Isolation and immunologic characterization of the human platelet alloantigen, P1A1

0161-5890/79/06014353 102.00/O

Mokcuhr Immunology, Vol 16,pp. 353-360. OPergamon PressLid. 1979. Printedm Great Bmm.

ISOLATION AND IMMUNOLOGIC CHARACTERIZATION OF THE HUMAN PLATELET ALLOANTIGEN, Pl*‘* THOMAS Milwaukee

J. KUNICKI

and RICHARD

Blood Center, Inc.. the Department of Biology, Marquette of Medicine, Medical College of Wisconsin. Milwaukee,

H. ASTER University. and the Department WI 53233, U.S.A.

Abstract --Platelets from patients with Glanzmann’s thrombasthenia. a congential disorder of platelet function, are deficient in the membrane alloantigen. PI Al (Zw). present on platelets of nearly all normal subjects. Because thrombasthenic platelets are also deficient in two membrane glycoproteins, designated Ilb and IIIa. it has been suggested that one or both of these glycoproteins may carry the PIAl antigenic determinant. To address this question. the PIA] antigen was isolated by sequential lectin affinity chromatography of sodium deoxycholate extracts of platelet membranes, by indirect immunoprecipitation of Nonidet P40 extracts of lactoperoxidase-iodinated intact platelets. and by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane preparations. By each of the three separatory procedures, the Pl~l antigenic marker was shown to be associated with glycoprotein IIIa. Studies of the effects of proteolytic enzymes. disulfide-reactive agents and temperature on PIAl activity andoftheeffect ofvarioussugarson thePIA]-anti-PIAl reactionindicatethatthePIA1 antigen, situatedona portion of the molecule susceptible to cleavage in situ by trypsin. but not by chymotrypsin. bromelain. or papain. requires intact disulfide bonds for its full expression. and is probably determined by a polypeptide sequence in GPIIIa. Virtually all of the PI ~1 activity of platelets appears to be located on the external plasma membrane. PIA) appears to be the first alloantigen to be assigned to a specific platelet membrane constituent and the third peptide-determined alloantigen to be assigned to a human cell membrane protein.

1978). The Pl~l content of intact platelets. isolated plasma membranes and solubilized membrane protein preparations was assayed by determining their ability to compete for PIAlspecific antibody and thereby inhibit the lysis of S’Cr-labeled target platelets added subsequently (Aster et al.. 1973: Liebert & Aster. 1977). Using this method. ng quantities of specific antigens are detectable with a standard error of 5-102,. The specificity of each Pl~l antigen assay was confirmed by demonstrating failure of the material tested to inhibit antibodies reactive with other alloantigens. platelet autoantibodies and drug-dependent platelet-specific antibodies (Kunicki & Aster. 1978). The specificity of PIAl antigen in a given preparation was expressed as the reciprocal of the amount of protein, determined by the method of Lowry et al., (1951). required to give 504<, inhibition of SICr release in a standardized inhibition assay (Kunicki & Aster. 1978).

INTRODUCTION

Recently, we reported an apparent deletion of the platelet-specific alloantigen PlAl from platelets in Glanzmann’s thrombasthenia, a congenital disorder of platelet function (Kunicki & Aster, 1978). SDS polyacrylamide gel electrophoresis (SDS-PAGE) of thrombasthenic platlets confirmed previous reports by Nurden & Caen (1974) and Philips & Agin (1977~) that thrombasthenic platelets are deficient in membrane glycoproteins IIb and 1Ila.t This combination of molecular and antigenic deficiencies led us to speculate that the Pl~l antigen might be located on one or both of these glycoproteins. We now report further studies which indicate that the PlAl determinant is associated with glycoprotein IIla. MATERIALS AND METHODS

Indirect protein

Assay of PIA’ anrigen activity

Antisera specific for Pl~l antigen used in this study were identical

to those

previously

described

(Kunicki

& Aster.

* Supported by Grant HL-13629 of the National Heart, Lung and Blood Institute, and by fellowship from Marquette University, Milwaukee, WI. t Membrane glycoprotein terminology: the terminology originally proposed by Phillips & Agin (19776) is used with the modification for the GPI complex proposed by Nurden & Caen (1978), that is: GPIbcc (membrane bound: M, = 143,000); GPIIb a (M, = 132,000); GPIIIa (M, = ll5,OOO); GPIV (also called GPIIIb; A4, = 97,000). M, values listed here are those determined in fully reduced (10% 2mercaptoethanol) samples. Under non-reduced conditions, the M, values for GPIIIa and GPIIIb are 143,000, 100,000 and 97.000. respectively (Philips & Agin. 1977b). 353 M,MM 16/f&_*

immunoprecipi~arion

of

“S1-laheled

membrane

Platelets obtained by differential centrifugation of freshly drawn whole blood anticoagulated with EDTA (Aster & Enright, 1969) were washed 3 times in ice-cold 0.01 M Tris-HCI; 145 mM NaCl; I mM EDTA. pH 7.5 (THEN 7.5) at 4°C. and suspended in the same buffer. Washed platelets were iodinated. (Nachman LJ~al.. 1973; Phillips er al.. 1977h) and indirect immunoprecipitation was performed as described by Degos LJ~al.. (1977). Sodium IzsI (2mCi; New England Nuclear) was mixed with a platelet suspension containing 20 x IO” platelets in I I ml of THEN 7.5. IO 111of 0.05 M KI and 250 pg lactoperoxidase (Sigma) were added and the mixture was incubated at room temp for 30 min with occasional shaking. At 0, 7. I4 and 21 min. 50 ~1 of 10 mM H,O, was added to the suspension. After 30 min. 30 ml of icecold THEN 7.5 was added. the platelets were washed six times with THEN 7.5 at 4°C. and made to a final volume of 4.5 ml in the same buffer. A half ml of 0.5”,, (v/v) Nonidet P40

3.54

T~HOLl,L\S J. KIJNICKI

(NP40) waq added to the washed platelet suspension. After incubation at room temp for 30 min with constant stirrlng. the msoluhle material was pelleted at 120.000 g for 2 hr at room temp. The supernatant. containing 50-60”,, of the platelet radioactivity and 25-35”,, ofthe total platelet protein, served as the solubleextract of PI 41 antigen. Preparations not used immediately were stored at ~ 20 C. Aliquots of the soluble extract (250 /ig protein in 100 1’1) were incubated with 80 ltl(400 /~g) ofIgG purified from either PI \I-specific serum or normal serum by ammonium sulfate fractionation and DEAE-cellulose chromatography (Whatman DES’). Theconcentration oflgCi was determined by quantitative radial immunodiffusion. The purity of the IgG preparations was established by immunoelectrophoresls and SDS-polyacrylamide gel clcctrophorcsis. After I hr at room temp. excess rabbit anti-human IgG (Miles-Yeda) was added and the mixture was incubated for I hr at 37 C. followed by 4X hr at 4 C. The precipitate was washed 4 times, Preliminary indirect immunoprecipitation curves defined the ratios of “‘l-extract: human IgG and human IgG: rabbit anti-human IgG that resulted in maximum specific precipitation of “sI-laheled material. Signiticant amounts of I “1 wcrc not precipitated in the absence ofadded rabbit antiI&. The washed precipitate was soluhllired in 3.3”,, SDS (0. I j-0.30 ml). 5O-,~l aliquots were removed and subjected to SDS polyacrylamide gel electrophoresis. The gels were then stained with Coomassie Blue R as described by Fairbanks (jr (I/.. (1971). and dried in a Slab Gel Dryer (Bio Rad Model SE540) on tilter paper. Individual samples were cut into 2 mm-wide slices and the individual slices were assayed for ’ “1 in a gamma counter (Nuclear, Chicago.).

Electrophoresis was performed as previously descrihcd (Kunicki & Aster. 1978; Phillips & .4gin. 19776) in a commercial slab-gel apparatus (Bio Rad Model 220) by the method of Laemmli (1970). The resolving slab gel contained 7.5”,, acrylamide and 0. I”,, SDS and was covered with a I -cm stacking gel. Electrophoresis was layer of 3”,, acrylamide carried out at 25 V for I8 hr at which time the tracking dye had reached a point 0.5-l .O cm from the bottom of the gel. Completely reduced samples were incubated in a sample buffer containing IO”,, 2-mercaptoethanol at IOO’C for 3 min prior to electrophoresis. Glycoprotein hands were visualized in electrophoresed gels by staining with PAS according to the method of Fairbanks et ul., (1971). Stained gels were scanned at 550 nm (0.05 mm slit width) using a Gilford 222A spectrophotometer equipped with a gel scanner (Gilford Model 2520).

Plasma membrane-enriched preparations were made from intact wjashed platelets accordmg to the glycerol-lysis method of Barber and Jamieson (1970). Washed platelets from 50-100 outdated platelet concentrates (about 5 x IO” platelets) served as starting material. An average yield of membrane material of I mg protein per platelet concentrate was routinely obtained. Plasma membranes were soluhilized by addition of I”,, sodium deoxycholate (DOC). After ICI5 min at room temp. insoluble material w’as pelleted at 120.000 R for 2 hr. The clear supernatant. containing on the average 70”,, of the total protein and 93”,, of the total Pl~l activity. was used as a source of soluble PI*! antigen. Prepmrutwn o/ rhc Lens culinaris

Ircrin column

The lectin was isolated from common lentil beans (obtained at a local supermarket) according to the method of Howardc’rul.. (197l).Thespecificityofthepurilied lectin was tested by agglutination of blood group 0 red cells and inhibition of agglutination by a-methylmannoside. The purity of the isolated lectin was confirmed by SDSpolyacrylamide gel electrophoresis. The purified lectin was

and RIC‘H,4RL> H ASTER attached to cyanogcn bromide-a~tl\ared Sepharvw 41~ (March 01 rrl.. 1974) as dexrlhed by PhIllip\ and :\gtn (I 9770). Coupling wa\ dctermlned to he 70&X0”,, complctc b> this method. based on O.lI1,,, of the unbound materlai.

The Scpharose heads con~amlng hound L cI~/~HN~I\ Icctln were washed extensicely with 0.05 .v Trls. pH 7.6. suspended in I”,, DOC. poured into a plastic column (Phxmacla K9 30) and washed succeasivelq with I00 ml of I”,, DOC’. IO0 ml of 2.5”,, r-methylmannosidc m 1”,, DO<‘. and tinally with I”,, DOC. The total volume of packed bead\ waq Ii ml. 5 ml ot I”,, DOC- added to the Con A column contaimng I5 ml packed heads and were allowed to flow through the column for 30 min. The columnwaswashedwith70mlofl”,, DOCand2,Omlfractionc werecollected at a flow rateof0.5 ml,min until theO.D.,,,, ot collected samples reached background. The column wat then perfused with 50 ml of S”,, 7-methylmannosidc in I”,, DOC to elute material which had hound to theCon A. .4liquotsofeach fraction were removed for PI,\’ antigen as\aq as described above. The ‘flow-through fractions (Con A I) and ‘adherent’ fractions (Con A II) were separately pooled and processed a~ described above.

Prelimmaryexperimenta were undertaken to determlne the extent to which PI 41activity wa\ affected by various enzyme, (Table I ). The enzymes utilized in these studies were obtained commercially (Sigma). DPCC-trypsin >(I). (Type chymotrypsin (Type I-S). papain (2 x c)\tallircd. lyophihzed) and bromelam (practical. grade II). W&led platelets were suspended al a tinal concentration of IO”’ ml in THEN 7.5. for trypsin or chymotrypsin dIgestIon: 111 THEN X.1 containing 5 m.M cysteme for papain digr\tion. and In PBS 7.4 containing 5 m,M cystelne. for hromelain digestion. Enzyme was added at the final concentrations Indicated in Table I and the reaction, were allowed to proceed at 37 C with occasional agitation Tryp$in hydrolysis was terminated by addition of 2 mg soybean trypsm inhibitor (Sigma) mg trypsln: chymotrypsin hydrolysis by addition of 0.5 mM phenyl methyl sulfonyl fluoride: and bromelain or papam hydrolysis by direct transfer to an ice bath. Enzyme treated platelets were wathed twice with THEN 7.5 and tested for PI&l actlvlty. Isolated plasma membranes at a tinal concentration of 1 mg protein/ml were treated with the above enzymes under the same conditions. Enzyme was added at the final conccntratlons indicated in Table I i-ollo~~ng treatment.

Characterization

of the Human

Table I. Effect of proteolysis on PIAl antigen activity. Washed platelets or platelet plasma membranes isolated by the glycerol lysis technique were incubated at 37 C with the following enzymes under the conditions described in the Materials and Methods section. The effect of proteolysis on the ability of these preparations to absorb antibody specific for PIAI antigen was then determined Proteolysis

Enzyme Trypsin

“,,PI,” activity

I

0.1

0. I

Papain Bromelain

0.1 0.6

Enzyme

platelets

Doseczsb Time (min)

Chymotrypsin

Proteolysis

of intact

__-__

of isolated

3 5 10 30

100+ 75 60 50 50

I 30

lOOf loo+

10

lOOf

IO

loo+

platelet

plasma membranes ‘j:, Residual ‘>,,P1~1 Dose(h) Time (min) protein activity

Trypsin

I.0 3.0

I5 I5

70 50

70 5

Chymotrypsin

0.01 0.10

15 15

65 56

lOOf lOO+

Papain

0.20 0.30 0.40

I5 I5 I5

59 61 55

lOO+ 100+ IOOf

Bromelain

I.0

I5

55

lOO+

‘olrng enzyme protein (h)mg enzyme protein

per IO” platelets. per mg membrane

membrane material was washed twiceat then tested for Pi~l activity.

protein.

12O.OOOg for 90 min.

Prepuruiive SDS-PAGE Isolated platelet membranes were solubilized and electrophoresed as described above. One sample of electrophoresed proteins was cut from the slab gel and stained with Coomassie Blue R, using an abbreviated version of the method described by Fairbanks cf crl.. (1971). to locate the positions of individual proteins including GPIIb and GPlIla. After correction for expansion during staining, the areas containing protein were cut from the unstained portion of the gel. Gel sections were added to 1-2 ml of PBS 7.4 in test tubes. ground into fine particles. and incubated for 2 hr at room temp. Gel particles were pelleted by centrifugation for 30 min at 1000 g and supernatant material was aspirated. Extracts obtained from individual gel sections were assayed for PI,%1 activity. RESULTS

Effect of proteolysis

on PIAl activity

Treatment of intact platelets or isolated membranes with chymotrypsin, bromelain or papain, reduced total membrane protein by up to SOo/, but enhanced the activity of the PlAl antigen (Table 1). After treatment with trypsin, however, up to 95% of P~AI activity was lost. The decrease in PIAI activity induced

Platelet

Alloantigen.

355

PI,\’

by proteolysis of intact platelets with trypsin paralleled the decrease in density of glycoprotein bands corresponding to Ilbcc and IIIa on PAGE noted previously by Okumura and Jamieson (1976). A similar but slower decrease in IIb and IlIa was chymotrypsin. treatment with observed after bromelain and papain. but no effect on PIAl activity was observed. Partial purification

of’ PIA’ anti,qw

I Tab/c 2 J

Platelet membranes prepared from pooled outdated platelet concentrates provided a convenient source of material for the isolation of PI*‘antigen. No significant loss or alteration of PI*) antigen was observed when platelets were stored at room temperature for 72 hr. The membrane fraction obtained by the glycerol-lysis method contained about SO,<, of the total protein of the intact platelets. About SO-9OU,{,of total platelet PIAl was recovered in the membrane preparations and its specific activity (ratio of antigen: protein) increased by IO-fold relative to intact platelets. This suggests that all or nearly all of the antigen is located on the external surface of the platelet plasma membrane. Detergent soluhili~ation of nwnhurw protein. A soluble suspension of Pl~l antigen could be prepared from isolated membranes using either DOC or NP40. Because DOC interfered with the S’Cr lysis assay to a lesser extent than NP40, the former detergent was used for affinity to prepare soluble Pl~l antigen chromatography. Using DOC at a final concentration of I:,(,. 70’::, of the total membrane protein and 93’!,, of the total PIAI antigen activity were obtained in soluble form. The specific activity of the soluble PI Al material increased I .33-fold relative to the isolated membrane fraction. Aj’nir~ chromatography. The DOC extract of plateletplasmamembrdneswassuccessivelychromatographed on L. culinaris lectin and Con A affinity columns in three independent experiments which yielded essentially identical results. Electrophoretic patterns are shown in Fig. 1. Recoveries of protein and PIAI antigen are shown in Figs. 2 and 3. The preliminary separation of membrane glycoproteins achieved with the L. culinaris Iectin column was identical to that previously described by Phillips and Agin ( 1977a). The -flow-through’ (Lens I) fraction was shown by electrophoresis to contain predominantly glycoproteins IIIa and IV (also called IIIb) and small but detectable amounts of Membrane

preparation.

Table 2. Purification

of PIA’ antigen _____

pi% Intact plrlelel\ Plasma membrane\ I” I, DOC-wluble ex,rac, Len\ I Con A I,

“‘Reciprocal SO:/, inhibition target platelets ‘*‘Total

‘ZK:;:,~

IX9 310 I5 I06

I

30 31 3 41 7

x.4 IX

41 I 166.7

from human

platelets

PIAl Unltlfh~

“,,yleld of PI,%’ ‘i.ntlgm

Purllicallon

56U 473 442

(100) 83 7x

(11 III.5 I4

350 300

62 53

14 56

of the amount of protein @g) required to give of anti-PI Al induced lysis of 51Cr-ldbeled

in a standardized inhibition assay (1). protein (mg) multiplied by specific activity.

“‘The values listed purification sequence, sequences.

here are derived from a single but are typical of several such

I tIOM,\S

.I. KUlcl(‘KI

and KIC‘HARD

H. AST‘Irll

2 is1.2

-

It $j

x

1.0 0.8

3

0.6

i=

0.4

$ 0.2 0 5

IO

I5

20

25

FRACTION

35

40

45

Fig. 2. Affinity chromatography of the wpernatant from sodium dcoxycholate-~oluhilized plarelct mcmhranes 01 ~turmal platclcts. PI:itelet nlernhr~illc~ wrc lsolatcd hy the glycerol-lqsis technique and x~lubilixd m I”,, sodium deoxycholatc (DOC). The wluhilized mcmhrane material was chromatographed on a column containing I. c~/rrrtrr/\ lectin coupled to Sepharose 48. I.?,, r-metll]vllnannosldc (1 MM) in I”,, DOC was added ah indlcatcd by the ax-row. The 0) of each fraction \+a determined and C).I).,.ii, (0 alrquots of each fraction acre ;rssayed for PI $1 antigen activity (mdicated (“y the shudcd area). -Lens I‘dcsignates ihc material not contatmng receptor\ for L. w/rw~~ lectin: ‘Len< Il‘dc~;lgnates the materwl speafically bound by the &tin and clutcd wth rMM

2

1

30 NUMBER

only the l’ractton containing fffa exhibited significant Pl~l activity (Fig. 3). The cllronlatogr~~phic separation of the Con A column produced a further 4litfd purification of Pl Al antigen. The overall purification from starting material (intact platelets) to the Con A If fraction was %-fold (Table 2).

Pofyacryfamide gel efectrophoresi~ under nonreduced conditions of ~P40-soluble extracts of ‘I ‘flabeled platelets (Fig. 4) rcvealcd three major radioactive peaks, two corresponding to mol. wt of 140,000 and 100.000, and a third presenting high tnol. gfycoproteins fbx and ffbx (Fig. 1). The ‘adherent’ fraction (Lens I I) contained predominantly gfycoproteins fhx and flbr and Icsscr ain~~unts of Iffa and IV (flib). Thed~stributi~~il oi‘Pf*\l antigen activity among the fractions coflcctcd (Fig. 2) suggested that the PIA! antigen is associated with glycoprotein flfa. No increase in the specific actlbity ofPf,jl in the flowthrough (Lens I) fraction was detected, since gfycoprotein fffa was disirihutcd bctwcen the Lens I and II fractions. tn ~rop~~l-tion to the total protein content of each fraction. To separate residual amounts of glycoprotcin Ifb and flla, the Lens I fraction was rcchromatographed on a Con

A affinity

column

(Fig.

3). The

adherent

only 2f”,, of the total protein, but about 90”,, ofthe PIhI activity of the total sample. ~lectrophoretic analysis (Fig. I ) of both the flowthrough’ (Con A I) and adherent (Con A II) and of the total sample (Lens 11 fractions, demonstrated that the Con A II fraction contained detectable amounts ofglycoproteins Iffa and IV (fflb) only, while gfycoproteins IIba and fbr appeared in the ‘flow-through’ fraction (Con A I). After essentially complete separation of gfycoproteins ffh and IIfa, fraction

(Con

A II)

contained

? (;

0.6

+CmA

I

t-C0nAiI-i

----I

R

0 5

IO

15

20

FRACTION

25

30

35

40

45

NUMBER

Fig. 3. Affinity chr~~rnuto~r~iphy on conc~tnt~valin A of the DOC-soluhie material recovered m the .tlo~~-tllrou~h’ fraction from the L.. r~ufiw~l.s lectin coiumn (Lens II. 5”,, xMM ,n I”,, DOC was added as indicated by the arroa The (O ~~ --0) of each fraction was detcrmincd and o.n.,,, aliyuots were assayed for PI ~1 antigen activity (shaded arca). The ‘flow-through’ fraction (Con A 1) contained material not specifically bound by Con A; specificially bound muterxat wa\ eluated with 5”,, xMM (Con A II).

Characterization

of the Human

Platelet

Alloantigcn,

357

PI\’

IU a,b

o/- , 5

1

/

/

,

,

/

,

IO

15

20

25

30

35

40

Al ,a,

ld,

$6 t$

WCE NUMBER

Mrlrlo~l

0

,

, 5

, IO

(

,

,

,

)

,

I5

20

25

30

35

40

SLICE NUMBER

Fig. 4. Sodium dodecyl sulf:~te-polyacrylamide gel electroextract of ‘**l-labeled phoresis of an NP40-soluble platelets. Intact platelets were subjected to iactoperoxidasecatalyzed iodination and then solubilized in 0.05’1,, (v/v) NP40. Following high speed centrifugation. aliquots of supernatant containing 100 i(g of solubilized protein were mixed with buffer containing SDS and electrophoresed on 7.5”,, polyacrylamide slab gels under nonreduced conditions. Gels were stained with Coomassie Blue R, dried on filter pqer. and cut into 2 mm-wide slices. The distribution of iodine label in the gel slices was determined. Identi~cation of membrane glycoprotein peaks is based on relative electrophoretic mobility.

material which barely entered the gel. Two minor peaks were apparent, with mol. wt of 66,000 and 55.000. Samples fully reduced in 2merc~~ptoethanol (Fig. 5) exhibited five major peaks corresponding to mol. wt of 152,000, 132,000. 120,000, 59,000 and 49,000. Five minor peaks were also seen. Identification of the ‘Z51-labeled peaks shown in Fig. 4 and 5 was based on ejaculated mol. wt for each peak and on comparisons with patterns of lZSI-labeled platelet membrane glycoproteins previously reported (Phillips & Agin, 1977h). The molecule bearing the Pl~l antigenic determinant was identified by incubating the solubilized protein with anti-PIAl antibody and then precipitating with anti-IgG as described in Methods.

wt

III0

Fig. 6. Sodium dodecyl sulfate-poiyacrylamide gel electrophoresis of precipitates formed by addition of 400 i&g of IgG obtained from serum specific for PInl antigen (e----e) or normal serum (0~.0) to an ~P40-soluble extract of iodinated platelets, followed by 1600 ittg of rabbit anti-human IgG. The washed precipitate was solubilized in 3.3’;,, SDS, and aliquots containing 400 1~8 protein were electrophoresed on a 7.5”,, polyacrylamide slab gel under i~onredu~ed conditions. Following electrophoresis. gels were stained with Coomassie Blue R, dried on filter paper, and cut into 2 mm-wide slices. The distribution of iodine label in the gel slices was then determined. The radioactivity peak in the center of the gel has an electrophoretic mobiiit~

eorrespondin~ to 8 molecular weight of IOO~OOO + 5000. Electrophoresis of precipitated material under nonreduced conditions (Fig. 6) revealed two radioactive peaks. one corresponding to a molecule of 100,000 mol. wt and the second representing high mol. wt material that barely entered the gel. The mol. wt of 100,000 is identical to that of non-reduced giycoprotein lila (Phillips & Agin, 1977b33.The high mol. wt material probably represents either nonspecifically bound radioactivity (since an equivalent peak of lesser density is present in material precipitated by normal IgG) and/or non-dissociated complexesofPlAl-anti-PIAl -rabbit anti-human IgG. Electrophoresis of precipitated material under fullyreduced conditions (Fig. 7) revealed a single radioactive peak of apparent mol. wt 120,000,

SLICE

Fig. 5. Sodium dodecyl SulFdte-polyacrylamide gel electrophoresis of an NP40-soluble extract of 1251-labeled platelets. Legend as in Fig. 4, except electrophoresis was performed under reduced conditions. in the presence of Zmercaptoethanol. Note decreased mobility of GPIlIa.

NUMBER

Fig. 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the same precipitates described in Fig. 6, except under reduced conditions. The radioactivity peak has an electrophoretic mobility to a mol. wt of 120.000~5000.

THOMAS

358

J. KLJNIC’KI

precisely &bat estimated for ~lycoprotein IIIa under reduced conditons (Kunicki & Aster. 1978; Phillips & Agin, 1977h). No peak was detected under nonreduced conditions with immunoprecipitatesobtained using normal human IgG, or serum specitic for the HLA-A2 membrane antigen (Figs. 6 and 7). The data shown in Figs. 4-7 are from one of two independent experiments which yielded essentially identical results.

Assay of Pl~i activity in extracts obtained from gel sections following preparative SDS-PAGE under nonreduced conditions. showed that only extracts containing protein of M, = 95,000 + lO.OO~exhibit~d PIjji activity. Under non-reduced conditions, this protein material represents both GPIlla and GPIIIb. In subsequent experiments, sections containing GPlllaand GPlIlb were removed. incubated in buffer at 25“C. 37’ C or containing IO”,, 2mercaptoethanol 56 C for 30 min and were again electrophoresed. Following reduction in this manner. GPlIIa and GPllIb were readily separated on SDS-PAGE. used for regardless of temperature However, reduction, none of the extracts prepared from these gels exhibited PI*’ activity. Treatment of isolated platelet membrane with 2 IO rnbl 2-amino ethyl isotlliourotlium bromide IAET), a potent disulfide reactive agent (Horvath c~fu/., 1972). resulted in total loss of Pl~i but did not affect HLA antigens in the same preparations. The sensitivity of Pl~i antigen to treatment of 2-ME and AET precluded the use of preparative SDS-PAGE as a method for further purification of the PIni antigen, since GPIlIa and GPliIb can only be separated under reduced conditions.

The PIti activity of isolated platelet membrane suspensions stored at 4 C in the presence of O.l”,, NaN, or frozen at -20 C. was determined over a period of SO days. At 4 t’, a linear decrease in Pl~i activity was observed such that at 30d. 60”$ of the activity remained and at 50d. 41”,, was present. At -2O’C, membranes stored for 50d retained 90”,, or more of their initial activity. ~~~ll~br~ines heated at 56 c‘ for 30 min, incubated at pH 3 or IO fi)~- 30 min, or lyophilized and stored for seven months at -20 C. retained full capacity to absorb antibody specific for PIAl antigen. PI,lt activity was completely lost when membranes were heated at 100 C for 3 min. No inhibition of complement-dependent lysis of i ‘Cr-labeled target platelets was observed when PlAi-specific sera were incubated at a limiting dilution with any of the following sugars (Sigma), at a final concentration of 1. 10 or 100 mM: u-glucose, t,-galactose. t,-mannose. L-fucose. u-glucuronic acid, r-methyl-n-mannose. x-methyl-n-glucose. ~~-m~nn[~saminc. .V-acetyl-~-~iucosaminc, V-acetylt,-galactosamine or TV-acetyl-lleu~drninic acid. DISCL-XION . .

The allelic platelet alloantigens, Zwa, and Zwb. were first identified by van Loghem er al., (1959) and Van

and RICHARD

H. ASTER

der Weerdt ct ui., ( 1963). respectively. using a platelet agglutination assay. Shulman (‘1 111. (1961). using complement fixation and serum obtained frotn two patients whodeveloped thrombocytopenia after blood transfusion, identified a platelet alloantigen present in 9X”,, of the general population which they called Pf-zr. Pl~l proved to be identical to Zw:l. The PI.41 (Zwd) antigen has been implicated in the pathogenesis of the syndrome of post-transfusion purpura (PTP) (Si!~lmann ct A., 1961) and appears to be the alloantigen that most often provokes isoimmune neonatal thrombocytopenia (Aster. 1977: Shulmann CI UI’.. 1964) Our observations on the effect of physical and chemical treatment of intact platelets and platelet membranes on the activity of the PInI antigen contirm and extend previous tindings by Shulman c’rul.. ( I961 ). Fractionation of platelets by the glycerol-fysis technique showed the antigen to be located almost exclusively on the plasma membrane; platelets heated at 56 ‘C for 30 min. incubated at pH 3 or 10 at 20°C for 30 min, frozen. or lyophilized, retained full capacity to absorb antibody specific for PlAi antigen: the antigen activity was not inhibited by any of I I sugars commonly found in glycoprotcins; and Pl~~i activity was totally lost when platelets were heated at 100 C for IO min. In contrast to the report of Shulman <‘Icri. (1961). we found the Pfhi antigen to be sensitive to proteofysis by trypsin. The susceptibiiity~~fthe~lntigen to destruction by heating at I00 C. to trypsin hydrolysis, and to treatment with reducing reagents, the failure of sugars to inhibit its combination with specific antibody. and the gene-dose dependency of its expression on platelets (Shufman ct trl.. 1961).suggest that PI Ai activity is dependent on theconformation of a polypeptide normally maintained by an intact disulfide bond(s). Philtips and hgin (iY77h) have shown that GPIlla contains at least two intrachain disuffide moieties. The differential effect of proteolytic enzymes on PI Al (Table I) suggests that. in the plasma membrane, the determinant is positioned on itscarrier molecule at a point proximal to the site(s) cleaved by chymotrypsin, papain, and bromelain but distal to or at a site of trypsin cleavage. When glycoproteins IIb and IIIa were completely separated from each other by chromatography on L,. ~rc/irzuris and concanavalin A (Figs. i-i), PlAi activity was recovered almost intact and only in the fraction containing glycoprotcin Illa. By indirect immunoprecipitatiou of “‘I-labeled NP40-sofubilized platelet membrane gtycoproteins (Figs. 4-7) it was found that precipitates obtained with antibody specific for PI 41 contained “‘I-labeled material with electrophoretic mobility corresponding to an apparent mot. wt of 100,000 under non-reduced conditions and 120,000 under reduced conditions. vatues identical to those of glyc~~protein 11Ia. Finally, PlAi activity was recovered only in the GPIIla band eluted directly from polyacrylamide gels. The association of PlAi with GPIIla observed by each of the three independent separatory procedures argues strongly that GPIII is the carrier of the Pti determinant. The well-characterized human alloantigens of the ABO. P. Lewis and Ii systems appear to be almost exclusively polysdcchar~de in nature (Giblett, 1977; Issit, 1978). However, only a small number of

Characterization

of the Human Platelet Alio~ntigen. Pl~l

alloantigen systems determined by peptide sequences have been assigned to specific integrai membrane proteins. Antigens of the HLA complex are known to reside on a set of proteins ranging in mol. wt from 34,000 to 55,000 (Cresswell et al., 1973; Humphreys et al., 1976; Sanderson & Batchelor, 1968). These markers are found on lymphocytes and possibly all other cells except erythrocytes on which they are expressed weakly, if at all. The erythrocyte alloantigens of the MNSsU system are now thought to be determined, at least partially, by peptide sequences Iocated on the red cell membrane proteins glycophorin A and B (Dahr et al., 1977; Lisowska & Kordowisz. 1977; Wasniowska et at., 1977). The Chido and Rodgers red cell markers appear to be determined, in part, by peptide sequences of the fourth component of complement (CJ) which is passively absorbed by red cells (O’Neill et al.. 1978) Our findings appear to permit the first assignment of an alloantigen to a platelet-specific membrane protein. If, as strongly suggested by the data presented here, PI A 1proves to be determined by a peptide sequence, its association with GPlIIa represents the third assignment of a protein-determined human alloantigen to a cell membrane protein. The significance of glycoprotein IlIa to platelet function is under investigation in a number of laboratories. The studies of Phillips (1972) and Okumura and Jamieson (I 976) suggest that the -major surface glycoprotein’ described by Nachman et al. (1973) and shown to be reactive with concanavalin A is, in fact. glycoprotein Ma. The role ofglycoprotein IIIa in platelet function remains to be elucidated but the severe dysfunction of platelets that lack IIia (Kunicki & Aster, 1978; Nurden & Caen, 1974; Phillips & Agin, 1977~) makes it likely that GPIIIa is critical to platelet-platelet adhesion and, possibly, to other aspects of platelet function. Identification of glycoprotein IIIa as the carrier of the antigenic determinant, PlAl, should facilitate further studies of the structure-function relationships of the human platelet membrane.

AckPlo~~ledge~nmrts-We wish to thank Dr. Jeffrey Winkelhake (Department of Microbiology, Medical College of Wisconsin) for his invaluable suggestions and criticism during the course of this work and Mrs. Ada Thundercloud for her assistance in preparing the manuscript.

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