analysis glycoprotein and protein composition bernard ... · two-dimensional sds-page analysis....

Analysis of the Glycoprotein and Protein Composition of

Bernard-Soulier Platelets by Single andTwo-dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel ElectrophoresisALAN T. NURDEN,DOMINIQUEDupuis, THOMASJ. KUNICKI, and JACQUESP. CAEN,

Unite 150 Institut National de la Sante et de la Recherche Medical, HopitalLariboisiere, 75475 Paris Cedex 10, France

A B S T R A C T Previous reports have described con-flicting results concerning the glycoprotein (GP) andprotein composition of Bernard-Soulier platelets. Inview of this controversy we have analyzed the plateletsof four Bernard-Soulier patients using improved singleand two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis procedures. An ab-sence of staining for carbohydrate of membrane GP lbwas characteristic for the platelets of each patient.Major periodate-Schiff staining bands correspondingto membrane GP lIb, Illa, and IlIb were clearly de-tected and their presence was confirmed by two-dimen-sional SDS-polyacrylamide gel electrophoresis. Theprotein content of the Bernard-Soulier platelets was in-creased two- to fourfold. However, analysis of their pro-tein composition using 7-12% acrylamide gradientgels showed normal polypeptide profiles. Lactoperoxi-dase-catalyzed 125I-labeling of the Bernard-Soulierplatelet surface proteins was followed by SDS-poly-acrylamide gel electrophoresis and autoradiography.No labeling in the Ib position was detected whereas theother major membrane GP, including Ia and Ila, werenormally located. In contrast, GP lb was clearly de-tected by periodate-Schiff staining and autoradiogra-phy when normal human platelets that had been ex-haustively treated with neuraminidase before the lacto-peroxidase-catalyzed iodination were analysed. Noabnormalities were detected in the GP patterns ofmembranes isolated from the patients' erythrocytes.Only a severe molecular abnormality or possible dele-tion of GP lb could account for this major plateletlesion in the Bernard-Soulier syndrome.

Dr. Kunicki's present address is the Blood Center of South-eastern Wisconsin, Milwaukee, Wis. 53233. Address reprintrequests to Dr. Nurden.

Received for publication 23 October 1980 and in revisedform 29 December 1980.

INTRODUCTION

The Bernard-Soulier (B-S)l syndrome is a con-genital platelet disorder with an autosomal recessiveinheritance that was originally characterised by theassociation of a prolonged bleeding time, the presenceof abnormally large platelets on peripheral bloodsmears, an often observed thrombocytopenia, and a de-fective prothrombin consumption test (1). Subsequentstudies have suggested that the bleeding tendencyarises from an abnormality in the platelet-vessel wallinteraction, or more specifically, in a diminished plate-let ability to adhere to subendothelium (2-4). B-S plate-lets are not agglutinated by bovine factor VIII (5, 6) orby ristocetin in the presence of normal human plasma(7, 8). A diminished binding of certain coagulation fac-tors to B-S platelets has been noted (6) whereas a re-duced binding of 125I-labeled thrombin to B-S plateletswas correlated with a decreased aggregation responsespecifically observed with this stimulus (9, 10). Finally,B-S platelets have been shown to lack the receptor forquinine- or quinidine-dependent antibodies (11).

Grottum and Solum (12) first reported that the plate-lets of two B-S patients had a reduced sialic acid con-tent and electrophoretic mobility. This observationsuggested the presence of a membrane glycoproteinabnormality. Nurden and Caen (13) subsequently lo-cated a severe reduction in the periodate-Schiff (PAS)-staining intensity of the then-termed glycoprotein (GP)I band following the analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ofmembranes isolated from the platelets of two B-S pa-tients. This abnormality was confirmed by the direct

' Abbreviations used in this paper: B-S syndrome, Bernard-Soulier syndrome; GP, glycoprotein; PAS reaction, periodate-Schiff reaction; PRP, platelet-rich plasma; SDS-PAGE,sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1431J. Clin. Invest. ( The American Society for Clinical Investigation, Inc. 0021-9738/81/05/1431/10 $1.00Volume 67 May 1981 1431-1440

analysis of unfractionated B-S platelets by SDS-PAGE(3). In contrast, Jenkins et al. (14) reported other find-ings after their studies on the platelets of a differentB-S patient. These authors detected a diffuse PASbandin the GP I position after SDS-PAGE and were un-able to detect PAS-staining in the GP II and III posi-tions. Furthermore, widespread changes in both theplatelet protein composition and the 125I-labeling pat-tern of the surface-orientated polypeptides were ob-served and emphasized. In recently published studies,Hagen et al. (15), and Shulman and Karpatkin (16)analyzed B-S platelets by crossed immunoelectro-phoresis using rabbit antisera raised against normal hu-man platelets (15) or isolated platelet membranes (16).Markedly different findings were reported. Hagen et al.(15) located a specific defect involving GP Ib and gly-cocalicin, a high molecular weight glycopeptide re-ported to be released from Ib as a result of Ca2+-de-pendent protease activity (17). In contrast, Shulmanand Karpatkin (16) noted numerous differences andsuggested that the abnormalities observed in the B-Ssyndrome and those of a second hereditary platelet dis-order, Glanzmann's thrombasthenia, reflected dif-ferences in endogenous membrane proteolysis.

In view of the apparent contradiction between ourinitial observations and those of Jenkins et al. (14), theincreased complexity of the human platelet membraneGPcomposition as revealed by the introduction of im-proved SDS-PAGEprocedures, and the lack of agree-ment in the reports on the analysis of B-S platelets bycrossed immunoelectrophoresis, we now describefurther studies performed on the platelets of four B-Spatients. Analysis of the GPand proteins of 125I-labeledplatelets of each patient by refined single and two-di-mensional SDS-PAGEprocedures has established thatB-S platelets possess a specific and characteristic mem-brane GP lesion. Furthermore, a comparison with theresults obtained for control platelets labeled with 125Iafter treatment with neuraminidase to remove exposedsialic acid residues has shown that a decreased sialicacid content cannot alone account for the observed B-Splatelet surface alterations.

METHODS

Materials

Dextran T 500 was obtained from Pharmacia Fine Chemi-cals, Uppsala, Sweden and sodium metrizoate solution (32.8%wt/vol, density 1.20 g/ml) from Nyegaard & Co., Oslo, Nor-way. All materials for SDS-PAGE were of electrophoresispurity grade and were obtained from Bio-Rad Laboratories,Richmond, Calif. High and low molecular weight proteinstandards used for molecular weight determinations follow-ing SDS-PAGE were also obtained from Bio-Rad Labora-tories. Schiff's reagent was prepared from Fuchsine Diament(Reactifs RAL, purchased from the Societe Chimique PointetGirard, Clichy, France). Neuraminidase (Vibrio cholera 500

U/ml) was purchased from Behringwerke, Marburg/Lahn,West Germany, Carrier-free Na125I (1 mCi/10 ,ul, in NaOHsolution, pH 8-11) was obtained from the RadiochemicalCenter, Amersham, England. Lactoperoxidase was obtainedfrom Sigma Chemical Co., St. Louis, Mo. Purified orosomu-coid was a gift from Dr. Graham Jamieson (American NationalRed Cross Blood Research Laboratory, Bethesda, Md.) Auto-radiography was performed using Kodak X-Omat MAfilm.

SubjectsFour patients with platelet function defects typical of the

B-S syndrome have been studied. N.V. is an adult womanpreviously studied by us (3); R.B. is an adult man whose casehistory was reported by Bernard et al. (18); W.B. is an adultwomanstudied previously by Devaraj et al. (19) and examinedby us through the courtesy of Dr. Frank Hill (The Children'sHospital, Birmingham, England); D.S. was an 11-yr-old boyat the time of study and together with N.V. and R.B. formedpart of the report by Rendu et al. (20). Normal human bloodwas obtained from healthy adult volunteers. Informed consentwas given and all studies were performed according to theprincipals of the Declaration of Helsinki.

Platelet isolationBlood (9 vol) was taken by venipuncture directly into an

acid-citrate-dextrose anticoagulant (1 vol) as described (3).Platelet-rich plasma (PRP) was prepared in either of two ways:(a) The blood was centrifuged at 120 g for 15 min at 15°C.This procedure was used for experiments with control bloodonly. (b) For the B-S patients and their appropriate controls,samples (2 ml) of blood were layered onto 5-ml vol of a solu-tion of sodium metrizoate and dextran T 500 as described bySolum et al. (21). The erythrocytes were progressivelyclumped by the dextran and sedimented. The PRP was re-moved and the platelet and leukocyte content enumerated byphase contrast microscopy. The PRPwas then centrifuged at250 g for 5 min and the platelet and leukocyte content of thesuspension re-evaluated. The sedimented cells were dis-carded. This process was repeated until the leukocyte con-tamination was <1% platelets (normally one or two centrifu-gations were sufficient).

The platelets in the PRPwere finally sedimented by centrif-ugation at 3,000 g for 15 min and resuspended in small vol-umes (10 ml buffer for the platelets sedimented from 50 mlPRP) of 0.01 M Tris-HCl, pH 7.4, containing 0.15 MNaCl,5 mMglucose, and 1 mMEDTA. The concentrated plateletsuspension was centrifuged at 2,600 g for 15 min in trans-parent plastic conical tubes (12 ml capacity). Any remainingerythrocytes settled to the base of the cone; the overlyingplatelets in the pellet were carefully resuspended andwashed twice with half the original PRP volume of theabove buffer.

Lactoperoxidase-catalyzed iodinationWashed control platelets were resuspended at 1 x 109 plate-

lets/ml in 0.01 MTris-HCl, pH 7.4, containing 0.15 MNaCland 1 mMEDTA. The platelets of the B-S patients were like-wise resuspended but at 5 x 108 platelets/ml. The protocolused for lactoperoxidase-catalyzed 1251-labeling was based onthe method of Phillips and Poh Agin (22). To 1 ml of eachplatelet suspension were added sequentially 0.5 mCi1251, 10,ul 0.25 mMlactoperoxidase dissolved in the plateletsuspension buffer, and finally at 10-s intervals, 5 x 12 ,ul 1 mMH202 also prepared in the platelet suspension buffer. The

1432 A. T. Nurden, D. Dupuis, T. J. Kunicki, and J. P. Caen

labeled platelets were sedimented and washed twice beforebeing resuspended for SDS-PAGEanalysis.

SDS-PAGEproceduresWashed unlabeled or 125I-labeled control platelets were re-

suspended at 2 x 109 platelets/ml in 0.01 MTris-HCl, pH 7.0,containing 0.15 M NaCl, 3 mMEDTAand 5 mMN-ethyl-maleimide. B-S platelets were similarly resuspended, but ata concentration of 5 x 108 platelets/ml. A one-fifth volume of12% SDS was added and the samples heated at 100°C for 5min. After cooling, aliquots were taken for protein analysis(23) and the rest was frozen at -20°C. Disulfide bond reduc-tion was perforned immediately before electrophoresis by in-cubating the samples at 37°C for 1 h in the presence of 5%vol/vol 2-mercaptoethanol.

GPanalysis. SDS-PAGEwas performed using 10.5 x 0.5-cm polyacrylamide rod gels and the Laemmli (24) buffer sys-tems. Each sample contained 400 ,ug platelet protein and wasmixed with 20 gl 50% vol/vol glycerol containing 0.0025% wt/vol bromophenol blue tracking dye before being applied to thegel. An internal standard for the PAS reaction, 5 ,ug oroso-mucoid dissolved in 2%SDS, was added to the sample beforeelectrophoresis and before reduction. Reduced samples wereanalyzed on 6%acrylamide (0.2% bis) separating gels, nonre-duced samples on 7% acrylamide (0.2% bis) separating gels.Each gel was overlayered with a 1.0 cm 3.5% acrylamidestacking gel. Electrophoresis was performed overnight at 25 Vand continued until the tracking dye approached within 0.5 cmof the base of the tube. After electrophoresis the gels werewashed overnight in 25%vol/vol isopropanol: 10%vol/vol ace-tic acid. GPwere located by the PAS reaction (25); the Schiffreagent was prepared fresh each month according to the pro-cedure of Segrest and Jackson (26). The stained gels werestored at 4°C in 7%acetic acid and densitometrically scannedwithin 24 h using a Vernon Recording Photometer (VernonIntegrating Photometers, Paris, France) equipped with agreen (Wratten No. 7) filter.

Polypeptide analysis. Samples were prepared and disul-fide bonds reduced exactly as described for the GPanalysis.SDS-PAGE was performed using 7.0-12.0% exponentialgradient acrylamide slab gels of dimensions 10 (height) x 14(length) x 0.15 cm (thickness). The ratio (g:g) of bis to acryl-amide was maintained at 0.027 throughout the gradient. A 1-cm 3.0% acrylamide stacking gel was also used. The buffersystems of Laemmli (24) were again used and electrophoresisperformed overnight at 25 V in a Bio-Rad model 220 verticalslab electrophoresis cell. Samples containing 100 ,ug proteinwere analyzed. Polypeptides were located using CoomassieBlue R250 (27). Stained gels were incubated for 1 h in 10%vol/vol acetic acid containing 1% vol/vol glycerol and dried ontofilter paper using a Bio-Rad model 224 Gel Slab Dryer. 125I.labeled polypeptides were located by autoradiography, thedried gels being left in contact with Kodak X-Omat MAfilm atroom temperature for up to 2 wk, after which the films wereprocessed according to the manufacturer's instructions.

Two-dimensional SDS-PAGE analysis. This was per-formed using an adaptation of the nonreduced/reduced twodimensional system of SDS-PAGE analysis as applied toplatelets by Phillips and Poh Agin (22). First dimension SDS-PAGEwas performed using 7% acrylamide (0.2% bis) tubegels and nonreduced samples as outlined in the GPanalysissection, 100 ,g SDS-solubilized platelet protein now being ap-plied to the gel. After electrophoresis the unstained gels wereagitated for 1 h at room temperature in the presence of 0.01 MTris, pH 7.0, containing 3 mMEDTAand 5% vol/vol 2-mer-captoethanol. The second dimension electrophoresis wasperformed using 7-12% exponential gradient acrylamide slab

gels as outlined in the section polypeptide analysis. One ex-ception was the substitution of the outer standard glass plate inthe gel forming apparatus with a plate with a bevelled upperedge (Bio-Rad Laboratories). The gel containing the now-re-duced polypeptides was layered onto the second dimensiongel, over the 3%stacking gel. The rod gel was fixed in positionby the addition of a small volume of 1%agarose dissolved bywarming in 10 mMTris, pH 7.0. A small plastic template wasattached at the right-hand side of the layered polyacrylamidetube gel before addition of the agarose. An aliquot (100 fi.gprotein) of reduced proteins of the sample being analyzed wasadded to the cavity in the agarose that formed when thetemplate was removed. After electrophoresis the gels werestained with Coomassie Blue R250. On occasion, PAS-stainingwas first performed to locate the position of the major GP. Thedetection of '25I-labeled proteins by autoradiography wasperformed as described in the section polypeptide analysis.

Molecular weight determinations. Apparent molecularweights of the platelet GPand polypeptides were determinedby comparing their relative migration during SDS-PAGEwith that of the following standard proteins: myosin heavychain (200,000 mol wt), f3-galactosidase (116,500 mol wt),phosphorylase b (94,000 mol wt), bovine serum albumin(68,000 mol wt), ovalbumin (43,000 mol wt), carbonic anhy-drase (30,000 mol wt), soybean trypsin inhibitor (21,000 molwt) and lysozyme (14,300 mol wt). Mixtures containing 2.5 ,ugof each protein were incubated with SDS and 2-mercapto-ethanol and SDS-PAGEperformed exactly as described forthe platelet samples. Measurements were made to the leadingedge of the stained bands.

Neuraminidase digestionWashed normal human platelet suspensions were resus-

pended at 1 x 109 platelets/ml in 0.01 M sodium phosphate,pH 7.4, containing 0.15 MNaCl. Preincubation at 37°C for 2min was followed by the addition of 0.05 vol neuraminidase(500 U/ml) in 0.05 Msodium acetate buffer, pH 5.5, contain-ing 0.154 M NaCl and 0.009 M CaCl2. The incubation wascontinued with agitation for 15 min, at which time the diges-tion was stopped by the addition of 4 mMEDTA. The plate-lets were sedimented and washed once in 0.01 MTris-HCl,pH 7.4, containing 0.15 M NaCl and 1 mMEDTAand thenresuspended for lactoperoxidase-catalyzed (1251) iodination.Total platelet sialic acid was estimated by the thiobarbituricacid method of Aminoff (28) following its release by hydroly-sis with 0.1 N H2SO4at 80°C for 1 h. Neuraminidase-releasedsialic acid was measured without prior acid hydrolysis. Opti-cal densities were read at both 532 and 549 nm and adjustedfor the presence of 532-nm-absorbing impurities (29).

RESULTS

B-S platelets. As is characteristic for the B-S syn-drome each of the patients possessed a low circulatingplatelet count at the time of study (Table I). The pro-tein content of the isolated platelets was increased,ranging from two to four times the normal amounts.Approximately 30% less sialic acid/mg protein was lo-cated in the platelets of the two patients where it wasestimated.

Platelet GP nomenclature. The four major PAS-staining membrane GP are termed Ib, IIb, IIIa, andIllb according to the criteria described by Nurden andCaen (30) and as previously used by us. GP Ig refers

Bernard-Soulier Platelet Glyco proteins 1433

TABLE ILaboratory Studies on B-S Platelets

Whole blood PlateletPatient platelet count Platelet protein sialic acid

103/cu/mm mgIlO9 platelets pg/mgprotein

N.V. (3) 88 16.5 5.0D.S. (1) 90 17.2 NDW.B. (1) 125 7.9 NDR.B. (2) 25 8.4 4.8Controls (20) 160-400* 3.85± 1.22§ 7.0±0.8§

The numbers in parentheses represent the number of timeseach patient has been examined. The values given for eachpatient are the mean of the results obtained, ND (notdetermined).* Range.§ MeantSD.

to the intracellular, granule localized GPthat has beentermed "thrombin sensitive protein" (31) or "thrombo-spondin" (32). Other membrane GP are given thenomenclature established by Phillips and Poh Agin(22) where it was clearly apparent that their electro-phoretic mobilities, with and without disulfide bondreduction, were comparable.

Platelet GP analysis. Preliminary studies estab-lished that the optimal separation of the major PAS-staining platelet GPwas achieved using the Laemmli(24) system of electrophoresis. Rod gels were used tofacilitate densitometric scanning. The optimal acryl-amide concentration for the analysis of reduced sampleswas found to be 6%acrylamide (0.2% bis). Fig. 1 com-pares the GP profiles of reduced samples of normalhuman platelets and those of the B-S patient (R.B.).The B-S platelet profile shows a specific and severereduction in the PAS-staining intensity of the lb band.Note that the absence of this band allows the full ap-pearance of a PAS-peak, band 4, present as a slowlymigrating shoulder to GP lb in the normal plateletprofile. Band 4 has an apparent molecular weight of160,000 and for reasons referred to later probably repre-sents the PAS stain of GP Ia and IIa.

As detailed by Phillips and Poh Agin (22), the plate-let membrane GP contain differing amounts of inter-or intramolecular disulfide bonds, thus their rate ofmigration during electrophoresis depends on the stateof reduction of these bonds. Fig. 2 compares the GPprofiles of nonreduced normal human and B-S platelets.A clearly apparent absence of staining in the Ib posi-tion was again a feature of the B-S platelet profile. Sim-ilar GP profiles were observed for the platelets of allfour patients studied. Inasmuch as the added orosomu-coid internal standard tended to migrate nearer to thetracking dye front on electrophoresis of nonreducedsamples, an increased 7% acrylamide (0.2% bis) con-

Eogc I Orosom. -P.o lg b~~~6 Il II

in 4.sV ' lb*2

c

200 150 100 SOmol Wt x 10-3

FIGURE 1 GP profile of B-S platelets after disulfide bondreduction. Washed normal human platelets and those isolatedfrom B-S patient (R.B.) were solubilized in 2% SDS as de-scribed in the Methods. Constituent disulfide bonds werereduced by incubating the samples for 1 h at 37°C in the pres-ence of 5% vol/vol 2-mercaptoethanol. Electrophoresis wasperformed using 6%acrylamide rod gels and the procedure ofLaemmli (24), 400,ug platelet protein was applied to each geltogether with 5 ,ug orosomucoid (Orosom). The GPwere lo-cated by the PAS reaction, the orosomucoid acting as an in-ternal standard. Typical densitometric profiles are illustrated.The separating gel origin is marked (t), the profile to theleft of this mark shows the PAS stain in the stacking gel.

centration was used for their analysis. When nonre-duced samples were analyzed, it was necessary toblock intrinsic membrane sulfbydryl groups with N-

E IfliClull~~~~b ll/

o 1iiU* Jrosom..

1.0

B-s

200 150 100 50mol wt x 10-3

FIGURE 2 GPprofile of B-S platelets in the absence of disul-fide bond reduction. Washed normal human platelets andthose isolated from B-S patient (W.B.) were dissolved by heat-ing at 100°C for 5 min in the presence of 2%SDSand 5 mMN-ethylmaleimide. SDS-PAGE was performed using 7%acrylamide rod gels as described in the legend to Fig. 1.Typical densitometric profiles of PAS-stained gels are illus-trated.


ethylmaleimide before heating samples at 1000C withSDS to prevent formation of high molecular weightaggregates of both membrane proteins and GPthat failto penetrate the separating gel. A similar observationhas previously been made (22).

Platelet protein composition. Analysis of the B-Splatelet proteins was performed on 7-12% gradientacrylamide slab gels allowing the detection of the majorpolypeptides within a molecular weight range of400,000 to 20,000. The polypeptide profiles of the plate-lets of two patients are illustrated in Fig. 3. No grossprotein abnormalities were observed. In particular,three large polypeptides with apparent molecularweights of 250,000, 230,000, and 200,000 were normallydetected. These polypeptides, which have been shownto be highly sensitive to degradation by an endogenousplatelet calcium-sensitive protease (33), have been re-ported to be modified in B-S platelets (14). As shown inTable I, the total protein content of B-S platelets was in-creased relative to nonnal human platelets. Fig. 3shows that this increase, whether it was two- (W.B.) orfourfold (N.V.) was the result of a general augmentationof the platelet proteins. Wesaw no sign of plasma pro-tein absorption. The position of GP lb was located byprior PAS-staining and is shown in Fig. 3. A faintCoomassie Blue band normally observed in this posi-tion was missing from the B-S platelet polypeptide pat-tern. In contrast, the Coomassie Blue band given by GPIlb, which is only separated from other comigratingpolypeptides on nonreduced gels (22), was present andpossessed a normal migration.

Analysis of 1251-labeled platelets. The surface pro-teins of platelets isolated from patients (N.V., R.B., andW.B.) have been labeled with 125I by the lactoperoxi-dase-catalyzed procedure and the labeled proteinsanalysed by SDS-PAGE. A comparison of different pro-cedures showed that the best resolution was achievedby autoradiography following the analysis of plateletsamples on 7-12% gradient acrylamide slab gels. Atypical analysis as performed on the platelets of patientW.B. is illustrated in Fig. 4. Identification of the labeledbands was aided (a) by correlating the peaks of radio-activity with the individual PAS-stained GPas locatedon slab or rod gels, (b) by the analysis of 1251-labeledplatelets of a patient with Glanzmann's thrombas-thenia, whose platelets were specifically deficient inIIb and IlIa (31), and (c) by direct comparison with theprofiles given by Phillips and Poh Agin (22). Analysisof reduced B-S platelet samples showed zones of radio-activity apparently normally associated with JIb andIIIa, the surface GPmaximally labeled with 125I by thelactoperoxidase-catalyzed procedure. However, theclose migration of Ib, Ia, and IIa prevented a preciseevaluation of their '251-labeling intensities. In contrast,analysis of nonreduced samples, which more clearlyseparated Ib from the other major membrane GP(com-pare also Figs. 1 and 2), showed a specific abnormalityin the Ib position.

Two-dimensional SDS-PAGEanalysis. The nonre-

NONREDUCED REDUCED Mol wt

NONREDUCED

a -'m'11b-- _i-

Con. B-S

1 (N.V.)

REDUCED mol wt

lb 1P lb

Cont. B-S Cont. B-S

1 (N.Y.) 2 (W. B.)

FIGURE 3 Protein profiles of B-S platelets. Washed plate-lets from two control donors and from B-S patients (N.V. andW.B.) were solubilized with SDSand aliquots (100 ,ug protein)subjected to SDS-PAGEafter (reduced samples) or in the ab-sence of disulfide bond reduction (nonreduced samples).SDS-PAGEwas performed using 7-12% gradient acrylamideslab gels and the procedure of Laemmli (24). Proteins were

detected with Coomassie Blue R 250. The position of GP Ibon the control patterns was detected by PAS-staining and isshown. The position of the protein band corresponding to JIbis likewise marked on the nonreduced profiles.

FIGURE 4 Single dimension electrophoresis of the 1251-la-beled surface proteins of B-S platelets. Washed suspensionsof normal human platelets and those isolated from the B-Spatient (W.B.) were incubated in the presence of 1251 and thesurface proteins labeled by the lactoperoxidase-catalyzed io-dination procedure as described in the Methods. Aliquots ofboth reduced and nonreduced SDS-solubilized protein (100,ug) were analysed by SDS-PAGEon 7-12% exponential gra-dient acrylamide slab gels as described in the legend to Fig.3. 125I-labeled proteins were detected on protein-stained,dried gels by autoradiography.

Bernard-Soulier Platelet Glycoproteins 1435

la I|- ; la/lbellb- Ealb s

Illa/b- _!1s_ ~lllb- w .

300200

300200

100

50

25

X 10-3

100

50

25

X 10-3A*..-

Control B-S

aCon l ...- S

Control B- S

duced/reduced two-dimensional system of SDS/PAGEof Phillips and Poh Agin (22) uses the different con-tents of inter- or intramolecular disulfide bonds withinthe individual membrane GPto improve the resolutionachieved using single dimension electrophoresisalone. Fig. 5 illustrates the analysis of 1251-labeled nor-mal and B-S platelets by this procedure. Autoradio-graphs of dried, Coomassie Blue-stained gels are

laI la

Illb

B-S Pt 1251

Ia l

lb lilb

W... .

*.0

R1-

CONTROgL P1 21

shown. An absence of '25I-labeling in the Ib position isthe striking feature of the autoradiograph of the B-Splatelet gel and confirms the finding illustrated for theplatelets of the patient (W.B.) in Fig. 4. Furthermore,zones of radioactivity corresponding to GP Ia and IIawere clearly detected. Two low molecular weight spotsgiven by the ,8-subunits of Ic and IIb (22) were also nor-mally present. Neither in our studies nor those reportedby Phillips and Poh Agin (22) has the ,8-subunit of GPIb been shown to be labeled with 1251 during lacto-peroxidase-catalyzed iodination of normal humanplatelets. It was, however, clearly detected by Coomas-sie Blue staining. No spot corresponding to the p-sub-unit of lb was located when B-S platelets were soanalyzed (data not shown).

Analysis of neuraminidase-treated platelets. Nor-mal human platelet suspensions were incubated withneuraminidase under conditions which gave riseto the maximal release of surface sialic acid residues.The surface proteins of the desialated platelets, whichhad lost -50% of their total platelet sialic acid content,were then labeled with 125I by the lactoperoxidase-cata-lyzed procedure. Fig. 6 shows a PAS-profile typicalof those obtained for neuraminidase-treated plateletsfollowing SDS-PAGE. A significant decrease in thePAS-staining capacity of Ib was observed, however,the GPwas still a major contributor to the PAS-profile.Only minor mriodifications in the PAS-staining capacity

Ec0In

In.IC

FIGURE 5 Two-dimensional SDS-PAGE analysis of 1251_labeled B-S platelet (PL) surface proteins. Washed suspen-sions of normal human platelets and those isolated from thepatient (N.V.) were labeled with 125I as described in thelegend to Fig. 4. The platelets were dissolved with SDS inthe presence of N-ethylmaleimide and samples (100 ,ug pro-tein) subjected to first dimension electrophoresis using 7%acrylamide rod gels as described in the legend to Fig. 2. Afterelectrophoresis, the unstained gel were incubated in thepresence of 5% vol/vol 2-mercaptoethanol and a seconddimension electrophoresis of the now reduced proteins per-formed on 7-12% gradient acrylamide slab gels. A singlesample of reduced 1251-labeled proteins (100 1Lg) of the samplebeing analysed was also applied to the right hand side of theslab gel. Protein-stained gels were dried onto filter paper andthe _251-labeled proteins detected by autoradiography. Twoweakly labeled low molecular weight polypeptides aremarked (+-) in the lower part of the gel, these correspond to the,p-subunits of Ic and Ilb respectively (22).

00.049

mol wt x 10-3FIGURE 6 GP profile of neuraminidase-treated normal hu-man platelets. Washed platelet suspensions were incubatedfor 15 min at 37°C in the presence or absence of neuramini-dase (25 U neuraminidase/109 platelets). Samples of the con-trol and neuraminidase-treated platelets were dissolved bySDS and analyzed without reduction on 7% acrylamide rodgels as described in the legend to Fig. 2. GPwere detectedby the PAS-reaction, typical densitometric profiles are shown.


of the other membrane GPwere detected. The residualIb exhibited a slightly slower rate of migration duringelectrophoresis whereas that of IIb was increased. Thealtered migration of lb suggested that the observedpeak represented desialated, or partially desialated, Ibrather than a GP fraction inaccessible to the action ofthe neuraminidase.

Analysis of the 125I-labeled proteins by autoradiogra-phy clearly showed an 125I-labeled band in the GP Ibregion of the gel although, as expected from the PAS-staining, the band had a slightly slower migration (Fig.7). The identity of this band as Ib was confirmed usingthe nonreduced/reduced two dimensional SDS-PAGEsystem (data not shown). Apart from mobility changes,remarkably little alteration was observed in the distri-bution of 125I among the surface proteins of the desi-alated normal platelets. The protein profiles of theneuraminidase-treated platelets also showed no modifi-cations, except for the altered rates of migration of lband IIb now more clearly apparent owing to the greatersharpness of the protein-stained bands.

DISCUSSION

Despite the fact that a number of investigators havereported studies on the GPand polypeptide composi-tion of B-S platelets (3, 11, 13-16, 21, 30, 34-36), theconflicting data reported in the literature has made itdifficult to precisely interpret the nature of the molecu-lar lesion(s) of these functionally defective platelets.Our current work has been designed to clarify the na-ture of the structural abnormalities of B-S platelets.

Characterization of normal human platelet GP. Inview of the difficulty in isolating well characterizedmembrane fractions from the platelets of patients withmoderate to severe thrombocytopenia (each of the pa-tients studied had a low circulating platelet count,Table I) our investigation has been restricted to theanalysis of unfractionated washed platelet suspensions.Preliminary studies compared the GP resolution ob-tained using SDS-PAGE procedures incorporating acontinuous SDS-phosphate buffer systerm similar tothat used for platelet GPanalysis by George et al. (37),with procedures incorporating the discontinuous SDS-Tris glycine buffer system as employed by Phillips andPoh Agin (22). Emphasis was placed on obtaining PASprofiles that allowed the quantitative evaluation of themajor PAS-bands by densitometric scanning. The bestresolution of the major PAS-staining GPwas achievedusing the discontinuous buffer system, reduced sam-ples, and 6%acrylamide separating gels. In comparisonwith the GPprofiles of reduced, whole platelets givenby George et al. (37), we have obtained an improvedseparation of GPIg from Ib and of GP IlIa from IIIb.

B-S platelet GP. The predominant feature of the GPprofile of the platelets of each of the four patients

x

PROTEIN

mol wi

20050_lb- v

150

100_

2

.io43'

- -

1251-

lb--w-llb- _

Ilia-.-p *of

Cont. + Neuram.

FIGURE 7 Protein and 125I-labeling profiles of neuramini-dase-treated normal platelets after single dimension electro-phoresis. Washed platelet suspensions were incubated in thepresence or absence of neuraminidase as described in thelegend to Fig. 6. The control (Cont.) and neuraminidase(Neuram.) treated platelets were then incubated in thepresence of 125I and the surface proteins labeled using thelactoperoxidase-catalyzed procedure. Aliquots (100 ,ug protein)of nonreduced SDS-solubilized platelets were analyzedby SDS-PAGEon 7-12% gradient acrylamide slab gels. Thegels were stained for protein, dried and analyzed by auto-radiography.

studied was the total absence of PAS-staining in theposition normally occupied by GP Ib. The characteri-zation of this defect was aided by the analysis of bothreduced and nonreduced samples. As defined by Phil-lips and Poh Agin (22) Ib is a high molecular weightGPcomposed of two subunits. Incubation of the parentmolecule with 2-mercaptoethanol results in the cleav-age of intermolecular disulfide bonds and the sepa-

ration of a small (22,000 mol wt) /8 subunit from a

large (148,000 mol wt) a subunit. In terms of thisnomenclature the PASband termed lb in the GPprofileof reduced, normal platelets in Fig. 1 is the a subunitof Ib. The labeled Ib in the GPprofile of nonreduced,normal platelets in Fig. 2 is the parent molecule.

Jamieson et al. (35) isolated membranes from theplatelets of two B-S patients and reported a specificdefect but that 40% of the PAS-stain was located in theGP I position following SDS-PAGE. Comparisons be-tween our results and those of Jamieson et-al. (35) may

suggest heterogeneity in the extent of the Ib lesion ob-served between different patients. A possible contribu-tion of Ia and IIa to the PAS-stain located in the GP Iposition was not, however, taken into account by Jamie-son et al. (35) who analyzed reduced samples by a single


Cont. + Neuram.

dimension SDS-PAGE procedure giving four majorPAS-stained bands. In our studies using reduced con-trol platelet samples an additional PAS-peak (band 4)was separated as a shoulder to the trailing edge of lb.This band was a marked feature of the reduced B-Splatelet profiles. Only using nonreduced sampleswere we able to clearly resolve Ib from other membraneGPduring single dimension SDS-PAGE.

125I-Labeling of B-S platelet surface proteins. Jenkinset al. (14) and Hagen and Solum (34) both studiedthe surface proteins of B-S platelets using lactoperoxi-dase-catalyzed iodination and reported differing re-sults. Jenkins et al. (14) found a highly abnormal profileof 125I-labeled proteins following SDS-PAGE, includ-ing the presence of numerous labeled low molecularweight polypeptides. In contrast, Hagen and Solum(34) described a normal 125I-labeling profile. Our resultswere consistent for the platelets of the three patientsstudied using this procedure; autoradiography fol-lowing single and two-dimensional SDS-PAGEshowed an absence of labeling in the Ib position. Theother major 1251-labeled surface GPas detailed by Phil-lips and Poh Agin (22) were all normally detected. Welocated none of the changes previously described forB-S platelets by Jenkins et al. (14).

In their studies Hagen and Solum (34) detected125I-labeled polypeptides by slicing PAS-stained rodgels and measured the radioactivity in the slices using agammacounter. Wefollowed the same procedure ini-tially but noted that accurate evaluation of lb was dif-ficult owing to the small percentage of the total radio-activity located in the GP I region. Subsequently, weperformed autoradiography after the separation of thelabeled proteins on gradient slab gels. Exposure timesof up to 2 wk amplified the detection of the less-stronglylabeled surface proteins. Furthermore, in view of theincreased protein content of the B-S platelets (Table I)and their increased size (1, 6), at least under the condi-tions of this study (38), we decreased the concentrationof B-S platelets during the lactoperoxidase-catalyzediodination (see Methods). With these procedures, aclear absence of radioactivity in the Ib position wasobserved either after the analysis of 125I-labeled B-Splatelets by the two-dimensional SDS-PAGEsystem orafter the analysis of nonreduced samples on single di-mension gradient acrylamide gels.

Relationship between Ib and glycocalicin. Solumand his co-workers (17, 39) have provided immuno-chemical evidence that lb is the membrane GPprecur-sor of glycocalicin, a large (148,000 mol wt) glycopoly-peptide composed of -60% by weight carbohydrate,that is solubilized from the platelet surface duringplatelet homogenization (40). Our present resultsindirectly support this finding in that Ib was the onlymajor PAS-staining GP to show a severe reduction instaining on analysis of B-S platelets, which have been

shown in several studies to lack glycocalicin (15, 16, 21,35). George et al. (37) have expressed a similar viewfollowing the analysis of normal platelets before andafter glycocalicin loss. In contrast, Nachman et al. (41)have claimed that lb and glycocalicin give differentpeptide maps following trypsin digestion and are dif-ferent GPalthough absolute proof for the identificationof Ib was not given.

Examination of neuraminidase-treated controlplatelets. In confirmation of the original report ofGrottum and Solum (12) the platelets of the two B-Spatients examined showed a decreased sialic acid con-tent. However, despite the removal of -50% of the totalsialic acid content of normal human platelets by neura-minidase treatment before 125I-labeling little changewas induced in the pattern of the '25I-labeled GPas ob-served after SDS-PAGE. A clear band or spot in the Ibposition remained on the autoradiographs. McGregor etal. (36) have suggested that there is either a severereduction or an altered glycosylation of GPIa and lb inB-S platelets. Our results point to the presence of Ia onthe B-S platelet surface as this GPwas apparently nor-mally labeled with 125I during lactoperoxidase-catalyzed iodination. In contrast, any structural defectsin Ib must be severe as we were unable to locate it byPAS staining, 1251-labeling, or, in previous studies, bycrossed immunoelectrophoresis (15). The presence of amolecular deficiency of Ib in B-S platelets must be con-sidered. Further studies are now being performed to ex-amine the state of glycosylation of GPIa in the plateletsof our patients, meanwhile its presence in their plate-lets has been recently confirmed by crossed immuno-electrophoresis.2

In agreement with McGregor et al. (42) we observedthat GPIb of normal human platelets migrated with anincreased apparent molecular weight after neura-minidase treatment (see Figs. 6 and 7). This slower rateof migration is probably due to the normal contributionof the anionic charge of sialic acid to the migration oflb during SDS-PAGE.

Possible origin of the B-S platelet defect. Our re-sults emphasize that B-S platelets contain a specific andcharacteristic membrane lesion. Wehave obtained noevidence for the large scale changes in polypeptidecomposition as attributed to B-S platelets by Jenkins etal. (14). Furthermore, we failed to detect signs of thepresence of widespread endogenous protease activityin B-S platelets as proposed by Shulman and Karpatkin(16). The possible presence of exogenous protease ac-tivity either in the bone marrow or plasma was alsoconsidered. If this was present then presumably the

2 Kunicki, T. J., A. T. Nurden, D. Pidard, N. R. Russell, andJ. P. Caen. Characterisation of platelet antigens giving rise toindividual immunoprecipitates in CIE. Manuscript submittedfor publication.


protease would be free to interact with the surface ofother blood cells. Analysis of membranes isolated fromerythrocytes of each patient, however, revealed nosigns of GP degradation (unpublished results). Ifprotease activity is the cause of the B-S platelet surfacelesion then the protease must have a narrow specificityand a particular affinity for platelet GP lb.

The alternative hypothesis, therefore, is that adirect genetic defect at a stage in the synthesis of lbmay be the cause of the B-S platelet membrane lesion.Studies on the megakaryocytes of B-S patients may berequired to locate the origin of the abnormality. Mean-while we emphasize that the B-S syndrome is a dis-tinct congenital platelet disorder characterized by adistinctive membrane GPabnormality.

ACKNOWLEDGMENTSThese studies were supported by grant CRL 78.5.128.1 fromInstitut National de la Sante et de la Recherche Medical(INSERM), France, and by a grant from the Thyssen Founda-tion, West Germany.

REFERENCES1. Bernard, J., and J. P. Soulier. 1948. Sur une nouvelle

variet6 de dystrophie thrombocytaire h6morragiparecong6nitale. Sem. Hop. Paris. 24: 217-221.

2. Weiss, H. J., T. B. Tschopp, H. R. Baumgartner, I. I. Suss-man, M. M. Johnson, and J. J. Egan. 1974. Decreasedadhesion of giant (Bernard-Soulier) platelets to subendo-thelium. Further implication on the role of the von Wille-brand factor in hemostasis. Am. J. Med. 57: 920-925.

3. Caen, J. P., A. T. Nurden, C. Jeanneau, H. Michel, G.Tobelem, S. Levy-Toledano, Y. Sultan, F. Valensi, and J.Bernard. 1976. Bernard-Soulier syndrome: a new plateletglycoprotein abnormality. Its relationship with plateletadhesion to subendothelium and with the factor VIII vonWillebrand protein. J. Lab. Clin. Med. 87: 586-596.

4. Baumgartner, H. R., T. B. Tschopp, and H . J. Weiss. 1977.Platelet interaction with collagen fibrils in flowing blood.II. Impaired adhesion-aggregation in bleeding disorders.A comparison with subendothelium. Thromb. Haemo-stasis. 37: 17-28.

5. Bithell, T. C., S. J. Parekh, and R. R. Strong. 1972. Plate-let-function studies in the Bernard-Soulier syndrome.Ann. N. Y. Acad. Sci. 201: 145-160.

6. Walsh, P. N., D. C. B. Mills, F. I. Pareti, G. J. Stewart,D. E. Macfarlane, M. M. Johnson, and J. J. Egan. 1975.Hereditary giant platelet syndrome. Absence of collagen-induced coagulant activity and deficiency of factor XIbinding to platelets. Br. J. Haematol. 29: 639-655.

7. Howard, M. A., R. A. Hutton, and R. M. Hardisty. 1973.Hereditary giant platelet syndrome: a disorder of a new as-pect of platelet function. Br. Med. J. 2: 586-588.

8. Caen, J. P., and S. Levy-Toledano. 1973. Interaction be-tween platelets and Von Willebrand factor provides a newscheme for primary haemostasis. Nature New Biol. 244:159-160.

9. Ganguly, P. 1977. Binding of thrombin to functionallydefective platelets: a hypothesis on the nature of thethrombin receptor. Br. J. Haematol. 37: 47-51.

10. Jamieson, G. A., and T. Okumura. 1978. Reduced throm-bin binding and aggregation in Bernard-Soulier platelets.J. Clin. Invest. 61: 861-864.

11. Kunicki, T. J., M. M. Johnson, and R. H. Aster. 1978. Ab-sence of the platelet receptor for drug-dependent anti-bodies in the Bernard-Soulier syndrome. J. Clin. Invest.62: 716-719.

12. Grottum, K. A., and N. 0. Solum. 1969. Congenitalthrombocytopenia with giant platelets: a defect in theplatelet membrane. Br. J. Haematol. 16: 277-290.

13. Nurden, A. T., and J. P. Caen. 1975. Specific roles for sur-face glycoproteins in platelet function. Nature (Lond.).255: 720-722.

14. Jenkins, C. S. P., D. R. Phillips, K. J. Clemetson, D.Meyer, M. J. Larrieu, and E. F. Luscher. 1976. Plateletmembrane glycoproteins implicated in ristocetin-in-duced aggregation. Studies of the proteins on plateletsfrom patients with Bemard-Soulier syndrome and vonWillebrand's disease.J. Clin. Invest. 57: 112-124.

15. Hagen, I., A. T. Nurden, 0. J. Bjerrum, N. 0. Solum, andJ. P. Caen. 1980. Immunochemical evidence for proteinabnormalities in platelets from patients with Glanz-mann's thrombasthenia and Bernard-Soulier syndrome.J.Clin. Invest. 65: 722-731.

16. Shulman, S., and S. Karpatkin. 1980. Crossed immuno-electrophoresis of human platelet membranes. Dimin-ished major antigen in Glanzmann's thrombasthenia andBernard-Soulier syndrome. J. Biol. Chem. 255:4320-4327.

17. Solum, N. O., I. Hagen, C. Filion-Myklebust, and T.Stabaek. 1980. Platelet glycocalicin: its membrane asso-ciation and solubilisation in aqueous media. Biochim.Biophys. Acta. 597: 235-246.

18. Bernard, J., J. P. Caen, and P. Maroteaux. 1957. La dys-trophie thrombocytaire hemorragipare congenitale. Rev.Fr. Hematol. 12: 222-229.

19. Devaraj, R., C. Prowse, J. Dawes, and F. Toolis. 1979.Platelet specific proteins in Bernard-Soulier syndrome.Thromb. Haemostasis. 42: 1349.

20. Rendu, F., A. T. Nurden, M. Lebret, and J. P. Caen. 1979.Relationship between mepacrine-labelled dense bodynumber, platelet capacity to accumulate 14C-5HT andplatelet density in the Bemard-Soulier and Hennansky-Pudlak syndromes. Thromb. Haemostasis. 42: 694-796.

21. Solum, N. O., I. Hagen, and T. Gjemdal. 1977. Plateletmembrane glycoproteins and the interaction betweenbovine factor VIII related protein and human platelets.Thromb. Haemostasis. 38: 914-923.

22. Phillips, D. R., and P. Poh Agin. 1977. Platelet plasmamembrane glycoproteins. Evidence for the presence ofnonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis. J. Biol.Chem. 252: 2121-2126.

23. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193: 265-275.

24. Laemmli, U. K. 1970. Cleavage of structural proteins dur-ing the assembly of the head of bacteriophage T4. Nature(Lond.). 227: 680-685.

25. Zacharius, R. M., T. E. Zell, J. H. Morrison, and J. J. Wood-lock. 1969. Glycoprotein staining following electro-phoresis on acrylamide gels. Anal. Biochem. 30: 148-152.

26. Segrest, J. P., and R. L. Jackson. 1972. Molecularweight determination of glycoproteins by polyacrylamidegel electrophoresis in sodium dodecyl sulphate. MethodsEnzymol. 28: 54-63.

27. Fairbanks, G., T. K. Steck, and D. F. H. Wallach. 1969.Electrophoretic analysis of the major polypeptides of theerythrocyte membrane. Biochemistry. 10: 2606-2617.

28. Aminoff, D. 1961. Methods for the quantitative estima-


tion of N-acetyl neuraminic acid and their application tohydrolysates of sialomucoids. Biochem. J. 81: 384-390.

29. Warren, L. 1959. The thiobarbituric acid assay of sialicacids.J. Biol. Chem. 234: 1971-1975.

30. Nurden, A. T., and J. P. Caen. 1979. The different glyco-protein abnormalities in thrombasthenic and Bernard-Soulier platelets. Semin. Hematol. 16: 234-250.

31. Baenziger, N. L., B. N. Brodie, and P. W. Majerus. 1972.Isolation and properties of a thrombin-sensitive proteinof human platelets. J. Biol. Chem. 247: 2723-2731.

32. Lawler, J. W., H. S. Slayter, and J. E. Coligan. 1978.Isolation and characterisation of a high molecular weightglycoprotein from human blood platelets. J. Biol. Chem.253: 8609-8616.

33. Phillips, D. R., and M. Jakabova. 1977. Ca2+-dependentprotease of human platelets. J. Biol. Chem. 252: 5602-5607.

34. Hagen, I., and N. 0. Solum. 1978. Further studies on theprotein composition and surface structure of normal plate-lets and platelets from patients with Glanzmann'sthrombasthenia and Bernard-Soulier syndrome. Thromb.Res. 13: 845-855.

35. Jamieson, G. A., T. Okumura, B. Fishback, M. M. Johnson,J. J. Egan, and H. J. Weiss. 1979. Platelet membraneglycoproteins in thrombasthenia, Bernard-Soulier syn-drome, and storage pool disease. J. Lab. Clin. Med. 93:642-660.

36. McGregor, J. L., K. J. Clemetson, E. James, E. F. Luscher,

and M. Dechavanne. 1980. A comparison of the majorplatelet membrane glycoproteins from Bernard-Souliersyndrome with normals after radiolabelling of sialic acidor terminal galactose / N-acetylgalactosamine residues.Thromb. Res. 17: 713-718.

37. George, J. N., R. K. Morgan, and P. C. Lewis. 1978.Studies on platelet plasma membranes. IV. Quantitativeanalysis of platelet membrane glycoproteins by ('251)diaz-otized diiodosulfanilic acid labeling and SDS-polyacryla-mide electrophoresis. J. Lab. Clin. Med. 92: 430-446.

38. Frojmovic, M. M., J. G. Milton, J. P. Caen, and G.Tobelem. 1978. Platelets from "giant platelet syndrome(BSS)" are discocytes and normal sized.J. Lab. Clin. Med.91: 109-116.

39. Solum, N. O., I. Hagen, and T. Sletbakk. 1980. Furtherevidence for glycocalicin being derived from a largeramphiphilic platelet membrane glycoprotein. Thromb.Res. 18: 773-785.

40. Okumura, T., C. Lombart, and G. A. Jamieson. 1976. Plate-let glycocalicin. II. Purification and characterisation. J.Biol. Chem. 251: 5950-5955.

41. Nachman, R. L., T. Kinoshita, and B. Ferris. 1979. Struc-tural analysis of human platelet membrane glycoprotein Icomplex. Proc. Natl. Acad. Sci. U. S. A. 76: 2952-2954.

42. McGregor, J. L., K. J. Clemetson, E. James, and M.Dechavanne. 1979. A comparison of techniques used tostudy externally orientated proteins and glycoproteins ofhuman blood platelets. Thromb. Res. 16: 437-452.


analysis glycoprotein and protein composition bernard ... · two-dimensional sds-page analysis....

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