Biochimica et Biophysica Acre, i 119 (1992) 312-321 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

BBAPRO 34133

Binding of glycoprotein Ilia-derived peptide 217-231 to fibrinogen and yon Willebrand factors and its inhibition

by platelet glycoprotein l ib / I l ia complex

Jacquelynn J. Cook i.,, Maciej Trybulec 1,**, Elizabeth C. Lasz 2, Shabbir Khan 3 and Stefan Niewiarowski 1,2

s Department of Physiology, Temple Unicersity School of Medicine, Philadelphia, PA (U.S.A.), 2 Thrombosis Research Center, Temple Unicersity School of Medicine, PhikuMphia, PA (U.S.A.)and .1 flqstar Institute, Philadelphia, PA (U.S.A.)

(Received 6 August 1991)

Key words: RGD: Fibrinogen; lntegrin; GPIlla-derived peptide; Platelet receptor; Von Willebrand factor

Based on previous reports in the literature and the high homology between platelet glycoprotein (GP) Ilia 217-231 and similar portions of other fl subunits of integrin receptors, we hypothesized that this region may participate in ligand binding. Using a polyclonal antibody against GPIIla 217-231(YC), we tested the interaction of a synthetic peptide representing this region with fibrinogen (Fg), in the enzyme-linked immunosorbent assay (ELISA) system. Results show a calcium-independent, dose-related, direct interaction between GPIlIa 217-231(¥) and immobilized Fg. This peptide also bound to von Willebrand Factor (vWF) and fibronectin (Fn), but did not attach to a 50 kDa Fn fragment which is deficient in the cell attachment site. In addition, purified GPl lb / l l l a displaced GPIIIa 217-231(Y) from Fg and vWF. Binding of IZSl.GPllla 217-231(Y) to Fg coated tubes was inhibited by soluble Fg and by the GPl lb / l l l a complex. We synthesized this peptide with several alterations; similar peptides with Pro-219 replaced with an Ala showed significantly reduced binding to Fg and vWF. The decreased binding of the peptides with Pro-219 substitutes suggests that the confirmation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands. In conclusion, the amino acid residues between 217 and 231 of GPIIIa appear to be involved in ligand binding and Pro-219 probably plays a significant role in this interaction.

Introduction

It has long been accepted that platelets are essential for normal hemostasis which involves the adhesion of platelets to injured vessels and :he subsequent aggrega-

* Present address: Rh6ne-Poulenc Rorer Central Research, King of Prussia, PA, U.S.A.

** On leave of absence from the Department of Pharmacology, Nicolaus Copernicus Medical School, Cracow, Poland.

Abbreviations: ADP, adenosine diphosphate; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; Fg, fibrino- gen; Fn, fibronectin; GP, glycoprotein; HPLC, high-performance liquid chromatography; lgG, immunoglobulin G; kDa, kilodalton; PBS~ phosphate-buffered saline; RIA, radioimmunoassay; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLC, thin-layer chromatography; Vn, vitroncctin; vWF, yon Wille- brand Factor.

Correspondence: S. Niewiarowski, Department of Physiology, Tem- ple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, U.S.A.

tion of activated platelets. The interaction of both Fg and vWF with the GPIIb/IIIa complex on the platelet surface plays a critical role in this process.

The GPIlb / I I Ia complex is the most abundant platelet cell-surface protein representing approx. 1-2% of the total platelet protein [1]. It is a member of the integrin superfamily of adhesion receptors and binds at least four ligands: Fg, Fn, vWF and vitronectin (Vn) [2]. These proteins and many other adhesive molecules which are present in the extracellular matrices and in the blood contain the tripeptide sequence Arg-Gly-Asp (RGD) as their cell recognition site [3].

Integrin receptors are noncovalent heterodimers of transmembrane glycop~-oteins which consist of a and fl subunits and. mediate many physiologically important functions. Recently, progress has been made towards the localization of ligand binding domains on these receptors. Reports from several laboratories which have utilized a variety of approaches including monoclonal antibodies [4], RGD-crosslinking to the/3 3 subunit [5,6] and analysis of enzymatic cleavage products of GPIlla

[7], indicate that the N-terminal portion of GPIl la is probably involved in the interaction of Fg (RGD) with activated platelets. However, discrepancies exist as to the identification of the specific binding area on this GP.

In our laboratory, we isolated a chymotryptic cleav- a g e product of GPIIIa, referred to as the "66 kDa component ' , which contains two subunits apparently linked by S-S bridges [7]. The low-molecular-weight (14-17 kDa) subunit begins with the NH2-terminal sequence of GPIIla and the larger component starts with residue 348 of GPIlla as deduced from a cDNA clone of this GP. In contrast to GPIIIa, this 66 kDa component did not bind to insolubilized GRGDSPK or Fg. These experiments suggested that Fg (RGD) bind- ing to GPIl la occurs between amino acid residues 130 and 347, which is the region of GPIIia deleted during chymotryptic degradation. Based on this evidence, we searched this portion of GPIIIa (f13) for regions of high homology with/31 and/3_~ integrin subunits. It is gener- ally believed that a function as important as ligand bi,nding would occur in a region which is similar among receptors. We hypothesized that GPIIIa 217-231, which shows 80% identity with the fl~ subunit and 67% with fl~, may participate in Fg binding to platelets. The purpose of the present project was to investigate GPI- lla 217-231 for its interaction with Fg and vWF and its potential competition with purified GPI Ib / l l l a for binding to these proteins. In addition, an analysis of the amino acid requirements for the binding activity of this peptide was initiated.

Materials and Methods

Peptide selection and synthesis The cDNA predicted sequences for fll [8], /32 [9]

and fl_~ [I0,11] integrin subunits were examined for regions of high homology between amino acid residues 130 and 347. The sequence corresponding to GPIIIa (f13) 217-231 (DAPEGGFDAIMQATV) displayed 80% identity with the/3~ subunit and 67% with the/~, subunit (Table I). A peptide representing this region, with a tyrosine residue added to the carboxy terminal for iodination, was synthesized using solid phase meth- ods by the Protein Chemistry Facility, Wistar Institute,

T A B L E I

Homology bem'een amino acid sequence of GPIIIa (ft.# 217-231 corresponding sequences of fl~ and fie subunits

62 A

63 A

P E G L M V A A

P E G F I A T V

TABLE I1

Sequences of GPilla-derired peptide.~

313

GPIIIa 217-231(Y):

Peptide A:

Peptide B:

Peptide C:

D A P E G G F D A I M O A T V (y)

D A P E G G F D A I M O A T

D A ~ G G F D A I M O A T

D A A E G G F D A I M Q A T

Philadelphia, PA and by Peninsula Laboratories, Bel- mont, CA. Once Fg binding activity of this peptide had been demonstrated, additional peptides with selected alterations were prepared: GPIIIa 217-230 (peptide A); GPIIIa 217-230 with Pro-219 and Glu-220 re- placed with an Ala and a Gin, respectively, (peptide B); and GPIIIa 217-230 with an Ala in place of Pro-219 (peptide C). All peptides were purified to homogeneity (greater than 95% purity) by reverse-phase high-per- formance liquid chromatography (HPLC) and the se- quences were confirmed by amino acid analysis (Wistar Institute). Peptide sequences are shown in Table 11.

Protein purification Purification of platelet membrane GPl lb / I I I a com-

plex was performed according to a modification of procedures described by Fitzgerald et al. [12]. Initially, outdated platelets were washed three times, and the platelets in the final pellet were lysed by resuspending them in 7 vols. of lysis buffer containing 1% Triton X-100. The lysates were then centrifuged at 20 000 gpm for 15 rain to remove cytoskelctal elements. Subse- quently, the lysates were passed over Concanavalin A-Sepharose (Sigma, St. Louis, MO) and the retained glycoproteins were eluted from the column with a buffer containing a-methyl-o-mannose. A heparin- Sepharose column was then used to remove throm- bospondin and a 160 kDa protein from the Con-A-re- tained glycoproteins. Following this step, we found that the preparation was highly contaminated with Fg and preliminary trials indicated that the contamination of GPIIb / I I Ia with Fg reduced G P l l b / I I l a binding to Fg coated ELISA plates. Because of this, we used a col- umn of anti-fibrinogen immunoglobulin G (IgG) cou- pled to activated Sepharose. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE)was performed according to Laemmli [13] and completed gels were stained either with Coomassie brilliant blue or silver reagent. No trace of Fg was detected following this final purification step.

The 66 kDa component obtained by the proteolytic degradation of GPIIIa was isolated in our laboratory according to procedures previously described [7]. Hu- man vWF was purified by a modification of the meth-

314

ods of Newman et al. [14] and Switzer et al. [15] and was provided by Dr. E. Kirby, Department of Biochem- istry, Temple University School of Medicine, Philadel- phia, PA. Human Fg was purchased from Kabi (Stockholm, Sweden), and Fn and the 50 kDa Fn fragment were purchased from Telios Pharmaceuticals (San Diego, CA).

Polyclonal antisera production GPIIIa 217-23~(YC) was coupled to Kehole limpet

hemocyanin through the terminal cysteine residue (Peninsula Laboratories, Belmont, CA) according to Lerner et al. [16]. Initially, New Zealand white (SPF) rabbits were injected with 500 #g of peptide-hemo- cyanin, in complete Freund's adjuvant. Subsequent booster injections (200 #g in incomplete Freund's ad- juvant) were administered every 7-10 days. Serum samples were tested periodically using ELISA and when antiserum reached an acceptable titer (l:1000 with peptide), the animal was exsanguinated and the serum was stored at -20°C. The antibody to the 66 kDa component of GPllla was also raised in rabbits as previously described [7].

Binding of GPllb ~Ilia complex and GPllla-dericed peptides to immobilized proteins

We have developed various protocols utilizing the standard ELISA system [17,18] which are effective for evaluating the interaction of GPIlb/I l la and GPIIIa- derived peptides with adhesive proteins. Initially, sin- gle-antigen ELISAs were used to confirm adequate coating of plates by protein, to determine peptide recognition and cross-reactivity of antibodies and to establish appropriate concentrations. The following an- tibodies were used to confirm coating of the plates by the adhesive ligands: anti-human Fg IgG (supplied by Dr. A. Budzynski, Temple University School of Medicine, Philadelphia, PA); anti-human factor VIII serum (Nordic Immunological Lab., Capistrano Beach, CA); and anti-human Fn serum (Collaborative Re- search, Bedford, MA). All antisera were raised in rabbits.

Direct interaction of the receptor complex and the short peptides with adhesive proteins (Fg, vWF and Fn) was demonstrated using a double-antigen ELISA with the microtiter plates coated with the selected protein (Fg and vWF = 1 p.g/well; Fn = 0.3 /~g/well) in carbonate-bicarbonate buffer (pH 9.2). Following coating, overnight incubation and blocking of nonspe- cific sites with bovine serum albumin (BSA), various concentrations of peptide or purified receptor (in phos- phate-buffered saline (PBS) containing 0.5 mM cal- cium, 1% BSA and 0.05% Tween-20) were added and incubated for 2 h at 37°C. After this process and between all subsequent steps, a careful washing proce- dure (PBS with and without 0.05% Tween-20) was

repeated several times. Next, the appropriate anti- serum, in PBS with 0.5 mM calcium and 1% BSA, was added and incubated for 2 h at 37°{2. Anti-CPllla 217-231(YC), at a dilution of 1:500, was us, e~ to identify peptide bound to protein on the plate. The antibody to the 66 kDa component of GPIIIa, at a dilution of 1:1000, recognized GPl lb / l l la complex. The sample was then incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase for 2 h at 37°C. Finally, the phosphatase substrate was added, incu- bated for about 20 min at 37°C and blocked with 2 M NaOH. Absorbance was read at 405 nm in a microtiter autorcader (Biotech Instruments, Winooski, VT). Since the antibodies used to identify binding did not crossre- act with the adhesive proteins, it was accepted that the enzymatic activity of the alkaline phosphatase corre- lated with the amount of peptide or receptor complex bound to the protein coated on the ELISA plates.

We also used a modified ELISA protocol to investi- gate the effect of purified GPllb/I l la on the binding of GPIIIa 217-231(Y) to Fg and vWF. Following the incubation of the peptide (3 #M) with adsorbed Fg or vWF, increasing concentrations of GPIlb/ l l la (2-222 nM) were added to the wells. Separate wells with identical conditions were tested with each antibody (i.e. anti-GPllla 217-231(YC) and anti-66 kDa) so that the amount of bound peptide and complex could be identified simultaneously. These experiments were valid because the antibody against the 66 kDa component of GPIIIa does not recognize the short peptide and be- cause the anti-peptide antibody was used at a concen- tration at which it strongly recognized the peptide but did not crossreact with GPIlb/llla. It is not surprising that the anti-GPllIa antibody which we used does not recognize the peptide since GPIIIa 217-231 is not contained within the 66 kDa component against which this antibody was raised [7].

Labeling of GPIIb / Ilia with t251 Two Iodobeads (Pierce, Rockford, IL) were placed

in a reaction vial. 2 p.l of 1251, containing 730/~Ci, was buffered with 200/~l of PBS and added to the reaction vial for 5 rain. Purified GPIIb/Illa, 50/~l at 4 mg/ml, was added and incubated for 15 min. After completion of iodination, the mixture was withdrawn and the vial was washed off with 100 #l PBS. To separate free iodine from the lab~: d complex, this mixture was applied to a PD-10 column (Bio-Rad) and eluted with PBS (pH 7.4) in 0.5 ml fractions. Labeled GPIIb/lIIa was tested for 125I incorporation by performing TLC and on SDS-PAGE, followed by autoradiography. Fractions with greater than 97% incorporation of ~2Sl were used in experiments: autoradiograph showed two labeled band.~ corresponding to GPIIb and GPIIIa. Specific radioactivity of the complex was 895000 cpm//~g.

Labeling of GPIlia 217-231(Y) with 1251 The GPIlla 217-231(Y) was repurified using re-

verse-phase HPLC (Waters, C18, Delta Pak, 100 ,~) and radiolabeled with 12sI using lodo-Beads (Pierce, Rockford, IL). In brief, 320/~Ci of 1251 was buffered in a reaction vial with 100 t~! Tris-HCI (pH 7.4). This solution was transferred to a second reaction vial con- taining two lodo-Beads and incubated at room temper- ature for 5 min. 500 g,I of GPIIIa 217-231(Y) (70 /zg/ml) was added to the ~econd reaction vial. After allowing 15 min for iodination, the solution was re- moved from the lodo-Beads and applied to a Bio-gel 1'2 column (Bio-Rad) equilibrated with 5 mM Tris-HCl, 0.1 mM NaCI and 0,025 mM citric acid (pH 7.4) in order to separate labeled peptide from free iodine. TLC was employed to determine percent radioactivity bound. Fractions containing more than 93% radioactiv- ity bound were utilized for further studies.

Binding of PSI-GPIIIa 217-231(Y) to immobilized fib- rinogen

Radio immunoassay (RIA) tubes (Sarstedt , Pennsauken, NJ) were coated overnight with 1 /xg Fg dissolved in 100 #1 carbonate-bicarbonate buffer (pH 9.2), blocked with 1% BSA and 5% dry milk in PBS for 1 h at 37°C. Subsequently, 100/zl aliquot of ~zSI-GPI- IIa 217-231(Y), in PBS containing 0.5 mM calcium, 1% BSA and 0.05% Tween-20, were added to the turves and incubated for various time interals at 37°C. Nonspecific binding was determined using a 10-fold excess of unlabeled GPIila 217-231(Y). The same system was used for assaying displacement of t251-GPl- lla 217-231(Y) by an excess of unlabeled GPl lb / I l l a complex. After each incubation, tubes were washed twice with buffer.

Platelet aggregation In an effort to isolate the immunoglobulin fraction,

the antiserum containing the anti-GPllla 217-231(YC) antibody was applied to a Protein A-Sepharose column (Sigma, St. Louis, MO) and IgG was eluted with glycine-HCl buffer. Since the resultant fraction did not show immunoreactivity with the original injected anti- gen (GPIIIa 217-231(YC)), an alternate isolation method was performed. Briefly, 6 vols. anti serum and 0.9% NaCI (1:5) were added to 4 vols. saturated ammonium sulfate and allowed to precipitate. The precipitate was added to PBS (pH 7.4), dialyzed and centrifuged. Presumably, the supernatant contained all immunoglobulins from the original antiserum.

Suspensions of washed human platelets were pre- pared according to a modification of the method of Mustard et al. [19]; a portion of the platelets were treated with ehymotrypsin, which has been previously shown to expose Fg receptors [20]. Platelets were counted electronically (Coulter Channalyzer, Coulter

315

Electronics, Hialeah, FL) and the concentration was adjusted to 3- 108 platelets/ml. Washed platelets (420 /xl) were incubated with the anti-peptide immuno- globulin (65 tzl, final concentration = 500 #g/0.5 ml), Fg (5 #1, final concentration = 100 /zg/0.5 ml) and ADP (10 #1, 60/~M) under stirring conditions at 37°C. Aggregation of chymotrypsin-treated platelets was ex- amined similarly, but did not require the addition of ADP. Platelet aggregation was studied using a Payton aggregometer (Scarborough, Ontario, Canada) and was quantified by measuring the initial slope of aggregation as demonstrated by changes in light transmittance. The effect of the antibody was compared to control aggre- gation performed under identical conditions but in the absence of the antibody.

Results

Utilizing the ELISA principle, we have developed various protocols to demonstrate the binding activities of both purified GPl lb / I l l a and selected synthetic GPllla-derived peptides. Experiments included the di- rect interaction of these molecules (GPl lb / l l l a and GPllla-derived peptides) with natural adhesive ligands (Fg, vWF and Fn), the calcium-dependence for their binding activity and displacement of the short peptide from the ligands by the receptor complex.

Initially, we demonstrated that both purified GPl lb / l l l a and the synthetic peptide representing GPIIIa 217-231(Y) bind to immobilized Fg and vWF in a dose-related manner (Fig. 1A and IB). The attach- ment of the G P i l b / l l l a complex and of synthetic GPIlla-derived peptides to albumin coated plates was minimal and was accepted as nonspecific binding. Al- though the short, synthetic peptide (GPIIIa 217- 231(Y)) bound to both adhesive proteins similarly, it appears that the receptor complex (GPl lb / l l la ) has a higher affinity for Fg than for vWF in this system. In fact, there was about a 250-fold difference between molar concentrations of GP l lb / l l l a and GPIila 217- 231(Y) required to approach the binding saturation. This difference most likely reflects a different K a for the binding of G P l l b / l l l a and GPilla-derived peptide to fibrinogen and to yon Willebrand factor. The well known calcium requirement for GPl lb / I l l a complex formation that is necessary for Fg binding activity was also observed in this model when calcium-free binding buffers were substituted for the normal calcium-con- taining buffers (Fig. 2A). However, it appears that GPIIIa 217-231(Y) binds equally well to Fg in the presence and absence of calcium (Fig. 2B). We also examined the interaction of GP l ib / l l l a and GPIlla 217-231(Y) with Fn and a 50 kDa Fn fragment (prod- uct of pepsin digestion) that does not contain the RGD sequence [21]. It can be seen that G P l l b / l i l a and the GPIila 217-231(Y) peptide bind to intact Fn whereas

316

L/3 o <:

1.5 t A F / / . , / ~ 1.2 [

0.9 I ~"" A" vWF t

i ] / ' • |j Q .s8

0.01 e:-- x I t - i l i i 0 20 40 60 80 100

GPllb/lllo nM

120

< 0.6 !

/ . A ~ NSB 0 . 3 - ~ ~ 0

0 . 0 , ,~ I i I I 0 5 10 15 20 25 30

GPlllo 217-231 (Y) ~aM

Fig. i. Binding of G P i l b / l l l a and GPIlIa 217-231(Y) to fibrinogen and van Willebrand Factor. Panel A: dose-related, direct interaction of purified G P l l b / l l l a (2-110 nM) to immobilized Fg and vWF (saturated, 1 #g/well) . Panel B: dose-related, direct interaction of the synthetic peptide representing GPIi la 217-231(Y) (0.3-30 p.M) with immobilized Fg and vWF. Binding to wells coated with BSA was minimal and was accepted as nonspecific binding. Values represent mean +_ standard error of 4 ( G P l l b / l l l a L 12 (GPIl la 217-231(Y) with Fg) or 6 (GPl l la

217-231(Y) with vWF) experiments. In some cases, variation was very small and, therefore, error bars do not extend beyond the symbol.

it3 o

2.5

2.0

1.5

1.0.

0.5,

0.0

~ 0

0 ~ 0 /

no calcium 0

. i i , e ~ I ! , , u

20 40 60 80 t O0

GPIIb/IIIo nM

120

1,5 . B

1.2

0.9

06

0.3 !

O0 :

no COICium I

i"

I~ NSB __----- A

210 410 610 810 100

GPIllo 217-231(Y) #M

Fig. 2. Calcium requirement for G P I l b / l l l a and GPIIIa 217-231(Y) binding to fibrinogen. Panel A: G P l l b / l l l a binds readily to immobilized Fg in the presence of calcium (0.5 raM), but does not bind in the absence of calcium, values represent mean of four experiments. Panel B: GPIIIa

217-231(Y) binds similarly to Fg both in the presence and absence of ca lc ium, , = 4, mean +_ standard error.

1.5

1.2

A

I O

FN A ~y

0.9

0.6 50 kDa FN

0 5 10 15 20 25 30

0.8-

0.6 ¸

ffl o ~- 0.4

0.2

0.0

FN ~O

O

I 0 40

NSB

I I I I 80 120 160 200

GPIIla 217-231 (Y) ~M GPllb/llla nM

Fig. 3. Interaction of G P i l h / l l l a and GPIIIa 217-231(Y) with fibronectin and a 50 kDa fibronectin fragment. Panel A (GPIIia 217-231(Y)), and Panel B ( G P l l b / l l l a ) show that both the G P I l b / l l l a c,~mplex and the synthetic GPllla-derived peptide bind to intact Fn but not to a 50 kDa

peptic fragment of Fn. n = 4, mean +_ standard error. In some cases error bars are not visible due to very small variation.

1400

1200

1000

800

600

400

200

o~ o

/

/ /,

f0

SB

C j / /

2 4 24 T i m e ( h o u r s )

Fig. 4. Binding of t!~l-GPllla 217-231(Y) to immobilized fibdnogen. Effect of various incubation times on the binding of radiolabeled peptide to immobilized Fg. SB, specific binding; NSB, nonspecific binding. SB was measured following incubation of 7.5 ng of the radiolabeled peptide (specific radioactivity 3-10 a cpm per Jag) in a volume of 100/xl. NSB was determined in the presence of unlabeled

peptide (80 ug)added together with 12SI-GPIlIa 217-231(Y).

neither GPIlb/IIIa nor GPlIla 217-231(Y) bind to the 50 kDa Fn fragment (Fig. 3A and B).

In order to validate indirect ELISA used in our study, we radiolabeled GPllb/IIIa complex and GPI- IIa-derived peptide with IZSl, and we determined asso- ciation of t25I radioactivity with the fibrinogen coated plates using ELISA plates with removable wells (A.H. Thomas, Swedesboro, NJ). 1251-GPIlb/llla (2-66 nM) or 125I-GPIIIa 217-231(Y) (0.1-30 ~M) were incu- bated with the plates processed for the immunoassay. A 50-fold excess of unlabeled G P i l b / l l l a added to the plates, (following 2 h incubation with radiolabeled lig- and) caused a marked displacement of the radioactivity associated with the plates after the final washing. In a similar experimental system, a 100-fold excess of unla- beled GPIIIa 217-231(Y) displaced the 12SI-GPIIIa 217-231(¥) bound to the plates (data not shown). Fig. 4 shows that tZSl-GPllla 217-231(Y) bound to immobi-

317

030 / I I I I I I ~ - - - -

| 0 . 2 5 ~

0.20 ~ , ~ ~

o< 0.15

0. I0 •

0.05

0 . 0 0 I I I I f I I

0 5 10 15 20 25 30 35 40 /zM pept ide

Fig. 5. Effect of GPIlla 217-231(Y) and GPIlla 217-230 (Ala-219. Gin-220) on interaction between GPl lb / l I Ia and fibrinogen. GPllb/IIIa (20 nM) incubated for 2 h with immobilized Fg was displaced by increasing concentration of GPlIIa 217-231(Y) (-o-): however, the complex was not displaced by GPIlla 217-230 (Ala-219,

Gin-220) i-e-).

iized fibrinogen in a time-dependent and reversible manner reaching saturation within 2 h.

Another series of experiments indicated that the GPIIb/IIIa complex competes with GPIIIa 217-231(Y) for binding to both Fg and vWF. Initially, we tested the ability of GPIIIa 217-231(Y) (7.0-35/.tM) to displace GPIIb/Illa (20 nM) bound to immobilized Fg. At this range of concentration of GPIIIa 217-231(Y), a part.of the GPIIb/Il la complex was displaced (Fig. 5). By contrast, the complex was not displaced by GPIIIa 217-230 (Ala-219, Gin-220). Subsequently, we tested the displacement of the short peptide bound to Fg by

1.2~

T • o.8 _o

~ 0 .4

0.8-

~ \ o . ~ ~ t

/

do ,;o 2;0

0.6 i •

I 0.6

.0.4 ~ • _~ 0.4-

• 0.2 "~ ~ 0.2, O ,~ I o

o.o

)K" o~

I

0.0~ 0.0 i 0 0 410 810 120 160

GPnb/lllo nM GPtlb/tltlo nM

0.4

0.2

-o I t 0.0 2OO

1.0

O~

o -0 .6

t~

-< v

o I o

Fig. 6. Displacement of GPIIIa 217-231(Y) from fibfinogen and yon Willebrand Factor by GPilb/ l l la . GPIlla 217-XH(Y) (3/.tM) incubated for 30 rain with immobilized Fg (panel A) or vWF (panel B) is displaced by the addition of increasing concentrations of purified GPIlb/IIIa. Each condition was tested in duplicate wilh both the antipeptide antibody and the anti-66 kDa (component of GPIIIa) antibody to simultaneously determine the amount of bound peptide and GPIib/lI la complex. Nonspecific binding of complex and peptide to albumin was minimal, less than

0.2 absorbance at the highest concentration. These graphs represent the results of four consistent experiments with each ligand.

318

! n

1000

500

I I I I

0 10 20 30 40 50 6Pllb/llla (nM)

Fig. 7. Displacement of x251-GPllla 217-231(Y) by various concen- trations of the GPl lb / I I Ia complex. Fg coated tubes were incubated with radiolabeled peptide for l h (-o-) and 4 h (-o-), respectively. Finally, GPIIb/ I I la was added to the tubes and incubated for 2 h: bound radioactivity was determined after extensSe washing. Results

represent three similar experiments.

GPIlb/IIIa. Results show that the addition of purified GPIlb/IlIa caused a decrease, in a dose-dependent manner, in the amount of the short peptide detectable on the Fg coated ELISA plates (Fig. 6A). Similar results were obtained with vWF coated on the plates (Fig. 6B). These studies were performed by measuring absorbance with the anti-peptide (GPIIIa 217- 231(YC)) antibody and the anti-66 kDa antibody simul- taneously and under identical conditions. These experi- ments suggested that GPIIb/IIla may either displace GPIIIa 217-231(Y) bound to Fg and vWF, or it may hinder the ability of the detecting antibody to recog- nize its respective antigen. In order to differentiate between these two possibilities, we radiolabeled GPI- lla 217-231(Y) with mSl. The binding of lzSI-GPIIla 217-231(Y) to Fg was reversed by GPllb/I l la complex in a dose-dependent manner (Fig. 7). In conclusion, these experiments demonstrate that the GPllb/IIla

TABLE II1 immunoreactirity of anti-GPIlla 217-231(YC) with synthetic GPllla-derired peptides

Absorbance =405 nm. Peptides (2 #g) in carbonate-bicarbonate buffer (pH 9.2) were coated on microtiter plates (Falcon 3915, Lincoln, Park, NY) and incubated overnight. Following blocking with BSA, anti-GPllla 217-231(YC) (1 : 1000) was added. Anti-rabbit igG conjugated with alkaline phosphatase was used as a second antibody. After phosphatase substrate had been added, samples were read at 405 nm. Between each step. washing procedures were performed

10 rain 20 rain

GPHIa 217-231(Y) 1.93 < 2.7 Peptide A 0.19 0.44 Peptide B 0.19 0.46 Peptide C 0.24 0.43

complex displaces GPIIla-derived peptide from its fib- rinogen binding sites. 1000-fold excess of soluble Fg also inhibited binding of the radiolabeled peptide to immobilized Fg (data not shown).

Based on this strong evidence that GP|IIa 217- 231(Y) is involved in the binding of adhesive proteins to GPIIIa, further efforts in this project were directed towards the identification of regions within this domain of GPIIIa which are critical for ligand recognition and /o r attachment. Three additional peptides (peptides A, B and C) were synthesized and tested for their interaction with Fg and vWF. The amino acid sequences of these peptides are shown in Table II. Prior to the measurement of the binding of these peptides to Fg and vWF in the ELISA system, it was necessary to determine if the antibody to GPIIIa 217- 231(YC) maintained recognition for these altered pep- tides. In addition, comparative studies could only be performed if the reactivity of the antibody was not significantly different between the three peptides. Table

0 . 5 ¸

0.4

03

0.2

0.1

0.0 0 2O

A /o

/ c

o I

410 610 8~0 100

041

b '3 3 z

:O

< i

T A/?

/-

/ / / " C

~ I ~f. / O

0 / / j . . O / / - / B A

0 . 0 @ - , , t ~ ,

0 50 ! O0 150 200 250 300

Pepbde /~M Per. ~ ~e ,uM

Fig. 8. Binding of 'altered' peptides to flbrinogen and von Willebrand Factor. Peptide A = GPIl la 217-230 (shortened by 2 amino acids from original sequence). Peptide B = GPIlla 217-230 with Pro-219 replaced by Ala and Glu-220 replaced by Gin. Peptide C = GPIIla 217-230 with Ala substituted for Pro-219. Peptide A binds to both Fg (panel A) and vWF (panel B): however, peptides B and C bind minimally to these ligands. Values represent mean + standard error, n = 4. Error bars are only noticeable at severe', points because of the extremely low variation.

III shows absorbance (405 nm) values for anti-GPIIla 217-231(YC) reactivity with the four GPIlla-derived peptides which were tested in this project. It is obvious that the deletion of the final two amino acid residues significantly reduces (10 × ) the immunoreactivity of these peptides. However, there is no significant differ- ence in the antibody recognition of the three shorter peptides, even though several amino acids in the N- terminal portion have been changed. Table II! also shows the absorbance values of the antibody recogni- tion of the three altered peptides following an in- creased incubation time, which was possible once we were no longer comparing the reactivity of these pep- tides to that of the original peptide. The binding of peptides A, B, and C to Fg and vWF at increasing concentrations is shown in Fig. 8A and B. These results demonstrate that the natural sequence of GPIIIa 217- 230 (peptide A) interacts more strongly with both Fg and vWF than either of the altered peptides (B and C). This difference is statistically significant at the two highest concentrations tested for Fg and at all except the lowest concentration for vWF (P < O.01 ). However, there was no significant difference between the binding activity of peptides B and C, although peptide B bind- ing to Fg was less than peptide C at the 100 /zM, P < 0.01. The data were analyzed using ANOVA with significant F values followed by Tukey HSD post hoc analysis. Although the relative binding pattern of these three peptides to Fg and vWF was similar, there was greater absolute binding to Fg than to vWF. it is noteworthy that high concentrations of peptide B (0-35 #M) did not displace G P l l b / l l l a complex from its binding sites from Fg (Fig. 5). By contrast, the same concentration of GPIIIa 217-231(Y) resulted in about 50% displacement.

Finally, we found that the anti-peptide (GPIlla 217-231(YC)) antiserum (50 #!/0.5 ml platelet sus- pension) and partially purified antibody (ammonium sulfate precipitate, 200 #g/0 .5 ml platelet suspension) had no effect on fibrinogen-induced aggregation of chymotrypsin-treated human platelets or ADP-stimu- lated washed human platelets (data not shown).

Discussion

Previous work in our laboratory suggested that a synthetic GPil la-derived peptide, GPl l la 217- 231(YC), interacts with Fg since the addition of this peptide to a suspension of washed human platelets increased the appearance of ~25I-fibrinogen in the platelet pellet [22]. However, these early results pro- vided only indirect evidence of the binding of this synthetic peptide to Fg. Therefore, we developed a system to demonstrate and characterize the direct binding of GPll la 217-231(Y), as well as purified

319

GPl lb / l l la , to Fg and to other natural ligands (vWF and Fn) of the GPI lb / l I l a complex.

Recently, investigators have used immobilized GPl lb / l l l a [23] or GPi lb / l l la -der ived peptides [24] in an effort to examine the interaction of adhcsive ligands with their binding domains on these integrin receptors. We have utilized a system with the adhesive ligand immobilized, similar to that of Nachman and Leung [17] with several modifications. The removal of all Fg from the preparations of purified GPl lb / I i Ia complex greatly improved the reproducibility of dose- dependent, saturable binding of purified GPl lb / I l l a to immobilized Fg. In addition, our studies of the interaction of GPllla-derived peptides, purified GPI lb / l l l a and the adhesive ligands were made possi- ble by the antibodies that we raised. That is, we could identify G P l l b / l l l a with an antibody against a 66 kDa cleavage product of GPIlla which did not recognize the synthetic GPllla-derived peptides. Conversely, our antibody against GPilIa 217-231(YC) reacted strongly with the short peptides, but did not crossreact with the GPl lb / l l l a complex. Our experiments demonstrated that a substantial amount of radioactivities of both ~251-GPIIla 217-231(Y) and t251-GPllb/llla remained on the plates following extensive washing and were displaced by an excess of cold ligands. The reversibility of these reactions validate the assay used in our study.

The calcium requirement for the binding of the GPI lb / l l l a complex to Fg, which has formerly been reported [17], was also demonstrated in our system. In parallel experiments, the peptide representing GPllla 217-231(Y) attached to Fg both in the presence and the absence of calcium. These findings are compatible with the concept that calcium binding regions are pre- sent in the larger subunit of GPIIb [25,26] and that calcium is required for the maintenance of the complex in the conformation necessary for ligand binding [27] rather than for the direct interaction of the adhesive ligands with the binding domain on the integrin molecule. However, it has recently been suggested that GPIIla also contains calcium binding domains sir, ce GPIIla 119 (Asp), an oxygenated residue, interacts with divalent cations [28]. It appears, therefore, that some sites on GPIlla bind to ligands independently of calcium (GPIIla 217-231) and that other domains on GPIIIa that are essential for ligand binding do interact with calcium (GPIlIa 119).

Several series of experiments in the present study indicate that the binding of GPIlla 217-231 to adhe- sive ligands is specific. In preliminary trials, and in conjunction with all other experiments, it was evident that the peptide did not bind nonspecifically to mi- crotiter plates coated with BSA. We also establishcd that although GPIlla 217-231(Y) binds to various in- tact adhesive proteins (Fg, vWF, Fn), when the cell recognition site is deleted from Fn by peptic digestion

320

[21], this peptide and the G P l l b / l l l a complex do not attach to the remaining 50 kDa fragment. Additionally, GPIlla 217-231(Y) did not bind to immobilized platelet factor 4 (data not shown), which is also an adhesive protein [29]. The finding that the GP l lb / l l l a complex (2-200 nM) displaces the peptides (3011{I riM), from both Fg and vWF also indicates that GPIlIa 217-23 ICY) is binding to a specific site on these proteins and not merely sticking non-specifically to the surface of a larger molecule. In addition, equilibrium binding to Fg is demonstrated when both G P l l b / l l l a and GPIlia 217-231(Y) are present; it appears that the affinity of the complex for Fg is several times greater than that of the peptide. Finally, evidence of specific binding is provided by the peptides which are similar, but not identical, to GPIIla 217-231(Y). That is, when the peptide is shortened from GPIIla 217-231(Y) to GPI- ila 217-230, binding is not affected. However, when Pro-219 is replaced with an Ala, the peptide loses the ability to bind to Fg and vWF. The replacement of Pro-219 with an Ala would not be expected to signifi- cantly change the hydrophobicity or the charge of this peptide, however, it can be assumed that the secondary structure would be altered by removal of the Pro residue. Therefore, the decreased binding of the pep- tides with Pro-219 substituted suggests that the confor- mation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands.

The displacement of GPIIla 217-231(Y) from Fg by GPl lb / l l l a indicates that this region of the/33 subunit may indeed represent a binding site for Fg. If GPIIIa 217-231(Y) was not attaching to a site on Fg that 'is naturally used by GPI lb / l l i a complex to bind to Fg, it is conceivable that this integrin complex could bind to Fg and leave the small peptide in place. Similar results suggest that this region of GPIlIa is probably also important for the binding ofvWF to GPl lb / l l la . Charo et al. [23] demonstrated that a similar GPIlla-derived peptide (GPIlIa 211-2221 inhibited the binding of Fn, Vn, vWF and Fg to immobilized GPl lb / l l la . More- over, these authors have also shown inhibition of ADP-induced aggregation of gel filtered platelets by a polyclonal antibody directed against GPIlIa 204-222. These combined results suggest that the amino acid residues between 217 and 222, which represent the area of overlap of the active peptides characterized by these two independent laboratories, arc probably criti- cal for ligand binding to GPilla. Our finding that the substitution of Pro-219 dramatically reduced peptide binding to Fg and vWF and displacement of GPl lb / l l I a bound to Fg also supports this theory.

it is also interesting that the polyclonal antibody raised against GPIlla 217-231(YC) did not block fib- rinogen dependent aggregation of washed human platelets. This finding is consistent with the theory that the NH,-terminal portion of this peptide is involved in

Fg binding since it appears that the carboxy terminus is the antigenic portion. This can be deduced from the observation that the immunoreactivity of the peptide is greatly diminished following the deletion of two C- terminal amino acids and that the antibody recognition is not reduced when N-terminal amino acid residues (Pro-219 and Glu-2201 are altered. Similar results were obtained by Charo et ai. [23] as an anti-GPIlla 211-229 antibody had no effect on ADP-induced aggregation of washed human platelets: however, an antibody to GPI- lla 204-222 inhibited platelet aggregation to approxi- mately 35% of control values.

Although GPIlia 217-231(Y) bound to RGD con- taining peptides, it appears that binding of GPIIIa 217-231(Y) to Fg and vWF is RGD independent. Binding of GPIIIa 217-231(Y) to Fg was calcium inde- pendent, whereas binding of RGD peptides to the GPl lb / l l l a complex is Ca -'+ dependent [30]. We mea- sured the effect of several potential inhibitory peptides on the binding of GPIIla 217-231(Y) to Fg. Although complete inhibition could not be demonstrated, we found partial inhibition (30-411%) by the tetrapeptide RGDS (10-250 #M) and by an RGD-containing snake venom derived peptide, echistatin ({I.1-1.0 /zM) [31]. However, similar concentrations of RGES, SDGR, GRADSP, the fibrinogen gamma-chain dodecapeptide [32] and (Ala-24) echistatin [31] also showed similar inhibition (unpublished observations). It appears, therefore, that nonspecific factors may be.causing the inhibition of peptide from binding to Fg. In their recent study, Charo et al. [23] demonstrated that bind- ing of GPIIIa 204-229 to microtiter plates coated with Fg was not inhibited by either RGDS or RGES.

Although several earlier studies propose a Fg bind- ing region on GPIIIa that includes the area which we investigated [4,7], other reports suggest that this region of GPIIIa (217-2311 is distal to the RGD crosslinking sites on the/33 subunit [5,6]. As previously mentioned, Loftus et al. [27] recently presented evidence that GPll la 119 (Asp) is essential for Fg binding by demon- strating that the single amino acid change of Asp-119 to a Tyr results in the loss of ligand recognition. Although these investigators clearly showed that this amino acid is required for Fg binding, it apparently is not sufficient for this interaction since a 66 kDa com- ponent of GPIIIa which contains Asp-l l9 does not bind to Fg or to GRGDSPK [7]. In addition, findings from our study and the results reported by Charo et al. [23], collectively implicate a region of GPIIla within amino acid residues 211 through 230 for ligand interac- tion. This attachment area may possibly be limited to the overlapping portion of the fibrinogen-interactive peptides, GPIIIa 217-222, studied in these two labora- tories. Overall, these investigations suggest that there are at least two binding sites for Fg on GPIIIa. It has also been suggested that RGD-binding sites on the

GPl lb / l l l a complex reside on both subunits, GPIib and GPIlla [33,34], and that the fibrinogen gamma- chain peptide crosslinks to GPllb 294-314 [35]. Also, an RGD peptide affinity labels the a subunit of the Vn receptor (a~/3.~) [36] as well as the /3 suhunit of this integrin [6]. Most recently, Kieffer and Phillips [37] hypothesized that the RGD recognition site resides within amino acids 109-171 of GPIIla and another Fg recognition site, presumably RGD independent, corre- sponds to residues 211-222 of GPIlIa.

in conclusion, this expanding knowledge concerning ligand interaction with integrins of the /3.~ subfamily seems to be suggesting a "binding pocket" with multiple sites required for ligand attachment and/or conforma- tional maintenance. These domains are present on both subunits of the integrin complex: some are spa- tially or functionally linked to calcium binding domains and some bind ligands independently of calcium.

Acknowledgments

The authors wish to thank Dr. I. Charo for sharing his manuscript prior to its publication and also to Cathy Spiotta for her skillful prcpaiation of this paper. This work was supported by NIH grants HL15226 and HL36579.

Note added in proof:

Most recently Lanza et al. (Blood (1991) 78, suppl. 1, abstr. 1570) described a new variant of Glanzmann thrombasthenia in which platelets expressed abnormal GPIIIa with Arg-214 substituted with Trp-214. Platelets of this patient did not hind fibrinogen upon stimula- tion. This observation is in keeping with a hypothesis that GPIIIa 211-221 region plays a role in fibrinogen binding to the receptor.

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