Ionophore a 23187 and thrombasthenic platelets : a model for dissociating serotonin release and thromboxane formation from true aggregation

THROMBOSIS OPergamon

RESEARCH 16: 453-462 Press Ltd.1979. Printed

in Great Britain 0049-3848/79~101-0453

$02.00/o

IONOPHORE A 23187 AND THROmASTHENIC PLATELETS : A MODEL FOR DISSOCIATING SEROTONIN RELEASE AND THROMBOXANE FORMATIONFROM TRUE AGGREGATION Sylviane Levy-Toledano, Jacques Naclouf, Francine Rendu, Michel Riga&' Jacques P. Caen

and

Unite 150 INSERM, Hdpital Lariboisiere, 6, rue Guy Patin, 75010 Paris, France r( Service de Biochimie, C.H.U. Dupuytren, 87000 Limoges, France (Received

21.3.1979; Accepted by

in revised Editor M.J.

form 26.6.1979. Larrieu)

ABSTRACT The calcium ionophore A 23187 (iono.) induced a change in light transmission (LT) of thrombasthenic (thr.) platelets (~1.) either in plasma or washed and resuspended in buffer. This was not due to aggregation, but was associated with the formation of a central contractile gel mass and central apposition of organelles ; lactic dehydrogenase detenninat' n showed no evidence of platelet lysis. After incorporation of c IS1 C -arachidonic acid into the platelet phospholipid pool, stimulation with the iono. showed similar profiles of oxygenated products for both normal and thr. pl:, providing evidence for a normal activity of phospholipase(s) in thr. pl. under these conditions. The iono. induced normal thromboxane (TX) synthesis and serotonin (5HT) release in thr. pl.; the TX formation but not the accompanying change in LT, was inhibited by aspirin. The action of the iono. on thr. pl. thus provides a natural model for studying TX formation and 5HT release in the absence of aggregation.

INTRODUCTION Packham et al (l), using washed platelets, have recently shown that the aggregation and the early release of serotonin after stimulation by the ionophore A 23187 and bovine thranbin were partially independent of adenosine diphosphate (ADP) release and thromboxane (TX) synthesis which are considered as the usual pathways of platelet aggregation. We have tried to test this observation by studying the effect of the same ionophore on thrombasthenic platelets, which : i) do not aggregate in the presence of ADP, collagen, thrombin (2) or arachidonic acid (3) ; ii) release little ADP or 5HT and synthesize only small amounts of TX (analyzed in terms of TX8 ) following the addition of ADP or collagen (3) ; iii) synthesize TXB2 this formanormal7y after addition of arachidonic acid or prostaglandin G * or no tion of TXB2 being accompanied by a normal release reaction bu?' little 453

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aggregation (3). It was therefore of interest to measure the reactivity of thrcmbasthenic platelets (change in light transmission, thromboxane synthesis and 5HT release) in the presence of ionophore. MATERIAL AND METHODS Preparation of platelet-rich plasma (PRP) from normal donors and thrombasthenit patients The three thrombasthenic patients had the usual diagnostic criteria of Glanzmann's disease (2). Normal donors and thrombasthenic patients had not ingested any drugs for 2 weeks before donating blood. Whole blood was collected by venipuncture and was anticoagulated with l/10 vol sodium citrate 3.8 % (w/v). After centrifuging for 15 min at 120 a and room temperature, the platelet-rich plasma (PRP) was transferred to a polypropylene tube with a plastic pipette, and was stored at room temperature. Preparation of washed platelet suspensions on metrizwside gradients Nine parts blood was anticoagulated with one part acid-citrate dextrose solution containing 4.48 g sodium citrate,, 2.0 g glucose, and 2.73 g citric acid in 100 ml distilled water. The metrizamide gradient platelets (MGP) were prepared according to the method described by Levy-Toledano et al (4) : 5 ml of PRP were deposited on a metriramide gradient composed of 1 ml at 25 % and 1 ml at 10 % and centrifuged for 15 min at 1,000 g and 18" C. The platelet layer was resuspended in 3-4 ml of calcium-free buffer, pH 7.4 (4). Platelet change in light transmission (LT) This was expressed in terms of the percentage change in LT of PRP or MGP (4) in a Labintec aggreganeter at 37" C with stirring (1 100 rpm). To 0.4 ml of PRP or MGP (250 to 300 x 109/l) was added 2.5 ~1 of ionophore A 23187 (gift from Lilly Laboratories, Indianapolis, USA) in solution in ethanol at final concentrations ranging from 0.06 M to 25 PM. When acetylsalicylic acid (aspirin) was used, 6 .4 ml PRP was incubated with buffered aspirin (8 &l) or saline for 1 min at 37" C before the addition of ionophore (25 pM). Results are expressed as percentage change in LT, 3 min after the addition of ionophore. Electron microscopic studi.es Three min after the addition of ionophore to the stirred normal or thrcmbasthenic PRP, platelets were imaediately fixed with the same volume of 0.125 % glutaraldehyde in 0.1 M phosphate buffer. The samples were then centrifuged for 3 min at 15 000 g in an Eppendorf centrifuge and subsequently fixed with 1.25 % glutaraldehyde for 30 minutes at room temperature. The pellets were then washed twice with the same buffer, post-fixed in osmium tetroxide, and submitted to the classical dehydratation in alcohol and inclusion in Epon 813 (5). Thin sections were stained with uranyl acetate and lead citrate before study in the Philips 301 electron microscope. Thromboxane synthesis. 0.4 ml of PRP or MGP (250 - 300 x log/l) was incubated with different concentrations of A 23187 as described above. Three minutes after adding the ionophore, the reaction was stopped with 2 ml of ice-cold 99.5 % ethanol. After centrifugation, the.supematants were removed and kept at -20' C until analysis by gas liquid chromatography - mass spectrometry as described elsewhere (6). The results are expressed in ng/ml TXB since the measurement of this stable compound is widely chosen as a reflect of the active but labile TXA2

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(3, 6, 7). Therefore TX synthesiswill be'mentionnedin terms of TXB2 although TXA2 is the active metabolite. Serotonin release and lactic dehydrogenase(LDH) loss 5-HT release was measured after prelabellingtitrated PRP with[14 5HT (0.5pM) (48 mCi/mnol) from CEA, Saclay, France) for 15 min at 37 C. Labe ed PRP was stirred in an aggreganetertube with the ionophore (25 p!!)or ethanol for 1 min. The reaction was stopped by adding ice-cold EDTA (0.1 M) and centrifudorf centrifuge.The ging imaediatelyfor 30 seconds at 15,000 g in an E results were expressed as per cent release of totalYl4C 5HT uptake in the platelets. For the LDH loss, samples of titrated PRP were incubated at 37" C with stirring and exposed either to 2.5 pl ionophore (25 p!) in solution in ethanol or to 2.5 11 of ethanol alone. After 3 min the platelet samples were removed, spun and the LDH in the supernatantdetermined (8). The results are expressed as a percentage of the LDH activity released from the platelets in a sample of PRP treated with Triton (0.1 %). Analysis of hydroxy acids from pre-labelledplatelets telets of both normal and thrombasthenicsubjectswere labelled with Th 1 -arachidonicacid (50 mCi/mnol ; Amersham, the RadiochemicalCentre) as escribed (9). After labelling, the plateletswere isolated on the metrizamide gradie t as described above, and resuspendedin calcium-freebuffer (4) (300 x IOB platelets/l Labelled platelet suspensions(5 ml) were stimulated with ionophore (6 PM 'for 3 min at 37' C, with continuousstirring as for the aggregationprocedure.The samples were acidified at pH 3 with citric acid and extracted with 3 volumes of ethyl acetate. After evaporationto dryness under nitrogen, in the dark, the samples were purified on silicic,acid column chromatography(9) and the hydroxy fatty acid-elutedfraction was analyzed after prior transfonation into methyl esters. The thin-layer chromatography was run with an organic phase of ethyl acetate/isooctane/water (50 : 100 : 100, v/v/v) ; the radioactiveprofile was obtained using a Chromelec 101 apparatus.An aliquot from the same experimentwas derivatizedby silylation and analyzed with a gas liquid chromate raphy-massspectrometer (LKB 2091) equiped with a glass capillary column 4OV-101) (10). The following compounds were identified : 12-hydroxy-5,8, IO-heptadecatrienoic acid (HHT), 12-hydroxy-5,8, 10, 14-eicosatetraenoicacid (HETE) and arachidonicacid ; their fragmentationcorrespondedto that described by Hamberg and Samuelsson (11). Since we did not have deuterated carrier, we performed a relative quantitation between normal and "hrombasthenicplatelets by the addition of a known amount ofI2- droxystearicacid to each sample before the derivatizahT standard. tion, as an externa RESULTS Ionophore-inducedmorphologicalchange in normal and thrombasthenicplatelets In the presence of ionophoreA 23187, thrombasthenicPRP underwent an increase in LT, but this was less than that of control PRP (Table I). MBP required IO to 20 times lower concentrationsof ionophore to produce a change in LT than did PRP. Again, the change produced in thrombasthenicplateletswas much less than in control platelets (Table I).

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TABLE I IONOPHORE-INDUCED CHANGE IN LT IN THROYBASTHENIC AND CONTROL PLATELETS in LT at-3 min (in per cent)

Increase Ionophore concentration ()tW)

",;;trol (mean ok;)

ThrombaRpthenic (meanGf

3)

Electron-microscopy revealed that ionophore-induced thrombasthenic platelet "aggregates" had not the same appearance as normal ones, which looked like those stimulated by collagen and thrombin (Fig. 1). When control PRP was stirred with 25 pM ionophore,large platelet aggregates were formed, in whi ch the organelles had moved into the platelet centers, encircled by a web of miicrotubules and microfilaments, as described by Gerrard et al (12).

FIGURE

1

Electron micrographs of normal (left side) and thrombasthenic (right side) PRP in presence of ionophore 25 u!l.

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Under the same conditions (25PM ionophore)this internal transformationalso occurred in the thrombasthenicplatelets, but the platelet "aggregates"were small and loosely packed. Furthermore,in thrombasthenicplatelets, a dense central mass appeared at this concentrationof ionophore,while in normal platelets this physical change only occurred at ionophore concentrationsabove 100 pM (12). Ionophore-induced 14C -5HT release and loss of LDH form normal and thrombasthenlc platelets The morphologicalchange indu ed by the ionophore in thrombasthenicPRP was accompaniedby a release of $14C -5HT in the same amounts as for normal platelets (Table II . Simultaneousmeasurement of LDH was performed : the concentrationof A h 3187 which was used (25 pti)did not result in platelet lysis as shown by the leakage of insignificantamounts of this enzyme during the incubationtime (3 min) (Table II). TABLE II RELEASE OF PLATELET C'"c]-5HTAND LOSS OF LDH (PER CENT) FOLLOWINGTHE ADDITION OF A 23187 TO PRP

Ionophore (PM)

!4&5HT

25

0

LDH

25

Normal

7

52

11

15

Thrombasthenic

5

52

12

16

Mean values for 3 experimentsare given Ionophore-inducedcyclooxygenaseactivity of normal and thrombasthenicplatelets Since the ionophorewas able to activate the thranbasthenicplatelets,as shown by the change in LT and the release reaction,we have also examined. the synthesisof endoperoxidesresulting in thromboxaneformation.The results in Table III show that A 23187 induced a similar concentration-dependent productionof TXB in normal and thraRbasthenicPRP and MGP. However, at low concentration!! of A 23187 (0.6 PM), enormous amounts of TXB (1 400 ng/ml) were detected in both normal and thrombasthenicMGP, far greatee than the concentrationsproduced in control PRP (280 ng/ml) or thrombasthenicPRP (310 ng/ml) by a 40-fold higher concentrationof ionophore (25 uM).

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TABLE III TX82 SYNTHESIS INDUCED BY IONOPHORE IN CONTROL AND THROMBASTHENIC PLATELETS TX62 (ng/ml) Ionophore concentration

;$rol

(mean oki)

ThryR;sthenic

(mean;;

3)

(F) 0.06 z-06 12:o 25.0

1 1 2 2

:5 280

43; 875 400 800

3; 310

1 2 2 2

95: 100 200 500

We have measured the effect of the cyclooxygenase inhibitor acetylsalicylic acid aspirin) on both TXB formation and increase in LT provoked by ionophore (2 5 pi4) in control and Zhrombasthenic PRP (Table IV ; TXB fomtation was reduced by 99 % in normal and 90 % in thrombasthenic PR P but ?he change in LT of normal PRP was not affected, while that of thrombasthenic PRP was even increased, althou h there was no evidence of any lytic activity (no LOH loss, data not presente 1 ). TABLE IV EFFECT OF ASPIRIN ON IONOPHORE-INDUCED LT AN0 TXB FORMATION IN CONTROL AND THROMBASTHENIC PiiP

Aspirin (mW)

Control LT change (per cent)

Thrombasthenic LT change (per cent)

0

98

320

34

278

8

92

1.2

60

25

Synthesis of hydroxy acids after ionophore stimulation -arachidonic acid Thrombasthenic platelets incorporated and released like normal latelets after stimulation with A 2318 normal and thrombasthenic W6 1 showed a similar profile of non-cyclired oxygenated products, identified as the normal C17- and C20-hydroxylated arachidonic acid derivatives (Figure 2). Analysis by multiple ion detection confirmed that the stimulation of thrombasthenic platelets by A 23187 was followed by the formation of hydroxy acids (HA), including HHT and HETE in the same amounts as for normal platelets.

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Radiowtivity cpm I 2000

tiA

459

AA

1000

t Origin

1 Front (Wcm)

FIGURE 2 Thin layer radiochromatogramof the hyd acid (AA) in normal and thrombasthenicl response to A 23187 (6 PM) stimulation.

acids (HA) and the arachidonic AA labelled isolated platelets in

DISCUSSION During the entire sequence of the basic platelet reaction : induction,tranSmission and execution, it has been shown that intracellularcalcium plays an important role In the propagationof events (13). Ionophoresrender membranes + and thereby allow the passage of cation from outside the ET;;'&: ;;2+-storing organelles into the cytoplasm.Pickett et al (14) have found that 10 pM A 23187 liberates arachidonicacid from the platelet hospholipidpool ; they have proposed that the ionophore activates a phosphoYlpase A2 by a calciun-depende Charo et al (15), using a specific antagonistof intracellularCa and Massini and Luscher (16) have shown the importanceof intracellularCa + in the functiens (aggregationand release reaction of normal platelets in the presence of ionophore.KinloughRathbone et al ( 17) on the other hand, have shown that A 23187 causes shape change and aggregationof thrombin-degranulatedrabbit platelets, indicating that these functions are independentof ADP release ; furthemmre, it has previously been shown that ionophore-inducedplatelet aggregationcannot be attributed to the formation of prostaglandinendoperoxidesbecause it is not inhibited by aspirin (18). Thrombasthenicplatelets provide valuable model for the study of these interrelationships,since they do not aggregate,undergo a release reaction or synthesize TXB2 in the presence of ADP or collagen (3), although ADP produces a shape change (19) and is normally bound by thrombasthenicplatelet membranes (20). Our data show that the ionophore has the same effect on thrombasthenic as on normal platelets as regards 5HT release and TX formation.

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Furthermorewe have extended the finding of White et al (18) since the ionophore induced a change in LT in thtwsbasthenicplateletswhich was not inhited by as irin and was thus independentof TXB synthesis.Aspirin even increased tf: e change in LT produced by the ionopKore in thrusbasthenicPRP, although no significantlysis was detected ; potentiationof ionophore-induced aggregationhas already been reported on normal platelets (21). Thromboxane synthesis is evidently not necessary for ionophore-inducedaggregationof normal platelets, since we found no correlationbetween aggregationand TX formation in PRP and MGP. Lower concentrationsof ionophorewere sufficient to produce both LT change and TXB induction in MGP than in PRP, suggesting a different mode of action of ion6phore and/or.a different platelet reactivity in the presence or absence of plasma proteins.The same observationholds true for both LT change and TXB2 synthesis in thranbasthenicM6P and PRP ; the effect of plasma proteins on the action of ionophorethus appears to be similar in nomal and thrombasthenicplatelets. Since thrombasthenicplatelets,when isolated on metrizamidegradients and resuspended in a calciun-freebuffer, undergo 5HT release and TXB synthesis like nonaal platelets, they are evidently able to mobilize the& internal Ca2+ stores in response to the ionophom and thus to increase cytopla@c calcium.levelsnormally.These results are consistentwith the normal Ca content (2) and storage capacity of calciutn-rich dense bodies (22) of thraabasthenic platelets,which have previouslybeen reported from t$is laboratory. Furthermorethe availableCa2+ allows the activationof a Ca +-dependentphospholipase ; we have found evident of nonaal activity of a phospholipasein these plateletson the basis of f4C -arachidonicacid incorporationin the nomtal phospholipidsof thrombasthenicplatelets (23) and subsequent release in the presence of ionophore ; the metabolism of the liberated arachidonic acid by the cyclooxygenasepathway .as well as by lipoxygenasealso appeared to be normal. Our data suggest that the requirawnts for arachidonicacid metabolism, namely fatty acid compositionof phospholipids.liberationby a phospholipase,oxidation by a lipoxygenase,cyclooxygenaseand a thromboxane synthase, are all present in thrombasthenlcplatelets and function nomally in the presence of ionophore. All these biochemicalevents induced by the calcium ionophore in thrombasthenit platelets were accaqmrciedby a change in LT which was not due to aggregation but to an internal transfomatfon : ultrastmctural observationsshowed loosely packed thro&asthenic plateletswhile nomal plateletswere associated in clusters.This internal transfomation in thra&asthenic platelets, with the appearanceof dense central masses of contractilecytaplasmicgel was essentiallythe same as the contractilewave described by Berrard et al in response to either arachidonicacid or A 23187 (24). Like the absence of aggregatesit may be related to the observed defect in Crane glycoprotein IIb/IIIa (25), since Gerrard et al (24) have presentedevidence for the identity of alpha-actininand glycoproteinIII and have suggested that alphaactinin may play a crftfcal role at the site of cell-cell adherence in anchoring contractionand orienting the contractilewave towards the centre of the aggre ate rather than the centre of individualcells. The deficiency of this pmte 9n would thus lead to the localisationof the contractilemass observed in thtiasthenlc platelets. These platelets thus provide a model for the explorationof secretion and TXB synthesisand the structuralchanges associatedwith them in the absence of aggregation. We thank Professor R.M. Hardisty for fruitful discussions,J. Breton-Gorius for helpul interpretationsof the electron microscopy and E. Savariau for the excellent technical assistance. This work was supportedby a grant from INSERM : A.T.P. A.S.R. 5

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