Identification of human platelet glycoproteins in SDS-polyacrylamide gels using 125I labelled lectins

IN

1DE:lTIFICAiIOII OF HU?!AN PLATELET GLYCOP?OTEINS SDS-POLYACRYLAMIDE GELS USING "'I LABELLED LECTINS. J.L. ClcGREGfiR", K.J. CLEKETSON', E. MES', XI, r? T GREENLAN3' , !I. DECHF,VAM!K . I!ISERC: iinite6;, 22 avenue du Doven Lepine 6950~~-3Ro~~: iaculte de MPdecine Alexis Carrel ' rue Guillawle Paradin 69OC8 LYOii-FRkE. !jniversityof Bern, Theodor Kocher Institut ’ W-3000 Bern 9 SI;!ITZERLA?ll'l INSERH Unit@ 51 CR 33 CNRS ' 1. place du Pr. Joseph Renaut 69371 LYON-FMZICE. (Received 15.6.1979; in revised form 27.;.1$*79.

Rcccikctd

Accepted by by- Esec~lt.ive

Editor Y..J. Edi rorial

Lnrrieu. Qi‘l’ico

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ifl5 IHlC r Glycoprotfins of whole platt,iets wliibilized in sodiilm di7dec]i: sutphatc (SUS) and sc;larc~ted iri? ;i T.5.: LaemmLi stab gel were idrn“‘T-Lens culinaris (LCH-B) and tificd by “51-L~~hell~d Izctins. 7-q ” I-zclncnn4vnlin A (Con A) intensei:; iabcllc2 four major plateltit 7eribrnne gLv$yproteins idcntiticd as : Gi’Ti,, T?S, ITI (111.x) an,! IV !IIIb). Located in Ii I-LCH-I3 intcnscl:i label Ied 2 giycapr<>tein platelet granules (GPG) also kn,w7 as thrombin sensitive protein (TSP) or thcomb;?sl~,~ntlin. “‘1-Vheat jierm a,g,:glutinin (WC+.) intensely l,~belletl one glycoprotein band identified as CPIb . No labelleti. bands were obtained’with “j I-Kicinus cornuniz (RCA). The “‘I-labelled lcctin S3S-palvacrvlamide o_rL techniaua offers A sirr.ptc 1~3:~ in identifying pl ltelet ~L&zoprotrins and in screenin>: L-;rtins which cailld be used in the isc,Lati,>n %-ii i:ldividual r,>t*ins ‘by affinity-chrornatogrlphy.

INTRODUCTION Th+ resolution of complex mixtures of zlycoproteins such as are found in hwnnn blood platelets is an extremely diEEicult problem, particularly in csnne,.tion with the definition of disorders caused bp ncLccnlar defects. have been strongly implicated in pla!
Lens culinaris haemagglutinin B (LG!-Sj, concanavalin A (Con J-), b’!leat germ agglutinin (WGl\) and Ricinus communis aggl.2tinin 12Q (KC.1 17rj) t;ere from Miles laboratories, Inc. Elkhart, Indiana, USA. Carrier-free !:a”sI (17 Ci/mg) was obtained from Kiev England !;uc?ear (2F.T) , Heidelberg, EX. Iodination of lectins Lectin samples were dissolved in ICO ,.l of phosphate buffered saline (PBS) (pH = 7.4) at a concentration of I ng/rz! or an a!i.quot taken having a similar concentration. The different lectin samples :r’crc then labelled with 0.5 - 1 .O mCi of I’sI by the addition :I_ 5? il ?E a solution of chloramine T at room temperatiire the in PBS (0.7 mg/ml) (12). Utcr shaking f,>r 7 r:i?. reaction was stopped by adding 100 ~1 of a solution of sodium metabisulfite in PBS (0.63 mg/ml). To each sample Gas then added I50 ~1 of a solution of bovine serum albumin (BSA) in PBS (5 y/ml) as a carrier protein, and the samples were transEerred to individual dia!;;sis bags and dialysed against several changes of PBS for at least 3 days at + :‘C. At the end af this time the percentage of radioactivity precipitated by 5Z TCA was determined. Samples with at least 952 of precipitable counts were used, others were dinlysed further until they fulfilled this conditi.in. Separation of platelet glycoprotein bands Platelets isolated from blood of a healthy consenting donor were washed as were then directly solubilized in (I 2). Washed platelets previously described sodium dodecyl sulfate (SDS) in the presence of dithiothreitol (DTT) and Slao gels (IOcm x 14cm x 0.15cr) electrophoresed as previously described (12). bearing 10 electrophoresed samples were sliced longitudinally, fixed and extensively washed (Sh) in 40X (v/v) methanol-iZ (v/v) acetic acid-water to remove SDS. Labelling glycoproteins in polyacrylamide gels using “sI-leccins. The technique used is similar to that described by Rostas et al (1977) Fixed gels were left for a minimumof 4h in (7) with minor modifications. This was followed by 3 washes of 20 min. using concentrated distilled water. (x 10) PBS. The sliced gels were then washed another 3 times with PBS leaving gel in a small glass tray the gels in each wash for 20 min. To each sliced was added 20 ml of PBS containing 20 1-g of iodinated lectins. The tray containing the gels was then rocked for I h after which the radioactive lfctin The labelled gels were washed 3 times 20 min. vith solution was removed. 500 m! PBS and left overnight. 40 ml PBS. Each gel was then imnersed‘in dried under vacuum and indirect Washed gels were placed on a filter pa?er, autoradiography carried out with Kodak Royal S-Gmat film (13). Controls were performed with lectins as above except that the appropriate sugar for each lectin at 0.2M concentration was used before and during lectin incubation and in all washing solutions.

c

3_,

i

TABLE

PLATELET GLYCOPROTEIM BANOS OBTAINED BY INDIRECT AGTORADIOGRAPHY OF POLYACRYLAMIDE GEL ELECTROPHOROGRAMS STAINED WITH “‘i LABELLED LECTINS.

GLYCOPRClTEIN/APPARENT

1251

_ABELLED LECTINS

LCH-6

(IIIa)

HHGP

1

2

266

245

4

5

G

Ia

206

194

169

147

3

268

,JGA HMWG

139

148

CON-A

= high

’ Authors bands.

241

222

200

molecular

assignement

157

192

weight

INDIRECT

130

114

131 138

glycoproteins,

of glycoprotein

III

Ib IIb

MOL.

WT

(~10~)

(Iiib)

(IV)’

I’;

V VI VII

VIII

106 91 81 7C

63

49

108 93 75 70

6:

48

107 90

61

G = granule

nomenclature

IX 37 42

35

glycoprotein.

to "'I

labelled

lectins

AUTORADIOGRAM

--J251-(Lc H-8) .-w~~~~-(CON-A) .-.~12~ I-(WGA)

Fig.

1

: Densitoqram

of indirect autoradiogram of polyacrylamide gel electrophoresis of whole platelets stained with “‘1 LCH, lzsI Con-A, "'1

i --

n

‘*A L_*, +

WGA.

-

AutoradiJgraphy sf ;olvscrvlamide g21i ::ncaining rl*crroshor*s2d ?:atelet samples treated with “’ I _ laballed LCE-2 sho;_ed the presence of lb glycJprotern bands (see Table I and Fig. 11. The most int2nsel:; labelled bacds were the granule glycoprotein (GPG) ( a l- 33 called thracbin sensitive prstzior thrombospondin) followed by glycoprotein III (IIIa), Ia, IIS and Ib. One glycoprotein situated between IIb and III (IIIa) was not numbered as it did not fit in the CUrrent nomenclature. The ::32 of “‘I lsbellrd con A on polyacrylazide gels allowed the identificatlan of 10 bands (~22 Table 1 and Fig. 1). The most intensely labelled bands L-ere giycoprotei;l III (ILLa) followed by 11’ (IIIb) ani IIb. Treatment with ‘I51 labelled Iv’GA shswed the pr2sznce of 10 bands (~22 Table I and Fig. 1). The most iatens2ly lab2lled glycoprotein was Lb. “‘I labelled RCA-170 did not bind to an:; glyzoprotein bands. The presence of the sugar specific for “‘1 labelled 12ctin prel,*ent2d it binding to gl:;copratci:~ bands on sliced ~~1s.

OISCClSSION A comparison of the glycoprotein compon2nts of intact platelets with those of isolated membranes showed the presence of a glycoprotein known as granule glycoprotein (GPG) in intact platelets and not in isolated membranes (Iii). This glycoprot2in is located in plateiet granules and is released in a soluble and membrane bound form through the caniculnr system as a result of thrombin treatment hence it is also given the name of thrombin sensitive protein (TSP) or thronbospondin (lh,lj). Najerus and Erodie (1972) (16) using ” ‘I labelled LCH-E observed a twofold increase in the number of LCH-B bi:lding sites on the platelet surface after thrombin stimulation. This increase in “51-LCH-B binding sites was attributed to changes taking place at the platelet surface and specifically to the presence of granule membrane glycoprotein (GPG) fused with the canicular membranes (Ii), in the process of granule release from platelets. No evidence was however available to indicate if LCH-B could bind to GPG. The present experiment shows that GPG has a high affinity to LCH-B. It confirms therefore that if GPG membranes are present at the platelet surfacs they would increase the number of LCH-B binding sites to the same extent as they do on SDS-polyacrylamide whole platelet autoradiograns (see Fig. 1). Two major membrane glycoproteins IIb and III (IIIa) have been shown by to be greatly diminished or absent in Glanzmann thrombasvarious techniques thenic (G.T.) platelets (18,19). The results obtained here indicate that these two membrane glycoproteins are major can h binding sites (see Table I and Fig. I). Firkin et al. (1978) (20) usin? “‘1 labelled con A showed that this lectin could agglutinate formalin-fk
ol.li;

,*Vo . j.'fl ,

:io bindin: was obtained with isdinated ?.CX. This may be due t5 the labilii' 1 .xashing .end snecificallv t.2 i 3~~s ty <,f galactose residues in methanol-acet th2 very limited number of galactose terminal sites as determined by surface labelling techniques (12). olatelet g1y;3pr17Clemetson et al. (1977) found that a minor part $2, many _ teins bound to Abrns precatorius lectin which has a similar spxificity to Ricinus communis. However this work was carried out with isolated membranes and it is possiblr that th2 dis crepancy vith th2 resiilts obtained ‘here using who12 platelets sslubilized directlv can 52 explained bv ioss of sialic acid exposing the pznultisate galactose residues during preparation pf m2mbranes. residues. ji977) (‘11 0ur results art? similar to those obtained ‘by Clzetszn 2: al. and Xachman et al. (1975, 1978) (2?,23) using lectin colummnity rhronatography. However Nachman et al. (1977) obtained two main glycoprotein using WGA (a ‘nigh molecular weigntglycoprstein and t’he GPIa + 15 complex) whereas w2 obtain 5 high colecular,weight glycoprJteins and ths Sl;;z:opro:ein Ia + Ib complex. Our results in that respect are similar to Ciemetson et al. (1977) (11). However those differences may be due to the different detergents (SDS or DOC) used for solubilization. ?;achman et al. (1975) showed that the main glycoprotein binding to a con h affinity column was GP III. Considering the poorer resolution obtainabl 2 with the gel electrophoresis system employed this fits well with the results obtained her2. i comparison with the results obtained with affinity chromatography on Lens culinaris lcctin (21) of solubilized platelet membranes also showsd that with the exception of the granule glycoprotein (not present in membranes) th2 results obtained her2 corresponded well. This indicates that this method Fill in all probability be very useful as a method of screening leetins for their binding to platelet glycoproteins so that they can be used for affinity-chromatography. This hns the advantage of using little material, either platelet membran2s or lectins and gives results in a relatively short time. The “‘1 labelled lectin SDS-polyacrylamide identifying more specifically those platelet ous surface’labelling and PAS techniques.

gel techniques offers glycoproteins detected

a way of by vari-

ACKNOP!LEDGMENTS E.F. Luscher The authors wish to thank Prof. are grateful to Miss F. Martin for secretarial partially supported by grants from “La Xission Soehringer Laboratories.

for

We

de

valuable discussions. assistance. This work was la R2cherche” and from

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glycopro-

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3. JEhKXINS, C.S.P., PHILLIPS, D.R., Ci_EI!FTSi":, K.J., >!EYER, D.,L.kRiIE.U, M.J. and LUSCHER, E.F. Platelet membrane glysoproteins iaplizated in rist~cttin-induced aggregation. J. CG:. I,YcL~. 57, !I?, 197;. 4. ROBINSON, P.J., BULL, F.G., ANDERSON, B.U. and ROITT, 1.X. Direst autoradiographic visualisation in SDS-gels of Lectin-binding components of the human erythrocyte membrane. Febs rletfzt 58, 330, 19?5. 5. BURRIDGE, K. Changes in cellular glycoproteins after transfomaation : Identification of specific giycoproteins and antigens in sodium dodecyl sulfate gels. ptr0c.Nat. AC&~. 5c.i. USA, 73, LA57, 1976. 6. ThhiiR, N.J.A. and ANSTEE, D.J. A method for the direct demonstration of the lectin-binding components of the hliman erythrocyte membrane. siCC!zCfls J.

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I?. PHILLIPS, D.R. and POH XGIN, P. Platelet membrane defects in Glanzmann's ehrombasthenia . 1. ceil:. Ii!v;I.!t. ij@, 533, 1977. '0. FLRKI?;, B.G., HEADRIX, L. and GOH, A. An investizaciac of thrsnbasthenic platelets with selected lectins. f:~:cv:>. 2~5. I?, 337, 107%. 21. CLEMETSOS. K.J., PFUELLER, S.L., LUSCHER, E.F. and JEX:ii!S, C.S.P. isolntion of the membrane glycoproteins of human blood platelets b:; lectin affinity chromatography. Gici!:bi. Kic*;:i::li. 4~ t: iSL, L93, ! 977.

71. \XH?!AX

P\.L .( TARASOV, E., WERSLER, B.S. and FERRTS, 3. i.:heat :;et-m aggluti:in affinity chromatography of human platelet membrane giycoproteins. Tk'mb. Rc?A. 12, 91, 1978.