Characterization of the Platelet Membrane GlycoproteinAbnormalities in Bernard-Soulier Syndrome andComparison with Normal by Surface-labeling Techniquesand High-resolution Two-dimensional Gel Electrophoresis

KENNETHJ. CLEMETSON,JOHN L. MCGREGOR,ELIZABETH JAMES,MARCDECHAVANNE,and ERNSTF. LUSCHER, Theodor Kocher Institute,University of Berne, CH-3000 Berne 9, Switzerland; Institut National de laSante et Recherche Me'dicale F-69500 Bron, France; Faculte de Medecine,Alexis Carrel, F-69008 Lyon, France

A B S T R A C T The platelets from three patients withBernard-Soulier syndrome have been analyzed by sur-face-labeling coupled with two-dimensional gel elec-trophoresis and compared with normals. As well as thepreviously described absence or deficiency in glyco-protein (GP) Ib(a) it could be shown that GP Ib,3 andan additional low molecular weight glycoproteinGP75.8-65 were not detectable using carbohydrate-la-beling methods or deficient to the same extent as theGPIba subunit. In addition, the thrombin cleavableglycoprotein could not be detected using carbohy-drate-labeling methods in two patients and was defi-cient in a third. This finding was confirmed in a fourthpatient by one-dimensional gel electrophoresis. Thus,the changes in the membrane of Bernard-Soulier plate-lets are more complex than previously thought.

INTRODUCTION

Bernard-Soulier syndrome is an inherited bleedingdisorder characterized by reduced adherence of theplatelets to subendothelium (1), the presence of "giant"platelets in blood smears (2) and a reduced plateletsurvival time in circulation (3) resulting in thrombo-cytopenia. Bernard-Soulier platelets aggregate nor-

A preliminary report on this research was presented at theVIlIth International Congress on Thrombosis and Haemos-tasis, Toronto, Canada and published in abstract form. 1981.Thromb. Haemostasis. 46: 108.

Address reprint requests to Dr. Clemetson, TheodorKocher Institute.

Received for publication 20 March 1981 and in revisedform 16 April 1982.

mally with collagen, ADP, epinephrine and arachi-donic acid (4) but do not aggregate with ristocetin/FVIIIR:WF (5) or bovine von Willebrand factor (4).Aggregation to, and binding of, thrombin is reducedcompared to normal platelets (6, 7).

Biochemical studies on Bernard-Soulier plateletsfirst showed a reduction in sialic acid content (3) andlater a deficiency in the membrane glycoprotein lbwas demonstrated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis with (8) or without (9)surface-labeling of platelets and has been confirmedin several additional studies (10-13) including crossedimmunoelectrophoresis (14). Glycocalicin, a readilysolubilized platelet glycoprotein derived from glyco-protein Ib was also shown to be decreased in Bernard-Soulier syndrome (10, 11).

Unlike Glanzmann's thrombasthenia, another plate-let disorder, involving principally glycoproteins Ilband Illa, where considerable heterogeneity has beenreported (14), little is known about heterogeneity andits relation to clinical symptoms in Bernard-Souliersyndrome, due largely to the rareness of this disorder.It has not yet been established if the genetically de-termined abnormality of the membrane of Bernard-Soulier platelets is restricted to glycoprotein lb or ifother membrane components are affected. A reporthas appeared that there are changes in glycosylationin Bernard-Soulier platelets compared with normal(15). Glycoprotein lb consists of two subunits, a and, linked by at least one disulphide bond (16). Since

previous studies had been carried out on relatively lowconcentration polyacrylamide gels in order to examinethe major glycoproteins, nothing was known about the

304 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/82/08/0304/08 $1.00Volume 70 August 1982 304-311

expression of the 13-subunit in Bernard-Soulier syn-drome.

To examine the question of heterogeneity in Ber-nard-Soulier syndrome and to see if glycoprotein lbis the only glycoprotein affected in this disorder, wehave surface-labeled Bernard-Soulier platelets and an-alyzed these by high-resolution two-dimensional poly-acrylamide gel electrophoresis.

METHODSFour patients fulfilling the diagnostic criteria of Bernard-Soulier syndrome were studied. All four had experiencedbleeding episodes and had increased bleeding times but haveled reasonably normal lives since being under medical con-trol. Patient V.S. (female, 28 yr old) from Switzerland hada bleeding time slightly longer than normal of 8 min (nor-mals 1.5-6 min) and a decreased platelet count of 39,000-72,000/ul. The Factor VIII complex components were nor-mal or increased, VIII:C, 100%; VIIIR:WF, 100%; VIIIR:Ag,170%. Aggregation of platelets to ADP, collagen and epi-nephrine were normal. Platelets did not aggregate to risto-cetin at 1.5 mg/ml. The peripheral blood smear showed typ-ical giant platelets.

Patient F.S. (female, 14 yr old) from France had a bleed-ing time in excess of 30 min and had had thrombocytopenicepisodes that were eventually treated by splenectomy. Since,the platelet counts have been within the normal range. Shealso had typical giant platelets in the peripheral blood smear(4.51 gm vs. 2.9-3.5 MmDiam for normals). Factor VIIIcomplex components were elevated; VIII:C, 248%; VIIIR:WF,145%; VIIIR:Ag, 555%. Platelets aggregated normally toADP, collagen, and epinephrine but failed to aggregate toristocetin at 1.2, 1.4, and 2.0 mg/ml.

Patient E.H. (male, 8 yr old) from France had a decreasedplatelet count and a bleeding time in excess of 30 min. Inthe peripheral blood smear the platelets were only slightlylarger than normal. Platelets from this patient respondednormally to ADP and collagen but showed a decreased re-sponse to thrombin. The initial slope of aggregation was 40%of the value obtained with normal platelets at the same plate-let concentration and the maximum aggregation was 63%of the value obtained with normal platelets. No response wasfound to ristocetin at 1.2 mg/ml but at 1.4 and 3.0 mg/ml-20% of the normal response was obtained. The initial slopeof aggregation was 20% of the value obtained with normalplatelets at the same platelet concentration and the maxi-mumaggregation was 15% of the value obtained with normalplatelets. Factor VIII complex was as follows: VIII:C, 140%;VIIIR:Ag, 100%; VIIIR:WF, 77%.

Patient M.S. has been previously described (13).Human blood platelets. The study was conducted ac-

cording to the principles embodied in the Declaration ofHelsinki. The isolation of a homogeneous preparation ofBernard-Soulier syndrome platelets was performed as pre-viously described by Jamieson and Okumura (7), with minormodifications. Blood (70-80 ml) was drawn from informed,consenting patients or normal donors, collected in 10-mltubes containing 1.4 ml acid-citrate-dextrose, 10 mMEDTAanticoagulant, and allowed to sediment (tubes tilted at anangle of 450, at room temperature (20°C), for 4 h. The plate-let-rich plasma was then gently removed and diluted withan equal volume of Hanks' buffered saline containing 10mMEDTAand no calcium or magnesium. The diluted plate-let-rich plasma (5 ml) was then layered on top of 4 ml ofFicoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden)

in 10 ml plastic or siliconized glass tubes and centrifugedat 800 g for 10 min. Erythrocytes and granulocytes sedi-mented to the bottom of the tube, while platelets and con-taminating lymphocytes were present as a narrow band atthe interface.

The interface was removed, mixed with 0.012 Msodiumcitrate, 0.03 M glucose, 0.12 M NaCl pH 6.5 containing 10mMEDTA to a volume of 8 ml and centrifuged at 80 g for5 min. Supernatant and pellet were examined by phase-con-trast microscopy or by bright field using a differential Wrightstain. The supernatant was then centrifuged repeatedly at100 g for 5 min, discarding the pellets until a homogeneousplatelet population was obtained.

Labeling. Platelets from each Bernard-Soulier syndromepatient and a healthy donor were immediately washed afterisolation and were labeled in parallel by lactoperoxidase-catalyzed iodination, periodate plus NaB3H4 or neuramini-dase, galactose oxidase plus NaB3H4 as previously described(8, 13). Isoelectric focusing and SDS-polyacrylamide gelelectrophoresis (5-20% acrylamide linear gradient gels) werecarried out as described (17). After fixing, the gels containing125I-labeled proteins were washed under running water for1 h, dried under vacuum and indirect autoradiography wascarried out with Kodak X-Omat R film (Eastman Kodak Co.,Rochester, NY) and Cronex Lightning Plus intensifier screens(Du Pont & Co., Inc., Wilmington, DE) as previously de-scribed (18). Gels containing 3H-labeled glycoproteins wereimpregnated with PPO, (Packard Instrument, Co., Zurich),dried under vacuum and fluorography carried out with Ko-dak X-Omat R film (19).

Incubation of labeled normal platelets in Bernard-Soulieror normal blood. Platelets from two normal donors with thesame blood group (Al Rh+) as the Bernard-Soulier patients(E.H. and F.S.) were isolated, washed, and surface-labeledby the neuraminidase, galactose oxidase plus NaB3H4 or per-iodate plus NaB3H4 methods. The pellets of labeled plateletswere resuspended in platelet poor plasma (3 ml), to which7 ml of whole blood was added (total volume 10 ml, plateletsadjusted to 250,000/,Ml). The platelet poor plasma and wholeblood were from the Bernard-Soulier syndrome patients orfrom the normal donors. The labeled normal platelets re-suspended either in Bernard-Soulier patient blood or in bloodfrom the same donor were incubated for 30 min at 370C,then isolated and washed twice in 0.01 M Tris, 0.005 MEDTA, 0.154 M NaCl, pH 7.4 buffer before solubilizationin SDS for SDS-polyacrylamide gel electrophoresis. Labelednormal platelet samples from the two donors were also di-rectly solubilized for gel electrophoresis without incubationin blood.

Immunoprecipitation was carried out by the method ofKessler (20) with some modifications. Platelets (10"), surface-labeled by the periodate plus NaB3H4 method were solubi-lized in 250 ,l of 0.15 MNaCl, 0.005 MEDTA, 0.5% NonidetP-40, 0.05 M Tris/HCl pH 7.4, (NET) buffer, followed bycentrifugation at 100,000 g for 30 min. The supernatant wasdivided into two aliquots of 100 ,ul that were mixed with 100Mil of antiglycocalicin IgG (1 mg/ml) (21), or 100 Al of an-tiglycoprotein IIb/Illa IgG (1.3 mg/ml) (21, 22), respec-tively, and left for 8 h at 4°C. Two aliquots (2 ml) of a 10%suspension of Staphylococcus aureus Cowan I (IgG Sorb, TheEnzyme Center, Boston, MA) were centrifuged for 10 minat 6,000 g and the supernatants discarded. The antigen-an-tibody mixtures were added to the pellets, mixed well, andleft for 1 h at 4°C. After centrifugation for 10 min at 6,000g the supernatants were discarded and the pellets werewashed three times with NETbuffer. To each of the washedsediments was added 200 ,l of 4% SDS, 4% dithiothreitol in

Membrane Glycoproteins of Bernard-Soulier Syndrome Platelets 305

water. The tubes containing the sediments were immersedin an ultrasonic bath (Branson Sonic Power Co. Danbury,CT) for 3 min, then treated at 100°C for 2 min, and finallycentrifuged for 10 min at 6,000 g. The supernatants wereanalyzed directly by polyacrylamide gel electrophoresis (17)followed by fluorography (19).

Identification of the thrombin-cleavable glycoprotein(GPV).' Platelets (10') surface-labeled by either the per-iodate/NaB3H4 or neuraminidase/galactose oxidase/NaB3H4methods, suspended in phosphate-buffered saline, pH 7.4,were divided into two aliquots. One was treated with throm-bin (1 U) for 10 min at 37°C, whereas the control was treatedsimilarly without addition of thrombin. The samples werecentrifuged at 1,100 g for 15 min, the supernatants werelyophilized and redissolved in 100 gl water and both thesupernatants and the pellets were prepared for gel electro-phoresis (17) followed by fluorography (19).

RESULTSPeriodate + NaB3H4 labeling. The platelets from

patients V.S. and F.S. showed distinct differences fromnormal (Fig. 1). GPIb (clearly identifiable by changesin molecular weight between unreduced and reducedin normals) was absent or present only in traceamounts. GPV (identified by its absence or decreasein thrombin-treated normal platelets, see also sectionon neuraminidase + galactose oxidase + NaB3H4 la-beling) could either not be detected (patient V.S.) orwas detectable at a very low level (patient F.S.). Inaddition, two other low molecular weight glycopro-teins, clearly visible in normals, could not be detected.One of these had an apparent molecular wt of 22,000and an apparent isoelectric point (pI) of 7.2-7.5 andcould be identified as the f3-subunit of GPIb by variouscriteria: This glycoprotein was not present or presentonly in trace amounts detectable after long exposuretimes if normal platelets were analyzed in the unre-duced state. It is precipitated along with the a-chainof GPIb by an antibody against glycocalicin (Fig. 2a)and can be demonstrated to split off from GPIb whenGPIb purified by lectin affinity chromatography onpeanut agglutinin was analyzed by two-dimensionalpolyacrylamide gel electrophoresis in the reducedstate. The second glycoprotein that could not be de-tected had a molecular mass of -17,000 daltons anda pl of 5.8-6.5, and was present in normal plateletsin both the reduced and the unreduced states in thesame position. Even after heavy overexposure of thefilm to the gel this glycoprotein could not be detectedin these patients. A third low molecular weight gly-coprotein, that labeled strongly with this techniqueand was present normally in Bernard-Soulier syn-drome platelets, could be identified as the #-subunitof lIb on the basis of the following criteria: It is pre-cipitated along with GPIIba and GPIIIa by an anti-

' Abbreviations used in this paper: GPIb, glycoprotein Ib;GPV, thrombin-cleavable glycoprotein.

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FIGURE 1 Fluorograms of two-dimensional separations, un-der reducing conditions, of platelets surface-labeled by theperiodate/NaB3H4 technique. (a) Normal platelets. (b) Lowerpart of gel with platelets from one normal donor. (c) Plateletsfrom Bernard-Soulier syndrome patient, F.S. (d) Plateletsfrom Bernard-Soulier syndrome patient, E.H. The nomen-clature used for the membrane glycoproteins is a simplifiedversion of that used in previous publications with minorchanges. The thrombin-cleavable glycoprotein is referred toas GPV in accordance with the nomenclature of Phillips etal. (20) and the earlier GPV now becomes GPVIa since itseems closely related to GPVIb. Unidentified glycoproteinsor those not earlier described are referred to by a nomen-clature based on the pl range as superscript and the molec-ular weight in kilodaltons (M. Wt. Kdaltons) as subscript.

306 K. J. Clemetson, J. L. McGregor, E. James, M. Dechavanne, and E. F. Luscher

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show that E.H. had -40±5% of the normal amountsof GPIb,B and of GP175865, using several other gly-coproteins, including GPIIb,B, as standards. Based ondensitometry of one-dimensional gels (not shown) E.H.had -30±10% of the normal amount of GPIba. Thedifferences in GPV were confirmed on one-dimen-sional gels run on platelets from patient M.S. (not

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body against GPIIb/IIIa (Fig. 2b) and is absent whenGlanzmann's thrombasthenia platelets were analyzedby two-dimensional polyacrylamide gel electropho-resis in the reduced state. Traces of a labeled glyco-protein were found in this position with unreducednormal platelet samples after long exposure times.

All of the glycoproteins not detected with patientsV.S. and F.S. could be seen with the platelets frompatient E.H. (Fig. Id). However, they were clearlypresent in lower amounts than normal. Because the x-

ray films used were flashed to increase their sensitivity,the response to light is approximately linear (19), anddensitometry of the spots on the film can be used toestimate the quantity of the component present on theplatelet surface. Although GPIba cannot be easily as-

sessed in this manner because of the proximity of otherglycoproteins and also the heterogeneity of GPIba inthe isoelectric focusing dimension, it was possible to

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Membrane Glycoproteins of Bernard-Soulier Syndrome Platelets

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shown). Differences in the GPIb region have alreadybeen described with this patient (13).

One of the normal controls showed a different pat-tern for GP175.8-6-5 (in the reduced state) compared withother single normals or to normal pool platelets. In-stead of the usual two major spots and one minor, fourspots were seen with the additional spots more acidicthan normal (Fig. lb).

Neuraminidase + galactose oxidase + NaB3H4 la-beling. The platelets from patients V.S. and F.S. againshowed marked differences from normal (Fig. 3).GPIba was absent or present only in trace amounts.The same two low molecular weight glycoproteinswere absent as with periodate/NaB3H4 labeling. Innormals the position of GPIb,B was not greatly affectedby neuraminidase but GP175865 was shifted to a morebasic pI, now running nearer to GPIb# at pI 6.1-6.8.The thrombin-cleavable glycoprotein termed GPVorSP85 (23-25) was not detectable. This glycoproteinwas identified by thrombin treatment of surface-la-beled normal platelets. Fig. 4 shows fluorograms oftwo-dimensional gels of surface-labeled normal plate-lets, normal platelets after treatment with thrombinand the supernatant after treatment with thrombin.The only glycoprotein affected is GPV, which is re-moved from the platelet surface. At the same time asmaller glycopeptide, which is a fragment of GPVtermed GPVf1 (26), appears in the supernatant. PatientE.H. again showed an intermediate situation with lev-els of GPIba less than normal but clearly visible(Fig. 3d), while densitometry gave values for GPIb#,GP176'1-6.8 and GPV(the thrombin-cleavable glycopro-tein) of -40±7% of normal.

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One-dimensional gels on platelets from patient M.S.showed that GPVwas virtually undetectable comparedwith normals (not shown). As with periodate/NaB3H4labeling, one control (the same one) gave a differentpattern of spots for GP1761-68 in the reduced stateshowing four major spots instead of the two major, oneminor generally obtained. The extra spots were on theacidic side of the normal and are apparently not dueto differences in sialylation as all four persist althoughthey are more basic after neuraminidase treatment(Fig. Sb).

Lactoperoxidase catalyzed iodination. The pat-terns of the labeled membrane proteins were virtuallyidentical between the platelets from the Bernard-Sou-lier syndrome patients and the normals and are there-fore not shown. There were, however, minor differ-ences that were noticeable with patients V.S. and F.S.but not with E.H. In these two patients GP lb wasabsent or shifted. This was only perceptible at rela-tively low exposure times as it was otherwise maskedby the heavy labeling of the neighboring proteins.With patient E.H. no clear differences could be seen.In all these patients several spots could be seen in theGPVregion, after long exposure times. However, therewere no differences from normal and GPVcould notbe definitively identified in normals by thrombin treat-ment presumably because it is very poorly labeled byiodination. The lower molecular mass (<40,000 dal-tons) regions also showed no difference from normal.

Incubation of labeled normal platelets in Bernard-Soulier or normal blood. Normal platelets were la-beled by either the periodate/NaB3H4 or the neur-aminidase/galactose oxidase/Na3H4 method and then

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FIGURE 4 Fluorograms of two-dimensional separations (7.5%acrylamide gels) under reducing conditions of: (a) Normalplatelets labeled by the neuraminidase/galactose oxidase/NaB3H4 technique. (b) Pellet from normal platelets labeledas in (a) after treatment with thrombin (1 U) for 10 min.The absence of GPV is indicated with an arrow. (c) Super-natant from normal platelets labeled and treated as in (b).The only labeled component present is the hydrophilic frag-ment of GPV, termed GPVfl (26). The cleavage of GPVbythrombin was similar with periodate/NaB3H4-labeled plate-lets.

308 K. J. Clemetson, J. L. McGregor, E. James, M. Dechavanne, and E. F. Luscher

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incubated in either normal blood or Bernard-Soulierblood. After reisolation and gel electrophoresis fol-lowed by fluorography the results of the different treat-ments were compared. Fig. 5 shows two-dimensionalseparations of periodate platelets + NaB3H4 labeled inthe reduced state. As can be clearly seen there are noobvious differences between the labeled normal plate-lets (Fig. 5a), the labeled normal platelets incubatedin normal blood (Fig. 5b), and the labeled normalplatelets incubated in Bernard-Soulier blood (Fig. 5c).In particular there were no changes in GPV or inGP1758-65. Densitometry of one-dimensional gels (notshown) also showed no difference between the var-iously treated platelets.

DISCUSSION

The Bernard-Soulier syndrome platelets examined inthe present study showed the classic membrane gly-coprotein defect of this disorder, a deficiency in GPIb, which has been described by several authors usingvarious techniques (8-14). In the majority of casesdescribed in the literature GPIb appears to be totallyabsent; however in some cases it appears to be presentin reduced amounts. It was, therefore, not entirelyunexpected that cases exist such as patient E.H. who

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shows an intermediate situation. Despite the fact thatthis patient does not have "giant" platelets, one of theclassic symptoms of this disorder, the poor responseto ristocetin and the biochemical differences from nor-mal clearly indicate that he should be considered asa less extreme case of Bernard-Soulier syndrome.

The ,3-subunit of glycoprotein Ib was either not de-tectable in Bernard-Soulier syndrome or present inamounts that correlated well with the a-subunit, im-plying that the synthesis or incorporation of both sub-units into the membrane is closely linked. Because lit-tle is known about disulphide-linked membrane gly-coproteins this conclusion was not necessarily apparent.

The unexpected findings of this study were the in-ability to detect, by carbohydrate-labeling techniques,a further low molecular weight glycoprotein, GP1758-6.5, in the platelets of both patients with fully developedBernard-Soulier syndrome, which was present in thesame relative amounts as GPIb13 in E.H., the inter-mediate case, and the deficiency or change in glyco-sylation in the thrombin-cleavable glycoprotein [GPV(23) or SP 85 (24)]. Again, E.H. appears to representan intermediate situation as the GPVwas detectable,but not as strongly labeled as in normals.

Previous reports have appeared claiming thatchanges in glycosylation could be observed in Bernard-

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FIGURE 5 Fluorograms of two-dimensional separations, un-der reducing conditions, of platelets surface-labeled by theperiodate/NaB3H4 technique. (a) Platelets from normal do-nor 1. (b) Platelets from normal donor 1, labeled then in-cubated for 30 min at 370C in the blood of normal donor1, then reisolated. (c) Platelets from normal donor 1, labeledthen incubated for 30 min at 370C in the blood of Bernard-Soulier syndrome patient F.S.

Membrane Glycoproteins of Bernard-Soulier Syndrome Platelets

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Soulier syndrome platelet glycoproteins comparedwith normal using metabolic labeling with sugars (15).The changes reported here do not appear so generalbut are nevertheless significant. Bernard-Soulier syn-drome has been used as a model for lack of responseto von Willebrand Factor/ristocetin and first indica-tions of the role of GPIb as the von Willebrand factorreceptor were based on the absence of this glycoproteinin Bernard-Soulier syndrome platelets (8, 9). Shulmanand Karpatkin (27) found extensive differences be-tween Bernard-Soulier and normal platelets usingcrossed-immunoelectrophoresis techniques and sug-gested that endogenous membrane proteolysis mightaccount for these. To see if proteases or glycosidasespresent in Bernard-Soulier blood might be responsiblefor the loss or degradation of the various glycoproteinsabsent in this disorder, labeled normal platelets wereincubated with Bernard-Soulier or normal blood. Noobvious differences were found (Fig. 5), which wouldexclude the presence of soluble enzymes but possiblynot that of membrane-bound enzymes.

Peculiarities in the response of Bernard-Soulierplatelets to thrombin have been reported (6, 7) thatwere ascribed to the absence of GPIb, which is knownto bind thrombin and has been claimed to be the high-affinity thrombin binding site on platelets (6, 28). Inone of these reports both the high affinity and the lowaffinity binding sites were reduced (7).

Considerable controversy exists on the nature of theplatelet thrombin receptor and two possible candidateshave been proposed. One of these is GPIb/glycocali-cin, which has been reported to have a high affinityfor thrombin (28). However, a-thrombin inactivatedwith reagents such as diisopropyl fluorophosphate orphenyl methyl sulphonyl fluoride does not activateplatelets though it binds with the same affinity as un-treated thrombin (29). This result led others to seekfor a receptor that was cleaved by thrombin and theevidence points to this being GPV (SP 85) (23, 24).

The changes in GPV in Bernard-Soulier syndromeobserved here might be a direct consequence of thedeficiency of GPIb if this indeed acts as a high-affinityreceptor for thrombin. In circulation the Bernard-Sou-lier platelets would be exposed to low amounts ofthrombin and in the absence of the high-affinity re-ceptor this would bind to and cleave GPV. If this tookplace at a sufficiently low concentration it would prob-ably not activate the platelets but their ability to re-spond to larger amounts of thrombin would graduallybe reduced. It is, of course, not yet possible to distin-guish whether GPV is affected in its entirety or onlyits carbohydrate moiety. In the latter case the changesin thrombin-induced aggregation in Bernard-Soulierplatelets might be caused by changes in conformationin GPVas a result of the defective glycosylation.

Since it is known that GPV is a peripheral plateletglycoprotein that can be removed by changes in ionicstrength (26) and in view of the transition that occursin Bernard-Soulier syndrome platelets when they be-come "giant-sized" the most likely possibility is thatGPV is lost from Bernard-Soulier syndrome plateletsduring the extensive washing that is part of the sur-face-labeling methods. This would also imply thatGPIb and GPVare associated on the platelet surfaceand that GPIb stabilizes the association of GPV withthe membrane, which would fit in well with the ideaof coupling of binding of thrombin to signal generationvia cleavage of a receptor (30).

One of the diagnostic characteristics of Bernard-Soulier syndrome is the tendency of the platelets tobecome giant on undergoing "shape-change." Theseplatelets are apparently normal-sized in circulationunlike those in May-Hegglin's disease (31). The rolethat the membrane glycoproteins play in this phenom-enon is still unclear but the deficiency or absence ofGPIb with its two-chain structure and of GP1758-65,which is probably also an integral membrane glyco-protein, must have a profound influence on the struc-ture of the membrane.

Thus, although the absence or strong depletion ofboth subunits of GP Ib remains the main biochemicalcharacteristic of Bernard-Soulier syndrome platelets thereare other changes in the surface glycoproteins, partic-ularly the absence or strong depletion of GP175.".5 withcarbohydrate-labeling methods, which may be equallyresponsible for some of the peculiarities that characterizeBernard-Soulier syndrome platelets.

ACKNOWLEDGMENTS

Weare grateful to Miss M-L. Zahno for excellent technicalassistance and to Mrs. P. Licini and Miss Y. Fuhrer for sec-retarial help. Wewould like to thank Professor E. A. Beckfor permission to study patient V.S. and for the help that heprovided in taking blood samples. This work was supportedby the Swiss National Science Foundation.

REFERENCES

1. Weiss, H. J., T. B. Tschopp, H. R. Baumgartner, I. I.Sussmann, M. M. Johnson, and J. J. Egan. 1974. De-creased adhesion of giant (Bernard-Soulier) platelets tosubendothelium. Further implications on the role of thevon Willebrand factor in hemostasis. Am. J. Med. 57:920-925.

2. Bernard, J., and J. P. Soulier. 1948. Sur une nouvellevariete de dystrophie thrombocytaire hemorragiparecongenitale. Sem. Ho^p. Paris. 24: 3217-3223.

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