromboxane (TXA2) formation (9, 10). However, this increase in TXA may be the consequence rather than the cause of aggregation, since others have $ound PAF-induced aggregation to be independent of ADP release or of cyclooxygenase products (11, 12, 13). Indeed, recent evidence has tied PAF to the activation of phospholipase C (141, inducing the proposed third pathway of platelet aggregation (15). Phospholipase C activation leads to the formation of 1,2 diacylglycerol and phosphatidic acid, both of which have been implicated in the mobilization of calcium (16). Nevertheless, the mechanism by which PAF exerts these effects remains elusive. The present study was undertaken and TXA in PAF-induced aggregation the effec 8 of calcium channel blockers gated. in order to assess the possible role of calcium in human platelet-rich plasma. In particular, in PAF mediated aggregation has been investi-

METHODS Platelet studies and TXB2 formation Platelet aggregation studies were performed in platelet-rich plasma (PRP) from five healthy volunteers (3 male, 2 female; aged 25 - 32 yrs.) before and 24 hours after administration of 500 mg ASA p.o. for five days. None of the participants had taken any drugs known to interfere with platelet aggregation 14 days preceeding the study. Venous blood was drawn directly into 5 ml syringes containing 10 % sodium citrate. PRP was generated by centrifugation at 200 g for 15 minutes. Platelet-poor plasma (PPP) was prepared from PRP by centrifugation at 2000 g for 10 minutes. Final platelet counts in PRP averaged 220,000 pll. Platelet aggregation was monitored by increase in light transmission according to the method of Born and Cross (17) using an Apact dual-channel aggregometer (LAbor, Hamburg, FRG). When reagents were added to PRP in vitro, they were mixed with PRP by constant stirring for 2 minutes at 37OC. In those cases where TXB release was monitored, the reaction was quenched at 5 minutes by the addition of O.!?ml ice cold ethanol to 250 ~1 PRP. After centrifugaqon, the supernatant was stored at -2O C in plastic tubes until analysis by RIA. H TXB was supplied by New England Nuclear (Dreieich, Germany). The unlabeled standar d? was kindly provided by Drs. U. Axen and J. Pike (The Upjohn Co., Kalamazoo, USA) and the TXB antibody was generated by Dr. J.B. Smith (Philadelphia, USA). The detection limi? of the assay was 1.5 ng/ml PRP. Chemicals In the in vitro studies, the following reagents were used: the calcium channel blocker nifedipin (Bayer, Leverkusen, FRG) and verapamil (Knoll, Ludwigshafen, FRG) (18), as well as the calmodulin antagonist trifluoperazine (TFP) (Sigma Chemie, FRG) (19) at final concentrations of 200 uM; the lipoxygenase inhibitors nordihydro-

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guaiaretic BW 755 C of 50 nM Beaufour,

acid (NDGA) (Sigma Chemie, FRG), at a final concentration of 15 NM and (Wellcome Research Laboratories, Beckenham, U.K.) at final concentration (20); the PAF-antagonist BN 52021 (gift of Dr. P. Braquet, lnstitut Henri France), at various final concentrations from 1 J.IM - 100 uM (21). acid (NuChek of I mM. Prep., Elysian, USA) was used as inducing agent at a

Arachidonic final concentration Tablets FRG).

containing

50 mg ASA were

supplied

by Merckle

GmbH

(Blaubeuren,

Statistical analysis All values are expressed assessed using Friedman s test

as means + SEM. Statistical evaluation of the data was for two-way analysis of variance by ranks. RESULTS

PAF induced a dose-dependent irreversible aggregation of human platelets in the range of 50 nM to 14uM, with a threshold dose of about 100 nM (Fig. 1, A). A biphasic curve could be observed at threshold concentrations, reminiscent of similar aggregation curves when ADP has been used as inducing agent. PAF did not cause a statistically significant elevation in TXB levels at any of the doses used, nor was there a trend favoring TXB2 increase with2PAF concentration (Table 1).

PAF (uM) 0 0.05 0.2 0.9 3.6 14.0

TXB2 FORMATION (ng/ml) < ( 7.8 5.2 9.1 9.6 1.5 1.5 + 2.4 + 2.5 T 2.7 + 8.5

TABLE I: Effects of PAF on TXB2 FORMATION. Platelet TXB levels were determined in PRP from five hea Tthy volunteers as described under Methods. All values are means + SEM. The p value was obtained by Friedma& test.

PO.05

After 5 repeated (Fig. not different inhibited when

days intake of ASA at a dose of 500 1, B). PAF continued to illicit full from control, although aggregation arachidonic acid (1 mM) was used as

mg/day, aggregation studies were irreversible aggregation at levels were totally and TXB formation inducer (zlata not shown).

Preincubation of PRP with the PAF antagonist BN 52021 produced full inhibition of the aggregation induced by 0.2 and 0.9 AIM PAF, when BN 52021 was used at a than PAF (Fig. 2). In other experiments, similar results ten fold higher concentration were obtained as long as the molar ratio of 10 : 1 (BN 52021 : PAF) was held constant (data not shown). NDGA, an inhibitor of 12-lipoxygenase (20) had no inhibitory effect on PAFinduced aggregation (Fig. 3), even at threshold concentrations of PAF. Similarly, BW 755 C was unable to significantly reduce the response to PAF, although at threshold concentrations, the irreversible reaction was made reversible.

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14

3.6

0.9 0.2

0.05

14

3.6

0.9

0.2

0.05

fiM PAF

FIG. I Platelet aggregation induced by PAF before (A) and after (81 ingestion of acetylsalicylic acid (ASA). Aggregation was monitored and ASA administered as described under Methods. These tracings are representative of duplicate experiments in five healthy volunteers.

VEHICLE

0.2 AM PAF

Platelets monitored

Inhibition b were preincu as described

of PAF-induced ated for under Methods.

FIG. 2 platelet

aggregation

by BN52021. and aggregation

was

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VEHICLE

NDGA

BW 755~

0.9 0.2

0.9 0.2

0.9 0.2

AM PAF

FIG. 3 Effect of nordihydroguaiaretic acid (Nmand BW 755 C on platelet aggregation induced by PAF. Aggregation was monitored as described under Methods. Final concentrations of NDGA and BW 755 C were 15 and 50 uM, respectively. These tracings are representative of duplicate studies in five volunteers.

0.2

0.9

0.2

0.9

0.2

0.9

02

0.9 pM PAF

FIG. 4 Effect of calcium channel blockers and trifluoperazine (TFP) on PAF-induced platelet ggregation. :ER, verapamil; NIF, nifedipin. The final concentration of all antagonists was 200 uM. These are representative tracings. Similar results were obtained in five experiments.

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The effects of the calcium channel blockers nifedipin and verapamil and the calmodulin antagonist TFP on PAF-induced platelet aggregation were determined for an above threshold concentration of PAF (Fig. 4). Preincubation with these agents caused the irreversible aggregation to become reversible. In one volunteer, the aggregatory response to PAF was completely abolished following pretreatment with verapamil. DISCUSSION In the present study, PAF induced dose-dependent platelet aggregation which could be blocked by BN 52021. BN 52021 is considered to be a specific PAF receptor antagonist (21). Our data also indicate that PAF induces aggregation utilizing a mechanism independent of the cyclooxygenase and lipoxygenase pathways. In fact, no correlation could be found between TXB2 production and PAF concentration. Further support for the independence of PAF aggregation from cyclooxygenase activity is given by the results of the ASA study. Following five days of ASA ingestion, at a dosage sufficient in completely blocking platelet cyclooxygenase, there was no change in the PAF-induced response. Cyclooxygenase dependent aggregation is associated with TXB2 levels in PRP between 500 and 1000 ng/ml (22,23) when arachidonit acid is used as inducing agent. Similarly, serum levels of TXB2 between 200 - 300 ng/ml have been reported in clotted blood (24). In contrast, in our study PAF caused no measurable increase in TXB2 production. An increase in TXB2 following PAF has been reported for rabbit platelets (9, 22). This discrepancy between these previous findings and our results might be related to species differences. Other groups (8, IO) have shown indomethacin to be capable of inhibiting the effects of PAF in human PRP. However, indomethacin at the concentration used can also block phosphodiesterase activity (23), thereby raising cyclic AMP levels. One can therefore not exclude the possibility that mechanisms other than cyclooxygenase blockade were responsible for these observations. Although the presence of IZlipoxygenase in platelets has been well documented, its pathophysiological significance is poorly understood (24). In the present experiments both NDGA and BW 755 C failed to inhibit the aggregation induced by PAF, suggesting that lipoxygenase products are probably not responsible for aggregation. The disaggregation observed after BW 755 C at threshold concentrations of PAF may be due to effects independent of lipoxygenase blockage. Recent studies indicate that calcium may play a central role in platelet aggregation and the release reaction (25). PAF has been shown to cause a substantial and rapid elevation in calcium levels in platelets without being dependent upon TXA production (26). Indeed, TXA2 does not seem to be involved in either the discharge oT internal calcium or the calcium influx induced by PAF (26). Our results with calciumchannel blockers further support this hypothesis. Furthermore, TFP, a calmodulin antagonist (191, also prevented the PAF-induced aggregation. Although TFP at the concentration used could interact with other enzymes, it is likely that its effect was due to calmodulin blockade since similar inhibitory effects have been reported for W 5 and W 7, other calmodulin antagonists (27). Thus, two different possibilities for the actions of antagonists of the calcium or calcium/calmodulin systems in preventing platelet aggregation prevail. In lower concentrations, all calcium blockers inhibit the influx of calcium (28). At higher concentrations, they very likely also interfere with the intracellular mobilization of calcium (29) and other calcium dependent enzymes. In summary, our data suggest that PAF induces irreversible platelet aggregation using a calcium-dependent mechanism, which can be successfully blocked by calcium neither products of the channel and/or calmodulin antagonists in vitro. Moreover, cyclooxygenase nor the lipoxygenase pathways appear to be primarily involved in platelet aggregation by PAF.

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ACKNOWLEDGEMENTS The authors would like to thank Dr. P. Braquet (Le Plessis Robinson, France) for his generous gift of BN 52021. We are further indebted to Ms. M.-T. Suchy for her technical assistance and Ms. R. Beyer for typing the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft. REFERENCES 1. BENVENISTE, J., HENSON, P.M., COCHRANE, C.G. Leukocyte-dependent histamin release from rabbit platelets: The role of IgE, basophils and a plateletactivating factor. J. Exp. Med. 136: 1356-1377, 1972. PAGE, C.P., ARCHER, C.B., PAUL, W., MORLEY, J. Paf-acether: a mediator of inflammation and asthma. Trends Pharmacol. Sci. 5: 239-241, 1984. BESSIN, P., BONNET, J., APFFEL, D., SOULARD, C., DESGROUX, L., PELAS, I., BENVENISTE, J. Acute circulatory collapse caused by platelet-activating factor (PAF-acether) in dogs. Eur. J. Pharmacol. 86: 403-413, 1983. CAILLORD, C.G., ty of PAF-acether MONDOT, S., ZUNDEL, J.L., JULOU, L. Hypotensive in rats. Agents Actions 12: 725-730, 1982. activi-

2.

3.

4.

5.

SANCHEZ-CRESPO, M., ALONSO, F., INARREA, P, ALVAREZ, V., EGIDO, J. Vascular actions of synthetic PAF-acether (a synthetic platelet-activating factor) in the rat: Evidence for a platelet-independent mechanism. Immunopharmacology 4: 173-185, 1982. CHIGNARD, M., LE COUEDIC, J.P., TENCE, M., VARGAFTIG, B.B., BENVENISTE, J. The role of platelet-activating factor in platelet aggregation. Nature 279: 799-800, 1979. NAMM, D.H., ANJANEYULU, S., TADEPALLI, S., HIGH, J.A. Species city of the platelet response to 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. Thromb. Res. 25: 341-350, 1982. specifi-

6.

7.

8.

CHESNEY, C.M., PIFER, D.D., BYERS, L.W., MUIRHEAD, E.E. Effect of platelet-activating factor (PAF) in human platelets. Blood 59: 582-585, 1982. SHAW, J.O., KLUSICK, S.J., phospholipase anal thromboxane sn-glyceryl-3-phosphorylcholine Acta 663: 222-229, 1981. MC MANUS, L.M., HANAHAN, lation by acetyl glyceryl ether 1981. HANAHAN, D.J. Activation of rabbit platelet synthesis by I-O-hexadecylloctadecyl-2-acetyl(platelet activating factor). Biochim. Biophys.

9.

10.

D.J., PINCKARD, phosphorylcholine.

R.N. Human platelet stimuJ. Clin. Invest. 67: 903-906,

11.

CAZENAVE, J.P., BENVENISTE, J., MUSTARD, J.F. Aggregation of rabbit platelets by platelet activating factor is independent of the release reaction and the arachidonate pathway and inhibited by membrane active drugs. Lab. Invest. 41: 275-285, 1979. FOUQUE, F., VARGAFTIG, cyclooxygenase-independent 1984. B.B. Triggering by Paf-acether and adenaline of platelet aggregation. Br. J. Pharmac. 83: 625-633,

12.

432 13.

PAF, CALCIUM AND PLATELETS

Vol. 43, No. 4

DAHL, M.-L. Aggregating and prostanoid-releasing effects of platelet-activating factor and leukotrienes on human polymorphonuclear leukocytes and platelets. Int. Archs. Allergy Appl. Immun. 76: 145-150, 1985. LAPETINA, let-activating E.G., SIEGEL, L. Shape change induced in human platelets factor. J. Biol. Chem. 258:7241-7244,1983. by piate-

14.

15.

VARGAFTIG, B.B., CHIGNARD, M., LE COUEDIC, J.P., BENVENISTE, two, three or more pathways for platelet aggregation. Acta Med. Stand. 642: 23-29, 1980. NISHIZUKA, Phospholipid 1984.

3. One, (SuppI.)

16.

Y., TAKAI, Y., KISHIMOTO, A., KIKKAWA, U., KAIBUCHI, K. turnover in hormone action. Rec. Prog. Horm. Res. 40: 301-345,

17.

BORN, G.V.R., CROSSf M.J. (London) 168: 178-195, 1963.

The

aggregation

of blood

platelets.

J. Physiol.

18.

MEHTA, J.L. Influence of calcium-channel arachidonic acid metabolism. Am. J. Cardiol. RAD, G.H.R., REDDY, K.R., WHITE, platelet aggregation and disaggregation.

blockers on platelet function 55: 158B-164B, 1985.

and

19.

J.G. Influence Prostaglandins

of trifluoroperazine on Med. 5: 221-234, 1980.

20.

SALARI, M., BRAQUET, P., BORGEAT, P. Comparative effects of indomethatin, acetylenic acids, 15-HETE, nordihydroguaiaretic acid and BW 755 C on the metabolism of arachidonic acid in human leukocytes and platelets. Prostaglandins Leukotriene. Med. 13: 53-60, 1984. BRAQUET, P. Treatment and prevention new series of highly specific inhibitors. Patent July 19, 1984. of paf-acether-induced illnesss by a GB Patent 8, 418, 424 July 19, US

21.

22.

DE CATERINA, R., GIANNESSI, D., GAZZETTI, P., BERNINI, W. Inhibition of platelet aggregation and thromboxane B production during aspirin treatment: Dependence on the dose of the aggregajory agent. Thromb. Res. 37: 337-342, 1985. ROSENKRANZ, B., FISCHER, C., MEESE, C.O., FRGLICH,/J.C. Effects of salicylic and acetylsalicylic acid alone and in combination on platelet aggregation and prostanoid synthesis in man. Br. J. Clin. Pharm., in press. WEKSLER, B.B., TACK-GOLDMAN, K., VALVANUR, B.A., SUBRAMANIAN, V.A., GAY, W.A. Cumulative inhibitory effect of low-dose aspirin on vascular prostacyclin and platelet thromboxane production in patients with atherosclerosis. Circulation 71: 332-340, 1985. SHAW, I.O., PRINTZ, M.Pd, HIRABAYASHI, K., HENSON, P.M. Role of prostaglandin synthesis in rabbit platelet activation induced by basophil-derived platelet-activating factor. J. Immunol. 121: 1939-1945, 1978. FLOWER, 36: 33-67, R.J. Drugs which inhibit 1974. prostaglandin biosynthesis. Pharmacol. Rev.

23.

24.

25.

26.

27.

BRASH, A.R. A review of possible Circulation 72: 702-707, 1985.

roles of the platelet

12-lipdxygenase.

Vol.

43,

No. 4

PAF, CALCIUM AND PLATELETS

433

28.

ARDLIE, N.C. Calcium Ther. 18: 249-270, 1982.

ions,

drug

action

and platelet

function.

Pharmacol.

29.

HALLAM, T.J., SANCHEZ, A., RINK, T.J. Stimulus-response platelets. Biochem. J. 218: 819-827, 1984.

coupling

in human

30.

LEVY, J.V. Calmodulin antagonists inhibit aggregation of human, guinea pig and rabbit platelets induced with platelet activating factor. FEBS. Lett. 154: 262264, 1983. VANHOUTE, P.M. Calcium-entry blockers culation (Suppl. 1) 65: 11-19, 1982. and vascular smooth muscle. Cir-

31.

32.

CHURCH, J., ZSOSTER, T.T. Calcium antagonistic Can. J. Physiol. Pharmacol. 58: 254-264, 1980.

drugs.

Mechanism

of action.