Characterization of an antibody raised against reduced glycoprotein IIIa of human platelets

THROMBOSIS RESEARCH 62; 43-%I,1991 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.

CI-IARACTERIZATION OF AN ANTIBODY RAISED AGAINST REDUCED GLYCOPROTEIN IIIa OF BUMAN PLATELETS

Annette J. Eckardt, Jacquelynn J. Cook and Stefan Niewiarowski* Department of Physiology, Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140. (Received

26.9.1990;

accepted

in revised form 2.1 .1991 by Editor E.I.B. Peerschke)

ABSTRACT against reduced and antibody polyclonal A vinylpyridylethylated human glycoprotein IIIa was raised in rabbits. Its reactivity with reduced GPIIIa was about 500 times higher than that of the antibody against native GPIIIa. The lowest amounts of purified reduced and native GPIIIa recognized by the antibody against reduced GPIIIa were 25 and 400 ng, respectively. The antibody did not recognize native GPIIIa (about l-2pg) in platelet extracts and chymotryptic degradation products of GPIIIa. It inhibited ADP-induced platelet aggregation but it did not inhibit fibrinogen binding to ADP-stimulated platelets. Our experiments suggest that the antigenicity of GPIIIa (Bs integrin) depends on the conformation of the molecule determined by numerous S-S bridges between cysteine residues.

INTRODUCTION It is well established that platelet fibrinogen receptors are associated with glycoprotein IIb/IIIa (GPIIb/IIIa) on the platelet surface (I)Platelet glycoprotein IIIa (GPIIIa) represents the subfamily of integrin receptors containing the 8, subunit, known as cytoadhesins, which include GPIIb/IIIa and the vitronectin receptor (2). GPIIIa consists of 762 amino acids and contains 56 cysteine residues, all of which appear to be disulfide iinked (3,4). Since these bridges cause native GPIIIa to be a highly folded molecule, it would be anticipated that different Key Words: glycoprotein IIIa, platelet aggregation, fibrinogen receptors *To whom all correspondance should be addressed. 43

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epitopes become available on the surface of this protein when it is in the reduced form. Over the past several years, polyclonal antibodies raised native or reduced GPIIIa have been used for against immunoprecipitation of the I3 integrin subunit synthesized by cultured cells and the recombinant GPIIb/IIIa complex (5,6,4). Differences between antibodies raised against native and reduced GPIIIa have been observed by a number of investigators. Bray et al. (5) reported that antiserum to nonreduced GPIIIa reacted poorly with the reduced protein and antiserum to reduced GPIIIa reacted poorly with the nonreduced protein. On the other hand, an antibody to reduced GPIIIa, in contrast to an antibody against nonreduced GPIIIa, precipitated a 35S translation product of a native GPIIIa precursor (5). In addition, Coller et al. (7) observed that an antibody raised against native GPIIIa which had been digested with pepsin reacted with native GPIIIa but it did not react with reduced GPIIIa. The purpose of this report is to characterize a polyclonal antibody which we raised in rabbits against reduced and vinylpyridylethylated human GPIIIa. This antibody was tested on GPIIIa and its chymotryptic degradation products (8,9). The developed antibody may represent an useful tool to study relationship between function and secondary structure of GPIIIa.

Reagents. Chymotrypsin, Concanavalin A-Sepharose, Protein ASepharose, methylmannoside and vinylpyridine were all purchased from Sigma Chemical Company (St. Louis, MO). Human fibrinogen was purchased from Kabi (Stockholm, Sweden). Collection of Blood. Blood was obtained from healthy individuals with the approval of the Institutional Human Experimentation Committee at Temple University School of Medicine, Philadelphia, PA. Human Washed Platelets. Platelets from freshly collected blood in the anticoagulant acid-citrate dextrose were washed in the presence of apyrase and heparin by the method of Mustard et al. (IO). Preparations of chymotrypsin-treated platelets were obtained as For preparative purposes, we used described previously (8). outdated platelet concentrates supplied by the American Red Cross, Philadelphia, PA. These platelet suspensions were washed with a solution containing 9 parts of 0.15 M NaCl and 1 part of 3.8% trisodium citrate (pH 6.5) and then with Tyrode's solution (pH 7.35). Platelet Count. The washed platelets were counted electronically (Coulter Channelyzer, Coulter Electronics, Hialeah, FL). Platelet Aggregation. Platelet aggregation was studied using a Payton aggregometer (Scarborough, Ontario, Canada). The effect of various antibodies on intact platelet aggregation was tested by the addition of 50 ~1 of different dilutions of the antibodies, 20 ~1 of fibrinogen (final concentration - 400/.&g/0.5 ml) and 10 ~1 of AD5 (60 j&M) to 420 ~1 of platelet suspension containing 3-4 X 10

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platelets/ml. Aggregation of chymotrypsin-treated platelets in the presence of our antibodies was examined the same way but did not require the addition of ADP to the platelet suspension. Antibody fragments were incubated under stirring conditions with platelet suspensions for 1 minute at 37OC prior to any other additions. Comparisons were made between the effects of the various antibodies by measuring the initial slope of aggregation as demonstrated by changes in light transmittance. All values were expressed as percent of control aggregation. Human fibrinogen was radiolabeled ~~~~i'is3~ ~~~~~~~ ~~~~~ Heights IL) utilizing Iodobeads (Pierce, Rockford Ill). Free iodine 'and labeled protein were separated by passage over a G-25 fine Sephadex chromatography column (Pharmacia, Piscataway, NJ). Specific radioactivit&of fibrinogen was about 1000 cpm per pg. Measurements of Ifibrinogen binding to platelets were studied according to procedures previously established in our laboratory (11) with minoi modifications. Aliguots of 400 ~1 of platelets (5 x 10 platelets/ml) were incubated with the selected antibody (final at room concentration 50 pg) and ADP (6OpM) for 5 miy@es I-fibrinogen temperature under nonstirring conditions. Finally, (100 pg) was added and allowed to incubate for an additional 5 Free and platelet-associated labeled ligand were minutes. determined following centrifugation through silicone oil and counting of the radioactivity i&athe platelet pellet and the I-fibrinogen to platelets was supernatant. Control binding of measured similarly, however, normal rabbit serum (IgG) was substituted for the antibodies being characterized. Finally, nonspecific binding was assessed in the absence of ADP stimulation an3 antibody. Binding was expressed as picomoles bound per 10 platelets and values were compared to control binding using the student's t test. Preparation of Native and Reduced GPIIIa. Purified GPIIIa was isolated by a modified method of Fitzgerald et al. (12). Platelet lysate from outdated platelet pellets suspended in 1% Triton X100 was adsorbed on Concanavalin A-Sepharose. Material containing GPIIb/GPIIIa was eluted with 100 mM methyl mannoside. A portion of the eluate was reduced and vinylpyridylethylated according to Huang et al. (13). Purified native and reduced GPIIIa were separated by preparative SDS-polyacrylamidegel electrophoresis and isolated by electroelution (14). SDS-PAGE and Western Blotting. SDS-polyacrylamide electrophoresis was performed by the method of Laemmli (15) using a 5% polyacrylamide stacking gel and either a 7.5% or 10% polyacrylamide running gel. Samples were dissolved in 2% SDS sample buffer. Molecular weight standards of myosin (200 kDa), B-galactosidase (116 kDa), phosphorylase B (97 kDa), bovine serum albumin (66 kDa) and ovalbumin (45 kDa) were run on each gel. Gels were stained with Coomassie brilliant blue reagent or subsequently transferred to nitrocellulose paper and immunologically characterized by the method of Towbin et al. (16). Polyclonal Antisera. Anti-GPIIIa and anti-reduced GPIIIa antisera were raised in rabbits injected with 75 pg of antigen emulsified

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of each in complete Freund's adjuvant. Booster injections of 40 l.(g antigen, suspended in incomplete Freund's adjuvant, were administered every 10 days until a high titer of antibody was detected by enzyme linked immunoassay. p@oclonal Antibody. GlO, which blocks platelet aggregation and I-fibrinogen binding to platelets by interacting with GPIIIa was kindly supplied by Dr. E. Kornecki (17). Preparation of IgG and F(ab')2. Antisera was applied to a Protein A-Sepharose column and IgG was eluted with glycine HCl buffer. The eluate was neutralized immediately to pH 7 with 0.2 M Tris-OH. A portion of the IgG was used to generate F(ab') (Immunopure F(ab') Preparation Kit; Pierce, Rockford, IL). IgE was incubated witi: immobilized pepsin. The F(ab')2 , Fc and undigested IgG were separated using an Immobilized Protein A column. ELISA. Enzyme linked immunoassay was performed according to a modified method of Engvall et al. (18). Briefly, an ELISA plate was coated with native or reduced GPIIIa overnight at 4OC. After blocking active sites on the plate with bovine serum albumin (BSA), antibody was incubated with antigen for 2 hours at 37OC. The plate was washed several times and the phosphatase-labelled second antibody was incubated for 2 hours at 37OC. The plate was again washed and then developed with phosphatase substrate. Absorbance was read at 405 nm. Since the antibodies being tested did not attach to BSA, it was accepted that the enzymatic activity of alkaline phosphatase correlated with the amount of antibody bound to the GPIIIa antigen on the ELISA plate.

RESULTS Figure 1 shows the pattern of migration in SDSpolyacrylamide gel electrophoresis of reduced and nonreduced GPIIIa and immunoblotting of these proteins with two polyclonal antibodies. It can be seen that the preparation of reduced GPIIIa stained with Coomassie blue showed one band (Fig la, lane 1) and that of nonreduced GPIIIa showed one major band (migrating with apparent Mr of 90 kDa) and one minor band with a mobility close to that of reduced GPIIIa (Fig la, lane 2). Antibody against reduced GPIIIa reacted very strongly with 400 ng of reduced GPIIIa and it recognized as little as 25 ng of reduced GPIIIa (Fig lb, The same antibody recognized 2,000 ng of lanes 1 and 2). nonreduced GPIIIa in the immunoblots and it showed two faint bands of this protein migrating with an apparent molecular weight of 93 kDa and 116 kDa (Fig lb, lane 3). These two bands had similar color intensity in immunoblots although the band with the slower mobility (116 kDa) corresponded to the minor band detectable by Coomassie blue staining (Fig la,lane 2). The lowest amount of native GPIIIa recognized by the antibody against reduced GPIIIa was 400 ng. By contrast, an antibody raised against nonreduced GPIIIa gave a high intensity band with 400 ng of native GPIIIa (Fig lb, lane 4). The lowest amounts of native and reduced GPIIIa detected by the antibody against nonreduced GPIIIa were 25 ng and 100 ng, respectively (data not shown).

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2

1

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3

4

FIG. 1 A) 7.5% SDS-polyacrylamide gel electrophoresis. Lane 1: 10 1.19 reduced GPIIIa, Lane 2: 10 pg nonreduced GPIIIa. B) Immunoblots of reduced and nonreduced GPIIIa. (transferred from 10% SDS-polyacrylamide gels). Lane 1: 200 ng reduced GPIIIa, Lane 2: 25 ng reduced GPIIIa, Lane 3: 2 pg nonreduced GPIIIa, Lane 4: 400 ng nonreduced GPIIIa. Lanes l-3 were stained with antibody against reduced GPIIIa diluted 1:200. Lane 4 was stained with anti nonreduced GPIIIa diluted 1:200.

In agreement with previously published data (8,9,19), we observed that the incubation of human washed platelets with chymotrypsin resulted in degradation of GPIIIa to the 66 kDa component which was detectable by the antibody against nonreduced GPIIIa (Fig 2, lanes 1 and 2). By contrast, the antibody against reduced GPIIIa did not detect this component in chymotrypsintreated platelets (Fig 2, lane 3). Preincubation of chymotrypsintreated platelets with 8-mercaptoethanol prior to the application on SDS-polyacrylamide gels did not result in the appearance of the 66 kDa component on immunoblots although reduced GPIIIa was detectable (Fig 2, lane 4). In addition, the antibody against reduced GPIIIa did not detect GPIIIa in intact platelets solubilized in SDS (Fig 2, lane 5).

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2

3

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FIG. 2 Immunoblots of the plvelet suspensions solubilized in SDS buffer& Lanes 1 and 5: 1 X 10 intact platelets, Lanes 2 aand3: 1.5 X 10 chymotrypsin-treated platelets, Lane 4: 3 x 10 chymotrypsintreated platelets preincubated with 1.5% B-mercaptoethanol. Lanes 1 and 2 were stained with anti nonreduced GPIIIa diluted 1:200. Lanes 3-5 were stained with anti reduced GPIIIa diluted 1:200.

Figure 3 compares the binding of these antibodies (against native and reduced GPIIIa) to the ELISA plates coated with reduced and alkylated GPIIIa. It can be seen that the antibody against reduced GPIIIa was at least 500 times more reactive with this protein than the antibody against native GPIIIa. Control experiments indicated that the antibody bound with the same reactivity to reduced and vinylpyridylethylated GPIIIa as well as to reduced GPIIIa that was applied on the ELISA plates immediately treatment with D-mercaptoethanol (data not shown). The after reactivity of the antibody against reduced GPIIIa with the native GPIIIa attached to ELISA plates was approximately 50 times lower than that of the antibody raised against native GPIIIa (data not shown).

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3.0

0 -0-o anti-reduced

2.5

CPIIIa

??

2.0

g *

1.5

4 1.0

c

0 /

0.5

.$A,

A

0.0 0.

I

/

anti-GPIIIa

/

0

/_/O/O

o-o/_9

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10

100

1000

IgG ng/ml

FIG.

3

Binding of anti-nonreduced GPIIIa and anti-reduced GPIIIa ELISA plates coated with 200 ng reduced GPIIIa per well.

IgG to

Figure 4 shows that the IgG and F(abt)2 fragments prepared from antiserum against reduced GPIIIa inhibited ADP-induced aggregation of suspensions of human washed platelets. Fifty percent inhibition was observed at 3 c(gof F(ab1)2 fragment. There was no effect of this antibody on the aggregation of chymotrypsintreated platelets. In control experiments, monoclonal antibody GlO blocked fibrinogen-induced aggregation of chymotrypsin-treated platelets. IgG prepared from the antiserum against nonreduced GPIIIa (3 - 12.5 pg) had no inhibitory effect on ADP-induced platelet aggregation (data not shown). Higher amounts of this antibody (25.0-50.0 pg) caused spontaneous platelet agglutination, an observation consistent with the report by Leung et al. (20).

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10

20

30 pugProtein

40

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60

FIG. 4 Effect of IgG and F(ab'), prepared from antiserum against reduced GPIIIa on intact and chymotrypsin-treated platelets. 0 intact platelets + IgG, ??intact platelets + F(abt)s, 0 chymotrypsin-treated platelets + IgG, 4 chymotrypsin-treated platelets + F(abt)a

Table 1 shows that purified &G prepared from antiserum against reduced GPIIIa did not block I-fibrinogen binding to ADP stimulated platelets. Neither 5 min preincubation of IgG with resting platelets followed by the addition of ADP nor preincubation of IgG with ADP-activated platelets for 5 min had any inhibitory effect on fibrinogen binding to platelets. TABLE 1 Effect of various antibodies on 1251-fibrinogenbinding to platelets IaG (50 Lea) Normal Rabbit Serum Anti Reduced GPIIIa Anti Nonreduced GPIIIa GlO (monoclonal) * picomoles/lO* platelets

1251-fibrinosen in platelet pellet , X + sd 2.8 2.5 2.4 0.3

+ 1.7 f 1.6 ?r1.3 + 0.5

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DISCUSSION Our data indicate that the antibody showing high reactivity against reduced GPIIIa recognized poorly purified GPIIIa, did not recognize GPIIIa in detergent solubilized platelet extract and did not recognize chymotryptic degradation products of GPIIIa. These observations are compatible with the contention that the specificity of polyclonal antibodies raised against GPIIIa depends on the conformation of the molecule, determined by numerous intramolecular S-S bridges, when injected for immunization. New epitopes are exposed following reduction of GPIIIa and the original conformation-dependent epitopes are destroyed. It has been found previously that glycoprotein IIIa has a large disulfide-bonded loop that is susceptible to proteolytic cleavage (21) and that degradation products of GPIIIa are linked with S-S bridges (21,9). The two bands, identified by the antibody against reduced GPIIIa in the immunoblots of "nonreduced" GPIIIa may correspond to the native, folded molecule and to a partially reduced and unfolded molecule. At present it is difficult to explain why the antibody against reduced GPIIIa did not detect GPIIIa in an extract of platelets solubilized in SDS althoggh it detected purified GPIIIa. The amount of GPIIIa in 1.5 X 10 platelets applied on the gel corresponds to about l-2 pg (22), a quantity detectable in the purified preparations, however, it is conceivable that some conformational changes occur in GPIIIa during its purification and additional epitopes are exposed. It appears that the polyclonal antibody isolated in our laboratory does not interact with a putative fibrinogen binding epitope on GPIIIa because it does not block fibrinogen binding to ADP-stimulated platelets and it does not inhibit fibrinogeninduced aggregation of platelets treated with chymotrypsin, which causes permanent exposure of the fibrinogen receptors (11,19). There is evidence that fibrinogen binding sites are present on the GPIIIa molecule. Calvete et al. (23) localized an epitope in the 23 kDa N-terminal fragment of GPIIIa for the monoclonal antibody P37 which blocks ADP-induced platelet aggregation. This antibody bound to fully reduced and carboxymethylated GPIIIa. Recently, Ramsamooj et al. (24), provided evidence that the conformationally dependent epitope on GPIIIa recognized by the monoclonal antibody cs-1, constitutes a region of the receptor which is involved in fibrinogen binding. This antibody did not react with reduced GPIIIa. It is conceivable that the conformation of the fibrinogen binding epitope on GPIIIa is altered following reduction and vinylpyridylethylation. This could explain the finding that the polyclonal antibody against completely reduced GPIIIa does not inhibit fibrinogen binding to platelets. The fibrinogen binding epitope on GPIIIa may remain intact during the partial reduction of this molecule. This may occur during the incubation of platelets with dithiothreitol or B-mercaptoethanol which leads to the exposure of fibrinogen receptors (25). It iS noteworthy that the antibody against reduced GPIIIa interfered with ADP-induced platelet aggregation although it did not block the binding of radiolabeled fibrinogen to ADP-stimulated platelets. This effect resembles that of the synergistic action Of two murine monoclonal antibodies (AP3 which is directed against GPIIIa and Tab which is directed against GPIIb) that inhibit ADP-

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induced platelet aggregation without blocking fibrinogen binding to platelets (26). Further studies on the mechanism of this inhibitory effect and its possible relation to the involvement of the GPIIIa molecule in platelet activation are warranted using a number of monoclonal antibodies recognizing various epitopes of reduced GPIIIa.

ACKNOWLEDGEMENTS This investigation has been supported in part by N.I.H. grants HL15226 and HL-36579. We wish to thank Mr. Weiqi Lu for computer assistance.

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Methods

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