BritishJournal offfaetnatology, 1979,41, 427-436.

Further Studies on a Specific Platelet Antibody Found in Bernard-Soulier Syndrome and its Effects on Normal Platelet Function G. TOBELEM, S. LEVY-TOLEDANO, A. T. NURDEN, L. DEGOS A N D J. P. CAEN

Department of Experimental Thrombosisand Haemostasis, University Paris VII, INSERM, Unit 150, C N R S ERA335, Hdpital Saint-Louis and Hdpital LariboisiPre,Paris, France AND

C. MALMSTEN A N D H. KINDAHL

Department of Chemistry, Karolinska Institutet, S-104

01

Stockholm, Sweden

(Received 18 May 1978; acceptedf o r publication 23 August 1978) SUMMARY. An IgG antiplatelet antibody found in a multitransfused patient with Bernard-Soulier syndrome (BSS), reacted with a normal platelet surface antigen of I S O 000 daltons which was similar to the glycoprotein missing from BSS platelets. The BSS platelet antibody (BSS-Pab) aggregated all control platelets which then released ADP and 5-HT and synthesized thromboxane. When mixed with the antibody, BSS platelets did not aggregate, did not release ADP and 5-HT and failed to synthesize thromboxane. The BSS-Pab was not inactivated by incubation with BSS platelet stroma. While the antibody did not aggregate thrombasthenic platelets, its aggregating activity was lost after incubation with their stroma. The USS-Pab did not provoke ADP or 5-HT release or thromboxane synthesis in thrombasthenic platelets or in the platelets of a patient with platelet cyclooxygenase deficiency or in normal platelets treated with indomethacin. The aggregating, release and synthetic responses of platelets after binding of BSS-Pab to its membrane antigen (probably glycoprotein I) requires the presence of glycoprotein IIb and/or IIIa and the normal metabolism of arachidonic acid. Tobelem et a1 (1976) have dpccribed some of the properties of an IgG antiplatelet antibody (Yab) found in a mukitransfused patient with Bernard-Soulier syndrome (BSS). In vitro, this antibody (BSS-Pab) induced a BSS like reactivity in normal human platelets. Platelets from patients with BSS lack a surface glycoprotein found in normal platelets (Nurden & Caen, 1975; Caen et al, 1976). The molecular weight of the antigen recognized by the BSS-Pab is about ISO ooo daltons, similar to that of the missing glycoprotein in BSS platelets, and the antigen is demonstrable on the surface of the normal human platelet by the lactoperoxidase-catalysed *251-labellingtechnique. These data strongly suggest that the BSS-Pab was formed against the Correspondence: Dr G. Tobelem, Laboratoire d’Himostase et de Thrombose Exptrimentale, Hbpital SaintLouis, z Place du Dr Alfred Fournier, 75475 Paris Cedex 10,France. 0007-1048/79/030~0427$02.000 1979 Blackwell Scientific Publications

427

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surface glycoprotein which is missing from or highly modified on the platelets of patients with BSS. Possible roles for the surface glycoproteins in human platelet function have been suggested (Nurden & Caen, 1975, 1976) but little is known about the sequence of intracellular events following their stimulation. A central role for prostaglandin endoperoxides (Malmsten et al, 1975)and thromboxanes (Svensson et al, 1975) during platelet aggregation has been suggested. In this paper the effect of an antibody acting on a specific site on the platelet membrane has been studied. The formation ofthromboxane B2 and the release of 14C-serotoninor ADP were used as measures of the platelet response. MATERIALS AND METHODS Patients The patient in whom the BSS-Pab was found (Tobelem et al, 1976) was the first case described by Bernard & Soulier (1948). The history of the patient with Glanzmann’s thrombasthenia has been described by Caen et af (1966) and that of the patient with platelet cyclooxygenase deficiency by Malmsten et af (1975). Collection of Blood Blood from healthy volunteers or from patients who had not taken any drugs for a t least I week was collected from the antecubital vein into 3.8% trisodium citrate or into a mixture of 4.48% sodium citrate, 2.73% citric acid and 2% glucose (one vol citrate to nine vols blood). Platelet rich plasma (PRP) was prepared by centrifugation at 120 g for 10 min a t room temperature. Platelet poor plasma (PPP) was obtained by centrifuging the remaining blood a t 27 soog for 15 min at I o O C . Gel filtered platelets (GFP) were prepared as described by Levy-Toledano et al(1972). The BSS-Pab was precipitated from the serum by ammonium sulphate, 40% w/v. The precipitate was washed and resuspended in saline and then dialysed overnight at 4°C against 0.005 M phosphate buffer, pH 8. I. The IgG was eluted from a DEAE cellulose column with the same buffer and then concentrated and dialysed against 0.15 M NaC1. The protein concentration was adjusted to 6 mg/ml. Platelet Aggregometry Platelet aggregation was performed on normal human, BSS, thrombasthenic or platelet cyclooxygenase deficient PRP by adding BSS-Pab ( I s o d 0 0 ,ug/ml final concentration) to 0.2 ml PRP in the cuvette of a Born MK I1 mini-aggregometer (produced by the Department of Pharmacology, Royal College of Surgeons, London) at 3 7°C under continuous stirring. The extent of aggregation was expressed as a percentage (Levy-Toledano et al, 197.2).Aggregation of GFP was tested similarly. Platelet aggregation in control human PRP was performed in the presence of PGEl (0.3 PM final concentration) or Indomethacin (2.4 x I O - M ~ final concentration). Absorption o f Antibody by Platelet Stroma After incubation of various concentrations of the BSS-Pab with

I

mg of platelet stroma

Platelet Antibody in Bernard-Soulier Syndrome

429

(normal, BSS or thrombasthenic), the supernatants were tested for residual aggregating activity using normal PRP as previously described (Tobelem et al, 1976). Release of 14C-jHT PRP was incubated a t room temperature for 45 min with 5 x IO-’ M 14C-serotonin creatinine sulphate (58 Ci/mol, Radiochemical Centre, Amersham, England). I ml samples were preincubated for 2 min a t 37°C followed by the addition of collagen (20 pg/ml) or BSS-Pab (600 pg/ml) final concentration. The release reaction was stopped 5 min after the addition of the different inducers by adding 0.2 ml ice-cold 0.1M EDTA, pH 7.4. After centrifugation for 1 5 min at 650g at IoOC, aliquots of the supernatant were removed for determination ofradioactivity (Mills et al, 1968).The released serotonin was expressed as YO of the total 14C-serotonin content of the platelets. Release ofADP The release of ADP was determined with the luciferase enzyme system (Strehler & Totter, 1954)following incubation with collagen (20 pg/ml) or BSS-Pab (600pg/ml) under the same conditions as described for the 14C-serotonin release. The release reaction was stopped by adding 0.2 ml ice-cold 0.1M EDTA, pH 7.4, followed by rapid mixing. The luminescence reaction was performed in 0.05 M glycyl-glycine buffer, pH 7.4, in a liquid scintillation detector as described by Stanley & Williams (1969).Firefly lanterns, ADP, glycyl-glycine HC1, pyruvate kinase and phospho-enolpyruvate were purchased from Sigma. Release of Lactate Dehydrogenase ( L D H ) Release of LDH was determined spectrophotometrically by measuring the oxidization of NADH in the presence of pyruvate (Wroblewski & LaDue, 1955). The incubations of PRP with collagen (20pg/ml final concentration) or BSS-Pab (600pg/ml final concentration) were performed and stopped as described for release of 14C-serotonin and ADP. The LDH released was expressed as YOof the total LDH in the PRP after sonication for 80 sin a M.S.E. Ultrasonic disintegrator (100W ) operating at an amplitude of 6 pm. Formation of Thromboxane B2 ( T X B , ) TXB2 formation in PRP incubated with BSS-Pab (600 pg/ml final concentration) or collagen (20 pg/ml final concentration) or arachidonic acid (0.6mM), was determined by the radioimmunoassay technique of Granstrom et al(1976).After incubation at 37°C for 5 min the reaction was stopped with 4 vol ice-cold 99.5% ethanol. The precipitate was removed by centrifugation and the amount of TXB2 in the supernatant was assayed. Collagen was purchased from Stago Laboratories, Asnii.res, France, and indomethacin from Sigma. PGEl was a gift from Dr John Pike of the Upjohn Company. RESULTS Platelet Aaregation Induced by the BSS-Pub BSS-Pab induced platelet aggregation in the PRP of each of 50 normal subjects tested. However, it did not aggregate BSS or thrombasthenic platelets regardless of antibody

G . Tobelem et al

430

concentration (Table I). The BSS-Pab also did not aggregate the platelets from a patient with cyclo-oxygenase deficiency. Indomethacin or PGEl treated control platelets were not aggregated by the antibody. BSS-Pab aggregated isolated gel filtered platelets from normal donors without addition of any plasmatic component (not shown in the Table I).

Adsorption of the Antibody by Platelet Stroma from Control, B S S or Thromhasthenic Platelets After incubation of various concentrations of the BSS-Pab with control platelet stroma ( I mg protein) the residual aggregating activity of the antibody was much reduced. A similar result was obtained when platelet stroma from two thrombasthenic patients (Thromb) was used. However, after incubation of similar amounts of BSS-Pab with the stroma ( I mg protein) isolated from the platelets of a patient with the Bernard-Soulier syndrome (BSS) all the antibody activity was recovered in the supernatant (Table 11). I4C-5-HT, ADP and LDH Release Induced by BSS-Pab When a strongly aggregating dose of BSS-Pab (600 pg/ml final concentration) was added to PRP from control donors, release of 14C-s-HT and ADP was maximal within 5 min. Release of 5-HT did not differ significantly from the release induced by collagen (Table 111) and the time course for the two inducers showed essentially the same pattern (data not shown). TABLE I. Platelet aggregation induced by the BSS-Pab in PRP from normal controls (mean of 50 different samples); BSS (Bernard-Soulicr patient); Thromb (thrombasthenia); PCO (platelet cyclo-oxygenase deficient patient); from PGE, (0.3 p~ final concentration PGE, treated control platelets; 2.4x I O - M ~ final concentration indomethacin treated control platelets. % Aggregation of platelets intensity in PRP

BSS-Pab (pgg/mlfinal conc.)

BSS Thromb. PCO PGE,

Control

Indom

1 SO

20

0

0

0

0

3 00

45 60

0

0

0

0

0

0

0

0

0

0

600

TABLE 11. Aggregating activity of BSS-Pab after incubation with 0.9% NaCl (Buffer), or with I mg ofplatelet stroma from patients with Bernard-Soulier syndrome (BSS), thrombasthenia (Thromb), or from a normal donor (Normal). (Mean values of three experiments.)

Platelet aggregating activity BSS-Pab (pgglmlfinal ronc.

Bufer

BSS

Thromb.

Normal

150

I00

I00

5

0

300

I00

30

25

600

I00

I00 I00

80

90

0

Platelet Antibody in Bernard-Soulier Syndrome BSS-Pab did not cause any release of 5-HT or ADP using platelets from patients with BSS thrombasthenia or cyclooxygenase deficiency (Table 111). Preincubation of the platelets with ~ ) or PGEl (0.3 ,UM) for 2 min suppressed the antibody indomethacin (Indom) ( 2 . 4 ~I O - M induced release from control platelets. Even with the highest concentration of antibody used (600 ,ug/ml final concentration), no significant LDH release was observed with either platelets from a normal control or a patient with cyclooxygenase deficiency or from a normal control platelet incubation with PGEl (0.3 ,UM) or indomethacin (2.4x I O - M) ~ for 2 min.

Thromboxane B2 ( T X B 2 ) Synthesis Induced by BSS-Pub Formation of TXB2 following aggregation by BSS-Pab of platelets from normal donors was found to be similar to that induced by collagen (Table IV). When BSS, thrombasthenic or cyclooxygenase deficient platelets were incubated with the antibody, TXBz formation was not induced. Normal platelets pretreated with indomethacin (2.4 x I O - M) ~ did not synthesize TABLE 111. BSS-Pab (final conc. 600 pg/ml) induced release of s-HT, ADP and LDH from platelets in PRP from a normal control and from each of one patient with Bernard-Soulier syndrome (BSS), thrombasthenia (Thromb) and platelet cyclooxygenase deficiency (PCO) and from platelets in normal control PRP treated with Indomethacin (Indom) or PGEI. The results are compared with the release induced by collagen (final conc. 20 pg/ml) from platelets in control PRP. Results are expressed in per cent (mean values of three experiments). Platelet rich plasma (PRP) Constituent Release 5-HT ADP LDH

Control

BSS

3827

o o

27210 8

-

Thromb.

PCO

Zndom

PCE,

Co1lag en control 30+10

0

0

3

1.5

0

4

0

I .8

8

8

8

-

TABLE IV. Thromboxane (TXB2) synthesis induced by BSS-Pab (600 pg/ml final concentration) in platelets in PRP from controls, patients with Bernard-Soulier syndrome (BSS), thrombasthenia (Thromb), cyclooxygenase deficiency (PCO), normal PRP treated with Indomethacin (Indom) and (PGE1) and normal PRP treated with collagen (20 pg/ml final concentration). TXBz was measured after 5 min incubation with antibody. The values shown are the mean of three experiments. T X B 2 synthesis (pg/ml) Control

BSS

Thromb. PCO Indom PGEi

Collagen control

8

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G. Tobelem et a1

TXB2 when incubated with BSS-Pab. PGEl decreased antibody induced synthesisof TXB2 in normal platelets. DISCUSSION Immunological studies of the BSS-Pab have shown that this antibody differed from the usual antiplatelet alloantibodies by giving a negative complement fixation test using a panel of control human platelets and a positive thromboagglutination test whatever the HLA and platelet types (Tobelem et al, 1976;Degos et al, 1977).The BSS-Pab did not activate the complement system or cause release of LDH during the aggregation of normal platelets and therefore it can probably be assumed that it did not cause cell lysis. Normal platelets treated with the antibody induced in vitro developed a specific BSS-like defect (Tobelem et al, 1976).Thus platelet adhesion to subendothelium was impaired and agglutination by ristocetin or bovine factor VIII was inhibited but aggregation induced by ADP or collagen was unaffected. Nurden & Caen (1975)and Caen et al (1976)have found deficiencies of the surface-orientated glycoprotein, GP I, in the platelets of two patients with BSS, one of which was the patient who had formed the BSS-Pab. This glycoprotein may exist both as a firmly-bound membrane fraction (GP Ib) and a more labile fraction termed glycocalicin (Okumura & Jamieson, 1976)or GP Is (Nurden, 1977).Both glycoproteins were missing from or highly modified in the platelets of BSS patients (Nurden & Caen, 1976).The platelets from four BSS patients have now been studied and shown to possess the same abnormalities (Nurden & Caen, 1978).The BSS-Pab has been shown to interact with a specific surface oriented platelet membrane antigen with a molecular weight similar to that of GP I (Degos et al, 1977)thus strongly suggesting that the BSS-Pab has been formed against one or both of glycoproteins. This conclusion is supported by the observation that while BSS-Pab aggregated all the normal platelets tested, it did not aggregate the platelets from three BSS patients (Tobelem et al, 1976).Furthermore, Nachman et al(1977)have obtained similar results with a rabbit antihuman platelet antiserum that had been adsorbed with chymotrypsin treated normal human platelets. This antibody specifically inhibited ristocetin induced platelet aggrcgation and it interacted with a platelet membrane surface protein with a molecular weight of 1 5 s 000. These observations are particularly interesting in that glycoprotein Ib and glycocalicin have been reported to be very susceptible to chymotrypsin digestion (Okumura & Jamieson, 1976;Okumura et al, 1976) and that chymotrypsin treated platelets were not agglutinated by risotocetin or bovine factor VIII (Okumura et al, 1976). Furthermore, chymotrypsin treated platelets are not aggregated by BSS-Pab (Tobelem, unpublished results). Stimulation of platelet TXB2 synthesis by antibodies has not previously been reported. TXB2 synthesis in control platelet aggregated by the BSS-Pab was similar in magnitude and time to that induced by collagen. BSS platelets are aggregated normally by collagen and arachidonic acid, release ADP and 5-HT normally and form TXB2 (Malmsten et al, 1977).The BSS-Pab did not aggregate BSS platelets and was not consumed following incubation with platelet stroma from patient with BSS. Furthermore, when added to BSS platelets the antibody did not induce ADP or 5-HT release or formation of TXB2. Since the BSS-Pab probably acts primarily on GP Ib-Glycocalicin, the foregoing observations suggest that arachidonic acid and collagen, which both induce a release response and thromboxane

Platelet Antibody in Bernard-Soulier Syndrome

43 3

formation in PRP from a BSS patient (Malmsten et al, 1977) do not interact primarily with the membrane site of that glycoprotein. The BSS-Pab antibody was consumed by thrombasthenic platelet stroma and it reduced the adhesion of thrombasthenic platelets to subendothelium (Caen et al, 1977). However, the antibody did not aggregate thrombasthenic platelets or induce within the release reaction or TXB2 formation. While collagen does not aggregate thrombasthenic platelets, it induces release of 5-HT and ADP (although much reduced) and TXB2 synthesis. Furthermore, arachidonic acid induces normal thromboxane synthesis in thrombasthenic platelets (Malmsten et al, 1977). All these data suggest that the BSS-Pab attaches to its antigenic site on the thrombasthenic platelet membrane but does not induce the stimulation of the TXBz pathway which can operate normally given precursor arachidonic acid. Thrombasthenic platelets contain glycoprotein I (Nurden & Caen, 1974) but appear to be severely deficient in glycoproteins IIb and IIIa (Phillips & Poh Agin, 1977; Nurden & Caen, 1978). It is possible, therefore, that the presence of GP IIb and/or IIIa are essential for the induction of both the release reaction and for thromboxane formation, depending on which inducer is used. Thrombasthenic platelets are totally unreactive with BSS-Pab, partially reactive with collagen and fully reactive with arachidonic acid. Thus, the mechanism of thromboxane synthesisper se is not impaired in thrombasthenic platelets (Malmsten et al, 1977). It cannot be argued that thrombasthenic platelets are unreactive to immunological stimuli since they are aggregated by platelet isoantibodies (Praga et al, 1972) or HLA specific antiserum (Heinrich et al, 1977). It is possible that following stimulation of glycoprotein I by the BSS-Pab, the events that lead to the release reaction, TXB2 synthesis and platelet aggregation require normal glycoprotein IIb and/or IIIa, underlining a possible functional relationship between the surface glycoproteins. The failure of BSS-Pab to produce aggregation, release and TXB, synthesis in platelets deficient in cyclooxygenase suggests that prostaglandin endoperoxide formation is necessary for BSS-Pab induced platelet aggregation, since cyclooxygenase is needed for the transformation of arachidonic acid to prostaglandins (Malmsten et al, 1975). Following the stimulation of glycoprotein I and the transmission of the stimulus by glycoproteins IIb and IIIa, the release of arachidonate from the phospholipids of cyclooxygenase deficiant platelets is probably normal but the cyclooxygenase deficiency prevents the transformation of the released arachidonate to prostaglandins and thromboxanes. Consequent on the cyclooxygenase deficiency, the release of ADP and 5-HT is impaired. Control platelets treated with indomethacin which blocks their cyclooxygenase activity (Malmsten et al, 1975),behave similarly. PGEl causes a rapid increase in the cyclic AMP level in PRP by stimulation of platelet adenylate cyclase and it has been proposed that the increase of intracellular CAMP was associated with an inhibition of platelet aggregation and the release reaction but that a decrease should have the opposite effect (Salzman, 1972). The mechanism of this effect on platelet function is not clear (Michel et al, 1976), though cyclic AMP inhibits prostaglandin and thromboxane formation (Malmsten et al, 1975). In the presence of PGE1, normal platelets do not respond to the BSS-Pab, in accord with the finding of Shulman et al (1973) that platelet aggregation induced by platelet antibodies is inhibited by PGE1. It has been suggested that antiplatelet allo-antibodies induce platelet aggregation and stimulate the release reaction in a way similar to that induced by non-immunological stimuli (Hirschman et al, 1973; Hirschman & Shulman, 1973; Shulman et al, 1973; Gockerman et al,

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G. Tobelem et a1

197s; MacDonald et al, 197s; Heinrich et al, 1977). Most of these reports concerned HLA antibodies. Two different mechanisms of platelet activation have been. considered: (i) a complement independent action of HLA antibodies on the platelet resulting in platclet aggregation and the release response (De Gaetano et al, 1970;Hirschman et al, 1973; Heinrich et al, 1g77), and (ii) a cell dependent alteration of platelets resulting in lysis (MacDonald et al, 1975). Shulman et al(1973) reported that both platelet aggregation and the release of platelet substances occurred in citrated PRP containing platelet antibody and that EDTA totally inhibited these reactions. The BSS-Pab also requires the presence of ionized calcium since EDTA totally inhibited the antibody induced aggregation. Shulman et al(1g73) and Heinrich et al (1977) showed that fibrinogen was essential for platelet activation by platelet antibodies. However, fibrinogen did not seem to be an essential cofactor for BSS-Pab induced platelet activation since platelets isolated by gel filtration were aggregated by the antibody in the absence of fibrinogen. The binding of the specific BSS-Pab to its platelet membrane antigen could lead to a conformational change of the platelet membrane allowing platelet activation and aggregation. Thus in normal platelets, if sufficient antibody was bound to the membrane, platelet aggregation could be induced. The effect of the BSS-Pab on the function of normal platelets seemed to be more specific than that of HLA antibodies since a subaggregating dose of BSS-Pab did not inhibit aggregation induced by ADP or collagen (Tobelem et al, 1976) while after attachment of HLA specific antibodies to the membrane, platelets were refractory to further stimuli (Heinrich et al, 1977). The first step in the surface stimulation of platelets by the antibody is probably its binding to glycocalicin and/or membrane GP Ib. The data of Levy-Toledano et al (1976) seemed to indicate that the carbohydrate part of the GP I molecule was involved in this binding. This first step appeared to be followed by an unknown mechanism involving the glycoproteins that were missing or abnormal in thrombasthenia (GP IIb and IIIa). These glycoproteins were different from the ADP receptor since the binding of 14C-ADP was normal in both thrombasthenic platelet membranes and control platelet membrane in the presence of BSS-Pab (Legrand & Caen, 1976).Glycoprotein IIb and/or IIIa could then be involved in the subsequent steps of platelet activation which lead to the platelet aggregation-disaggregation and the specific release response. ACKNOWLEDGMENTS

This work was supported by grants from the Swedish Medical Research Council (project no. 0 3 x-217) from INSERM (project 3 I 76 63/4) and from DGRST (project 76 7 1448). REFERENCES

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Further studies on a specific platelet antibody found in Bernard-Soulier syndrome and its effects on normal platelet function.

BritishJournal offfaetnatology, 1979,41, 427-436. Further Studies on a Specific Platelet Antibody Found in Bernard-Soulier Syndrome and its Effects o...
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