Abnormal phospholipid organization in Bernard-Soulier platelets

THROMBOSIS RESEARCH 31; 529-537, 1983 0049-3848/83 $3.00 + .OO Printed in the USA. Copyright (cl 1983 Pergamon Press Ltd. All rights reserved.

ABNORMAL PHOSPHOLIPID ORGANIZATION IN BERNARD-SOULIER PLATELETS

Perret, B.", Levy-Toledano, S."', Plantavid, M.", Bredoux, R.", Tobelem, G.'", Douste-Blazy, L.' and Caen, J.P.‘”

Chap, H.",

‘INSERM U 101, Biochimie des Lipides, H8pital Purpan, 31059 Toulouse, France. O” INSERM II 150, ERA 335 CNRS, Assoc. Claude Bernard, H8pital LariboisiPre, 2, rue A. Par-d, 75010 Paris, France. Received Received

10.6.82; in

final

Accepted in original form 22.6.82 Editor H.C. Hemker form by Executive Editorial Office

by 19.5.83

ABSTRACT Platelets from 3 patients with Bernard-Soulier syndrome have been examined for their phospholipids. Results showed a 4-5 times increase of total phospholipids in the larger pathological platelets, where sphingomyelin and phosphatidylserine were significantly decreased. One patient displayed some unusual accumulation of lysophosphatidylcholine. None’of these changes was related to plasma lipid disturbance and thus appeared as an intrinsic membrane modification. Platelets from one patient were investigated for phospholipid sidedness in the plasma membrane using treatment with phospholipases. Total phospholipids present on platelet outer surface were lowered, whereas the proportion was increased at the expense of phosphatidylethanolamine and -serine of sphingomyelin. The latter changes might explain the increased availability of “platelet factor 3” previously described in BernardSoulier syndrome.

INTRODUCTION Bernard-Soulier syndrome is a rare congenital bleeding disorder chararcterized by the presence of abnormally large platelets, which fail to aggregate to ristocetin in the presence of human von Willebrand factor and to bovine factor VIII. The functional defects of these platelets involve an impaired in vivo adhesion to vascular rabbit subendothelium (1,2).

platelet

Several recent studies membrane structure.

Key words asymmetry.

: Bernard-Soulier

have focused on the modifications Nurden and Caen (2-4) have reported

syndrome

; Platelet

529

membrane

of the a severe

; Phospholipid

530

ABNORWL

PHOSPHOLIPID

ORGANIZATION

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deficiency of membranous glycoprotein I, concerning its particulate component (GPIb) together with the peripheral soluble one (GPIs) ; both of them contain large amounts of sialic acid and their decrease accounts for a reduced electrophoretic mobility of the whole cell (5). As shown by freeze-fracture electron microscopy (6), these molecular defects are reflected in structural changesof the plasma membrane, which exhibits an altered transmembranous distribution of particles, with a special decrease of those located on the external part of the membrane. Finally, the membrane abnormalities may also concern some functional aspects, involving a particular behaviour of these platelets during their isolation (which accounts for their large size), probably related to a defect in the mechanisms controlling cell shape change (7). Several experiments have provided evidence for an asymmetric distribution of phospholipids across the bilayer plasma membrane of normal platelets: the outer leaflet is essentially made of choline-containing phospholipids (sphingomyelin, phosphatidylcholine), whereas the other classes (phosphatidylethanolamine, -serine and -inositol) are mostly located on the inner face (8-10). Although the origin of such a repartition is still unknown, membrane proteins are very likely involved in maintaining the lipid arrangement. The purpose of this paper is to elucidate whether the selective deficiency of a major protein (GPI) in the platelet membrane of Bernard-Soulier syndrome may lead to a different phospholipid organization. MATERIALS AND METHODS Patients. v... is a 37 years old woman. She had all the features of a BernardSoulier syndrome, clinical history (11) and biological findings including platelet membrane glycoprotein abnormality have been already published (2). She had a splenectomy 15 years ago. For this study, she was examined 3 times and informed consent was obtained according to the principles of the declaration of Helsinki. K ... is a boy 7 years old ; he has a cutaneous mucous bleeding history. Laboratory findings were in agreement with the diagnosis of Bernard-Soulier syndrome (50 000 platelets per ul, giant platelets, increased bleeding time, no response of patient platelets in the presence of ristocetin or bovine factor VIII). B ... is a man who presented also the typical characteristics of the Bernard-Soulier syndrome. His case history was already described (11).

platelet isolation. Blood was drawn by venipuncture using acid-citratedextrose as an anticoagulant (12). The whole blood was allowed to settle for 2 hours at room temperature in plastic tubes inclinated at 45', so as to obtain the platelet-rich plasma ; then, platelets were isolated by centrifugation and washed twice in calcium-free Tyrode buffers according toArdlieet al (13), except that the centrifugation speeds were lowered to 500 g for 10 min., owing to the larger size of the cells. During the washing procedure, platelet suspensions were submitted to brief centrifugations (500 g for 1 min.), so as to eliminate red and white blood cells. Under these conditions, white pellets were obtained and no contaminating white blood cells were observed during platelet counting. Non-lytic

degradation of platelet phospholipids. Platelets were finally suspended in a Tyrode buffer, pH 7.35, lacking albumin and containing 11 mM glucose, 1 mM MgC12, 0.25 mM CaC12. The cells (10 mg/ml, wet weight) were submitted to the sequential action of bee venom phospholipase A2 and of

355 (b)

17.8 ?I0.8 38.4 + 1.6 11.2 + 0.9 5.0 + 1.3 27.6 f 2.0 traces traces

ND

14.4 38.6 9.4 6.7 24.5 5.7 0.5

V ...

ND, non determined NC, non calculated NS, non significant

Taken from Perret et al. (IO), results of 6 determinations :ba; Result of 1 determination

Phospholipid content (nmoles/l09 platelets)

Phospholipid composition (percentage of total phospholipid) - Sphingomyelin - Phosphatidylcholine - Phosphatidylserine - Phosphatidylinositol - Phosphatidylethanolamine - Lysophosphatidylcholine - Lysophosphatidylethanolamine

Mean + SD

Normal Platelets (a)

1630

15.1 43.3 8.2 4.4 28.9 traces traces

B ...

1336

16.1 41.9 9.2 6.1 25.8 1.0 traces

K ...

NC

15.2 + 41.3 + 8.9 + 5.1 t 26.4 + NC NC

0.9 (p < 0.01) 2.4 NS 0.6 (p < 0.01) 1.2 NS 2.3 NS

Mean f SD

Bernard-Soulier Platelets

PhosphoZipidCompositionand PhosphoZipidContent of Bernard-SoulierPZateZetsComparedto Normal Platelets

TABLE I

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ABNORMAL PHOSPHOLIPID ORGANIZATION

sphingomyelinase C from Staphylococcus aureus, as previously reported (10). Indomethacin (20 @!M)was added as an antiaggregant. After 60 min. incubation, the cell suspension was centrifuged at 1500 g for 8 min. ; the supernatant was assayed for lactate dehydrogenase activity so as to estimate cell lysis, as described elsewhere (10) ; the pellets were used for phospholipid analysis. Phospholipid analysis. After inhibition of phospholipase activity by EDTA (15 mM), total lipids were extracted and analysed as already reported (IO), using the extraction procedure of Reed et al. (14) for the platelets which were processed for phospholipase treatment. For phospholipid compositions, the platelet suspensions were sent frozen from Paris to Toulouse (6 hours) and lipids were directly extracted according to Bligh and Dyer (15). RESULTS Data from Table I clearly show an important increase in phospholipid content of Bernard-Soulier platelets. As platelets from B... and K... were processed after mailing from one laboratory to another, platelets from a normal control were treated in the same way and gave a value in good accordance with previously published data (16,17). Moreover, Table I shows some significant variations in phospholipid composition of pathological platelets compared to normal ones. These include a decrease of sphingomyelin and phosphatidylserine, with a counterbalancing higher level of phosphatidylcholine. However, the latter variation was not statistically significant. Also, a relatively higher percentage of lysophosphatidylcholine was repeatedly found (three times) for one patient (V...), whatever the method of lipid extraction used. Two of the patients (V... and B...) were also explored for their plasma lipids. As shown in Table II, total cholesterol, high-density lipoprotein cholesterol, triglycerides and total phospholipids were in the normal range, as well as their plasma phospholipid composition. Only some increase in lysophosphatidylcholine was observed for B...

TABLE II Plasma Lipid Compositionof Two Bernard-SoulierPatients Bernard-Soulier patients V. . . B ... Total cholesterol @moles/l) HDL-cholesterol (mmoles/l) Triglycerides (mmoles/l) Total phospholipids (mmoles/l) Phospholipid composition (molar percentages) - Lysophosphatidylcholine - Sphingomyelin - Phosphatidylcholine - Phosphatidylserine + phosphatidylethanolamine HDL, high density lipoprotein.

6.3 2.3 0.42 3.2

5.2 1.8 0.59 2.8

7.9 20.2 66.2

14.5 15.3 64.9

5.8

5.2

Normal values

3.8 1.2 0.5 1.5 -

7.7 1.9

1.7 3.2

61t2 20 * 2 66 + 5 8+2

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Platelets from one patient (V...) were prepared in sufficient amount to perform non-lytic degradations of phospholipids by purified phospholipases. In Table III are summed up the results of hydrolysis for each phospholipid class upon non-lytic treatment by exogenous phospholipases. It must be recalled that this experimental model allows a complete degradation of all the phospholipids forming the outer leaflet of the membrane, as long as cell lysis is avoided. Under such conditions, one can see that total platelet phospholipids were apparently much less exposed to the enzymatic degradation in BernardSoulier platelets (16.8 % and 12.3 X, respectively, versus 25.4 % in normal platelets). This decrease was reflected in a lower hydrolysis of the cholinecontaining phospholipids (phosphatidylcholine and sphingomyelin) ; in the opposite, the other phospholipids appeared to be degraded nearly to the same extent as usually measured, hydrolysis values being even higher for phosphatidylethanolamine and phosphatidylserine. Taken together, these results indicate that the phospholipid composition of the membrane outer surface is somewhat different in Bernard-Soulier platelets. This is clearly illustrated in Fig. I, which shows a higher level of phosphatidylethanolamine and phosphatidylserine (26 % and 6 %, respectively, versus 12 % and 2 % in normal platelets). In the opposite, the relative contrib-of choline-containing phospholipids (sphingomyelin and phosphatidylcholine) is lowered to 39 and 26 %, respectively, versus 49 and 35 % in normal platelets.

TABLE III Non-LyticDegradationof Phospholipidsby PhospholipaseA2 (bee venom) and 5phingomyeZinaseC (S. aureusl in Bernard-Soulierand Norma2 PZateZets Normal Platelets (a) Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidylethanolamine Total phospholipids

69.5 23.4 4.5 10 10.8 25.4

% % % % % %

(b)

Bernard-Soulier Platelets Experiment I Experiment 2 39.2 15.6 10.1 6.7 14.6 16.8

% % % % % %

38.5 5.2 7.4 6.2 14.3 12.3

% % % % % %

(a) Taken from Perret et al. (10) ; (b) Percentages of hydrolysis. DISCUSSION Beside other membrane abnormalities described in previous studies (2-7), Bernard-Soulier platelets display important phospholipid modifications compared to normal platelets. These include a higher phospholipid content, a different phospholipid composition and an altered transmembrane distribution. The first two modifications might be due to the higher size of the cells, which are known as giant platelets containing numerous granules (especially dense bodies) and a dense tubular.system widely developed (18,19). The decrease in sphingomyelin, an usually predominant phospholipid of plasma membranes (9,10,20) might reflect a relative wealth of these platelets in intracellular membranes at the expense of the plasma membrane. This would also fit with the lower hydrolysis of total phospholipids obtained upon incubation of intact cells with phospholipases under non-lytic conditions. It is also interesting to notice that the

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same variation of sphingomyelin relative to phosphatidylcholine was recently described in platelets from thrombocytosis due to myelo-proliferative disorders (21).

NORMALS

5Oum

50

0

sp

(iii;llpc

40

IZZPE

cl

s y

-ps 30

mp1

iz % * 20

10I-

0

C

-

FIG. 1 Phospholipid composition of plasma membrane outer leaflet in normal and pathological platelets. Results are taken from Perret et aZ. (101 for normal platelets and are the means of two experiments for Bernard-Soulier platelets. They are expressed as molar percentages of total phospholipids present in plasma membrane outer leaflet as deduced from non-lytic degradations by bee venom phospholipase A2 and S. aureus, sphingomyelinase C. Abbreviations : SP, sphingomyelin ; PC, phosphatidylcholine ; PE, phosphatidylethanolwnine ; PS, phosphatidylserine ; PI, phosphatidylinositol.

An intriguing finding was the observation of a high level of lysophosphatidylcholine in platelets from one patient (V...). From the data of Table II, such an increase cannot be attributed to plasma lipid modifications. V ... underwent splenectomy in 1961, which might be responsible for this additional change. However, an altered platelet phospholipid composition upon splenectomy was never reported. The third important modification of phospholipids in Bernard-Soulier platelets is an altered transmembrane distribution. The amounts of blood available for this study were not sufficient to isolate the plasma membrane from pathological platelets. This procedure allowed us formerly to estimate the proportion of plasma membrane phospholipids relative to total cell phospholipids and to propose an asymmetric distribution of phospholipids through the membrane (9, 10). It thus remains impossible to state whether the lower accessibility of sphingomyelin in Bernard-Soulier platelets only reflects the relative wealth of intracellular membranes or whether sphingomyelin displays a more internal location. But the fact that phosphatidylethanolamine and phosphatidylserine are more accessible to phospholipases in intact patholo-

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gical platelets promotes the view of an altered transmembrane distribution of phospholipids. This is evident from data of Fig. 1, showing a lower abundance of choline-containing phospholipids (sphingomyelin, phosphatidylcholine) at the expense of phosphatidylethanolamine and phosphatidylserine in the plasma membrane outer leaflet. It is noteworthy that the increase of the latter on the platelet outer surface occurs despite the lower content of phosphatidylserine in total platelets (Table I). The modifications of phospholipid organization in Bernard-Soulier syndrome make these platelets an interesting model to study the role of lipidprotein interactions in maintaining phospholipid asymmetry. Such a function has been proposed for integral membrane proteins in preliminary studies using reconstituted phospholipid-glycophorin vesicles (22) or erythrocytes lacking the major sialoglycoprotein (23). Furthermore, spectrin has been shown to act as a stabilizer of phospholipid asymmetry in the human erythrocyte membrane (24). It might thus be attractive to correlate the modifications of phospholipid distribution in Bernard-Soulier platelets with the recently described defect in the mechanisms controlling cell shape (7). On the other hand, the procoagulant activity of platelets displays an absolute requirement for anionic phospholipids (25,26). The very low occurrence of phosphatidylserine on the outer surface of normal platelets might explain the total latency of "platelet factor 3", which becomes available only upon cell lysis or under certain conditions of activation where phosphatidylserine becomes translocated from the outer to the inner leaflet of the platelet membrane (27). The presence of slightly higher amounts of phosphatidylserine on the external surface of Bernard-Soulier platelets might thus explain the increased availability of "platelet factor 3" described in Bernard-Soulier syndrome (18) and would justify further investigations.

ACKNOWLEDGEMENTS This work was supported by INSERM (CRL 78.5.148.3). Thanks are due to Prof. L.L.M. van Deenen and Prof. R.F.A. Zwaal for providing the purified phospholipases and to Mrs Y. JonquiPre for reading the manuscript. REFERENCES I.

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