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Characterization of the Fcγ receptor on human platelets
CELLULAR
IMMUNOLOGY
128,462-479 (1990)
Characterization MARIANNE
of the Fey Receptor on Human Platelets’T2
KING, PATRICIA MCDERMOTT,
AND ALAN
D. SCHREIBER
University of Pennsylvania Graduate Group in Immunology and Cancer Center, University of Pennsylvania School ofMedicine, Philadelphia, Pennsylvania 19104 Received October 4,1989; accepted January 13, 1990 IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fey receptor on the platelet surface. We studied the platelet Fey receptor and characterized its interaction with IgG ligand and anti-Fey receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. L&id-binding studies indicated that normal human platelets express a single Fcr receptor (FcrRII) with 8559 + 852 sites per cell, Kd = 12.5 f 1.7 X lo-* M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-FcyRII were consistent with each Fey receptor expressing two epitopes recognized by the antibody. The number of Fey binding sites and affinity of binding were unchanged by the presence of 2.0 mMMg*+ or 10 pg/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fe-r binding sites or the affinity of binding. However, platelets preincubated with 5 &dexamethasone expressed a decreasednumber of Fcr binding sites as well as decreasedIgG-dependent platelet aggregation. Platelets from patients with Glanzmann’s thrombasthenia and from patients with the Bernard Soulier syndrome expresseda normal number and affinity of Fcr binding sites. The data suggest that platelet FcyRII binding of trimeric IgG occurs independent of actin filament interaction, Mg*+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fey-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 M, subunits. 0 1990 Academic Press, Inc.
INTRODUCTION Platelets contain a number of factors that may be involved in inflammation (l-2). In immune complex disorders, platelets are thought to interact with IgG-containing immune complexes through an Fey binding site on the platelet surface (l-6). Only immunoglobulin aggregatesof the y isotype have the ability to stimulate platelets, suggesting that the critical site(s) on the immunoglobulin molecule responsible for platelet activation resideson the heavy chain ($6). These data have led to the concept of a platelet Fey receptor. i Supported by NIH Grants HL-28207 and HL-40387. ZPresented in part at the National Meetings of the American Association of Immunologists and the American Society of Hematology. 462 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
Fcr RECEPTOR
ON HUMAN
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The association of thrombocytopenia with diseasestates in which circulating immune complexes occur further suggeststhat immune complex-platelet interaction may represent a mechanism by which platelet destruction is induced. There is evidence that some patients with chronic immune thrombocytopenic purpura (ITP) have circulating immune complexes which correlate with the degreeof thrombocytopenia (7-9). Thus, in some immune platelet disorders platelet clearance may be induced by immune complexes which bind to the platelet Fey receptor in such a manner that Fey domains remain available for macrophage detection within the reticuloendothelial system. In this study we further characterized the interaction of the platelet Fey receptor with IgG ligand and anti-Fey receptor monoclonal antibody. In addition, we examined the effectsof several possible modulatory signals on the expression of this receptor, since the regulation of this Fey receptor may have pathophysiologic significance. Finally, we attempted to ascertain the biochemical identity of the platelet Fcr receptor through both ligand binding studies with glycoprotein-deficient platelets and through direct isolation of the platelet Fey binding protein. The data suggest that human platelets have a single surface Fe-r receptor which is distinct from platelet glycoproteins IIb/IIIa, and Ib, V, and IX and which migrates on PAGE at M,
= 220,000. METHODS
Patients We studied 25 normal platelet donors ranging in age from 25-50 years who were not on any medications and who were aspirin-free for at least 2 weeks. We studied in a single experiment two individuals with type II Glanzmann’s thrombasthenia known to be deficient in GPIIb/IIIa, who were kindly provided by Dr. Margaret Johnson, Wilmington, Delaware. We also studied three patients from a family with Bernard Soulier syndrome kindly provided by Drs. Scott Murphy and Ingerman Wojensky of Jefferson Medical College. The platelets from these patients have been previously reported and have been demonstrated to be deficient in GPIb (10).
Preparation of Platelets Platelets were isolated daily from the blood of normal human donors and were used in experiments immediately following their isolation. Briefly, blood was drawn into 0.3 ml of 5% disodium ethylene diaminetetraacetate (EDTA) per 10 ml blood, unless otherwise noted, and centrifuged for 15 min at 18Ogat room temperature in a Sorvall centrifuge (Beckman Instruments, New York). The platelet-rich plasma (PRP) was aspirated and centrifuged a second time at 18Ogto remove any remaining red cells or leukocytes. Leukocyte contamination was lessthan 0.0 1%.The platelets were then centrifuged at 1OOOgfor 13 min and washed three times with phosphate-buffered saline containing 12.5 mM EDTA and 1.0 mM adenosine. The washed cells were suspendedin the appropriate assaybuffer, quantitated and adjusted to a concentration of l-2 X 109/ml.
Preparation of Human IgG Human IgG was isolated from fresh human serum by ammonium sulfate precipitation followed by ion exchange chromatography (QAE-Sephadex, Pharmacia, Piscata-
464
KING,
MCDERMOTT,
AND
SCHREIBER
way, NJ) and/or gel filtration chromatography (Sephacryl S-300, Pharmacia, Piscataway, NJ). Oligomeric IgG was prepared according to the method of Segal and Titus (11). Briefly, purified IgG (70 mg/ml in 0.2 MTris-HCl buffer, pH 8.4) was incubated with a 16-fold molar excessof dimethyl suberimidate (Sigma Chemical Co., St. Louis, MO) for 1 hr at 30°C. The reaction was then quenched by the addition of a fivefold molar excessof glycine (with respect to dimethyl suberimidate) and the mixture immediately applied to a tandem column of Sephadex G-200 (Pharmacia, Piscataway, NJ) and Ultrogel AcA 22 (LKB Instruments, Inc., Gaithersburg, MD) and fractionated into oligomers of defined molecular weight. Pools of each oligomer were made and concentrated to 5- 10 mg/ml. Fey and Fab fragments of IgG were prepared by papain digestion of IgG monomer according to the method of Porter ( 12). Heat aggregated IgG was prepared each day by incubating IgG monomer for 20 min at 63°C. IgG was radiolabeled with ‘251-Na(New England Nuclear, Boston, MA) to a specific activity of l-2 X lo5 cpm/pg protein (13). Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis (PAGE) was performed using a modification of the Laemmli system (14). The stacking gel contained 3.0% acrylamide in 0.125 A4 Tris-HCl buffer, pH 6.8, and the separating gel 5.0-10.0% acrylamide in 0.375 M Tris-HCl buffer, pH 8.8. Samples were dissolved in 0.01 M Tris-HCl, pH 6.8, containing 0.1% SDS and incubated for 10 min in a boiling water bath before application to the gels. Reduced samples also contained 0.1% 2+mercaptoethanol. Gels (16 X 20 cm) were run in a Bio-Rad Protein II vertical gel apparatus (Bio-Rad, Richmond, CA) at 4°C overnight at 65 V. After electrophoresis, gels were fixed and stained in methanol-water-acetic acid (5:5: 1) containing 0.25% Coomassie blue for l-2 hr. Gels were destained in the same solution in the absenceof Coomassie blue. IgG Binding to Platelets The ability of oligomeric IgG to aggregate platelets necessitated that all binding studies be performed in the presence of EDTA. Assayswere performed in Dulbecco’s calcium and magnesium-free PBS (GIBCO, Grand Island, NY) containing 0.2% human serum albumin (Sigma Chemical, St. Louis, MO), 2 mM EDTA and 15 mA4 sodium azide. Washed platelets were suspended in this buffer at a concentration of l-2 X lo9 cells/ml and 1 X 10’ platelets were incubated with increasing concentrations of radioiodinated IgG oligomer in a total volume of 0.5 ml in the presence or absence of a loo-fold excessof unlabeled heat-aggregated IgG. Incubations were performed at either 37°C for 1 hr or 4°C for 3 hr at which time equilibrium of binding was achieved. The reaction mixture was layered over silicone oil (15 ml silicone + 80 ml biphenyl silicone; William F. Nye, Inc., New Bedford, MA), the platelets sedimented and the amount of cell-associated ‘251-IgGquantitated in a Beckman gamma counter (Beckman Instruments, New York). The concentration of free ‘25I-IgG was defined as the amount of radioactivity which remained unbound. Specific binding was defined as the cell-associated radioactivity not inhibited by the presence of a 1OOfold excessof unlabeled IgG and was analyzed by Scatchard plot. For these calculations a molecular weight of 150,000 for IgG was employed assuming that each IgG subunit binds to a single receptor (15). The number of IgG-binding sites and the
Fey RECEPTOR
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affinity of specific binding to platelets was determined by a linear regression program using the least squares method. Preparation of Purified Anti-FcTRII Monoclonal Antibody Anti-FcyRII monoclonal antibody IV.3 was isolated from ascites fluid using protein A (Affi-Gel Protein A Maps II, Bio-Rad, Richmond, CA). Briefly, 1.5 ml ascites fluid (kindly provided by Dr. Clark L. Anderson, Ohio State University, Columbus, OH) was applied to a 5-ml protein A-agarose column. The column was washed with 15 vol of buffer and the IgG eluted with pH 2.8 buffer. The eluted protein was dialyzed against PBS, concentrated to 7 mg/ml and radiolabeled with “‘1 (New England Nuclear, Boston, MA) using chloramine T ( 13) to a specific activity of 200,000 cpm/ pg. Experiments of equilibrium binding to platelets were performed using ‘251-antiFcyRII and unlabeled anti-FcyRII in a manner similar to the human IgG ligandbinding studies above. Fab fragments of the monoclonal were prepared by incubating purified IV.3 antibody with immobilized papain (Pierce, Rockford, IL) for 4 hr at 37°C. The Fab fragments were separated from the Fc fragments and undigested IgG on a protein A column as described above. Purity of the Fab fragment was confirmed by Ouchterlony analysis and SDS-PAGE. Protein concentration was estimated by measuring the absorbance at 280 nm (E f’?&= 14.0) and by the Lowry method. Platelet Aggregation Studies Platelets used in aggregation studies were isolated from PRP containing ACD by gel filtration. Approximately 4 ml PRP were applied to a 50-ml Sepharose4B (Pharmacia, Piscataway, NJ) column and the platelets eluted with Tyrodes buffer containing 0.1% glucose and 0.35% BSA. The platelets were adjusted to 2 X 108/ml and used immediately. Since IgG-dependent platelet aggregation requires the presence of extracellular Ca*‘, CaC12was added to the cell suspension to a final concentration of 2 mM. The platelet suspension (450 ~1) was pipetted into an aggregometer cuvette and incubated in a dual channel recording aggregometer(Chrono-Log Corp., Havertown, PA) at 37°C with stirring for 1 min. Fifty microliters of platelet-aggregating agent (heat-aggregatedhuman IgG, thrombin, or ADP + fibrinogen) were then added and the response was recorded for 10 min. Results were expressed as percentage of aggregation and were determined graphically from the aggregometercurve. Preparation of Fey-Sepharose 4B Column Fifteen grams of freeze-dried cyanogen bromide-activated Sepharose 4B (Pharmacia, Piscataway, NJ) were swollen and washed with 2 mM HCl and then resuspended in a coupling buffer consisting of 0.1 M NaHC03 + 0.5 M NaCl, pH 8.3. Isolated Fey fragment (10 mg/ml), solubilized in the same buffer, was immediately added to the gel in the proportion of 20 ml Fcr fragment solution/55 ml CNBractivated Sepharose 4B. Following a 2-hr incubation at room temperature, the gel was washed once with coupling buffer and then incubated an additional 1.5 hr at room temperature with 0.2 A4 glycine, pH 8.0. The gel was then washed with four cycles of alternating pH with each cycle consisting of one wash with 0.1 M acetate buffer, pH 4.0, and one wash with coupling buffer, pH 8.3. The gel was then sus-
466
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MCDERMOTT,
AND
SCHREIBER
pended in PBS with 0.02% sodium azide and stored at 4°C until use. The residual protein present in the coupling buffer supernatant following the initial incubation and in the subsequent wash solutions was determined and indicated that the coupling efficiency for the Fcr fragment was 98%.
Isolation of Platelet Fey-Binding Protein Platelets were isolated from 3-4 units of blood obtained from normal donors as described above. The cells were washed 5X with PBS containing 12.5 mM EDTA and 1.O mM adenosine and pooled to yield approximately l-2 X 10” cells. The washed cells were pelleted, resuspended with 7.5 ml PBS containing 1%NP-40, 1 mA4 EDTA, 0.05 mMN-carbobenzoxy-L-glutamyl+tyrosine, l/3 units/ml aprotinin and 2 mMphenylmethyl-sulfonyl fluoride (all from Sigma Chemical Co., St. Louis, MO), and incubated on ice for 30 min. The detergent-solubilized cells were then centrifuged in an ultra centrifuge (Beckman Instruments, Inc., Palo Alto, CA) for 1 hr @ 100,OOOg. The supematant was removed and incubated with 42.5 ml ofgel consisting of Fey fragments (3.8 mg Fey fragment per milliliter Sepharose4B) coupled to cyanogen bromide-activated Sepharose4B (Pharmacia, Piscataway, N.J.). The incubation was performed overnight in a sterile flask with shaking at 4°C. The gel was then poured into a 50-ml column and washed with at least 25 vol of PBS containing 1 r&4 EDTA and 0.5% NP-40. Bound protein was eluted from the column by washing with 2 vol of 0.5 N acetic acid containing 0.5% NP-40. One-milliliter fractions were collected into 0.2 ml 2 MTris baseand examined at OD 280 nm. Protein-containing fractions were immediately dialized against 0.01 A4 Tris-HCl, pH 6.8, or PBS with 0.05% NP-40, pH 7.0.
WesternBlot Analysis Western blots were employed to identify platelet Fey binding proteins isolated from intact platelets. Proteins electrophoresed on polyacrylamide or agarose/polyacrylamide gels were transferred to nitrocellulose (Schleicher and Schuell, Inc., Keene, NH) using Bio-Rad’s Trans-Blot electrophoretic transfer cell (Bio-Rad, Richmond, CA). Gels were first preequilibrated in transfer buffer (25 mA4 Tris, 192 mM glycine, 20% methanol) for 20-60 mitt, overlayed with nitrocellulose and sandwiched between Whatman 3-mm filter paper and gel pads as described in the Trans-Blot protocol. The transfer was performed at 4°C overnight. The efficacy of the transfer procedure was assessedby staining the polyacrylamide gel slab and a portion of the nitrocellulose with Coomassie blue. Following the transfer, the nitrocellulose blots were incubated at room temperature with 3.0% bovine serum albumin in Denhardt’s borate-buffered saline (166.5 mM H3B03, 27.5 mM NaOH, 150 mM NaCl with 6 mM NaNa,, 1 g/liter Ficoll, 1 g/liter polyvinylpyrrolidone, 15 g/liter BSA, and 0.1% NP-40) for at least 3 hr to minimize nonspecific binding of proteins to the paper. The blots were then incubated an additional 2 hr with ‘*‘I-IgG trimer (200,000 cpm/lO ml Denhardt’s). Unbound radioactive protein was removed by washing the blots five times in Denhardt’s buffer or until any background radioactivity was eliminated. The blots were air-dried and exposed to Kodak X-Omat AR film for 3-7 days for development of the autoradiograph.
Fey RECEPTOR ON HUMAN
PLATELETS
467
125 I - IgG Added (pg)
FIG. 1. Binding of ‘251-IgGto human platelets. Isolated ‘251-IgGmonomer, dimer, and trimer were added in increasing concentrations to 1 X 10’ platelets and incubated in the presence and absence of a IOO-fold excessof unlabeled heat-aggregatedIgG at 4°C for 3 hr. A representative experiment illustrating the number of molecules of ‘*%IgG specifically bound is shown.
RESULTS Characteristics of IgG Binding to Normal Platelets We first examined the ability of normal human platelets to bind both radiolabeled monomeric and oligomeric IgG. We observed no significant specific binding of IgG monomer to platelets (less than 150 molecules IgG/platelet) over a wide range of monomeric IgG input. We then compared isolated IgG oligomers of varying molecular weight in their binding to platelets and we observed that the amount bound increasedas the degree of IgG polymerization increased (Fig. 1). Oligomer binding was inhibited by isolated Fcr but not by Fab fragments. We employed IgG of M, = 450,000 (IgG trimer) in all subsequent binding studies to avoid inconsistencies which might result with the use of larger, more heterogenous oligomer preparations. When platelets were incubated with radiolabeled IgG trimer at 4°C binding of lz51IgG ligand reached equilibrium within 3 hr. Furthermore, platelet-bound IgG trimer was displaced by the addition of heat-aggregatedIgG. Aggregated IgG was five times more effective than monomer in displacing the bound trimer. After 3 hr at 4°C platelet binding of IgG trimer was saturable and Scatchard plots of the data were linear, consistent with a single class of binding sites for IgG trimer on platelets (Fig. 2). Efect of Ionic Strength We further characterized the optimal conditions for IgG trimer binding by studying the effect of changesin ionic strength. We varied the ionic strength of the assaybuffer, while maintaining constant osmolarity, by the addition of either dextrose, D-mannose, or D-mannitol. The results were similar for each of these three sugars. Specific binding of IgG trimer under equilibrium conditions was saturable at ionic strengths
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AND SCHREIBER
125 I - I gG Added (pg) FIG. 2. Equilibrium binding of IgG trimer to platelets. A representative experiment illustrates the equilibrium binding of I25I- I gG trimer to platelets. Washed platelets were incubated with increasing concentrations of IgG trimer in a final volume of 0.5 ml for 3 hr at 4°C. The Scatchard plot of the data (inset) suggests that only one class of binding sites is present.
ranging from P= 0.07 to CL= 0.15 and Scatchard plots of the data were linear. When the ionic strength was progressively lowered from p = 0.15 to p = 0.07, we observed an increase in the total number of IgG binding sites expressedper platelet (Fig. 3). At ionic strength P = 0.07, 10,200 f 2741 (mean + SD) IgG binding sites/platelet were expressed (N = 5), while at P = 0.15, 4045 f 1195 (mean f SD) binding sites were expressed per platelet (N = 5). The affinity of IgG trimer binding to platelets was similar at p = 0.07 and 0.15. We also observed that this effect of ionic strength was reversible.
Normal Platelets We next studied the platelets obtained from 25 consecutive normal adults for the number of binding sites and affinity of binding for IgG trimer. The platelets from these normal donors expressed 8559 + 852 (mean +- SEM); range = 2825 - 18,290) binding sites for IgG trimer at ionic strength P= 0.07. The affinity of binding was Kd = 12.5 + 1.7 X 1O-’ M (mean + SEM) and did not differ substantially for the platelets expressing high or low numbers of Fcr binding sites.
Fcr RECEPTOR ON HUMAN
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I23
2
4
6
8
IO
12
14
1251- IgG Added (pg) FIG. 3. Effect of ionic strength. Platelets were incubated with %IgG at ionic strength of rr = 0.15 (A) or p = 0.07 (B). At r.~= 0.07, more ‘*51-IgGspecifically bound to platelets at equilibrium. Scatchard plots of the data (insets) demonstrate an increase in the number of IgG-binding sites expressed per platelet at low ionic strength. In this representative experiment at p = 0.15 (A), 2700 binding sites/platelet were expressed; at Jo= 0.07 (B), 5200 siteswere expressed.The affinity ofbinding was unaffected by change in ionic strength.
Efect of Divalent Cations We explored the divalent cation requirement for the binding of IgG to platelets. Since the above studies were performed in the presence of EDTA and in the absence of free Ca*+ and Mg*+, we determined whether the binding of IgG trimer by platelets was altered in the presence of Mg *+. We examined the effect of Mg*+ by substituting EGTA for EDTA at equimolar concentrations. We compared the effect of 2 mM EDTA, 2 m1J4EGTA, and 2 mM EGTA containing 2 mit4 Mg*+. Equal numbers of IgG-binding sites and affinities of binding were observed under each of these three conditions (Table 1). Efect of Cytochalasin B The presentation of a relatively fixed array of IgG to the platelet surface appears to be important in platelet binding and stimulation by IgG and suggeststhat the bridging of some cell surface molecules, e.g., the Fcr receptor, may be necessaryfor effective interaction of platelets with IgG oligomer. Such an interaction may require an intact platelet membrane cytoskeleton. We examined the effect of cytochalasin B, an inhibitor of cytoskeleton function, on the ability of platelets to bind IgG trimer. Platelets were incubated with lo-100 &ml of cytochalasin B for 30 min at 37°C and the number and affinity of IgG binding sites were determined in the presence of cytochalasin B (Table 1). Cytochalasin B did not alter the number or affinity of plateletbinding sites for IgG trimer. Thus, an intact cytoskeleton does not appear necessary
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TABLE 1 Effect of Mg’+, Thrombin, and ADP on Platelet Fey Receptor Expression Number of binding sites/cell’
Kd
Experiment
Control-EDTA (2 mA4) EGTA (2 mM) EGTA (2 mA4) + Mg*+ (2 mM)
6153 5872 6012
6.6 x lOmEM 7.6 X 1O-8M 6.6 X 1O-8 M
Control’ Thrombin (1 unit/ml)
5798 5522
9.1 x 10m8M 8.3 x 1O-8 M
Control’ ADP(lO&) ADP ( 10 PM) + fibrinogen (100 &ml)
2825 2326 2551
9.5 x IO-* M 6.9 X 1O-8M 11.1 X IO-*M
’ Represents the maximum number of IgG molecules bound per platelet at saturation (B max). ’ Platelets were preincubated in either 2 mM EDTA/PBS (control) or 2 m&f EDTA/PBS containing 1 unit/ml thrombin for 30 min at 37°C. Similar results were observed with 10 units/ml thrombin. ’ Platelets were incubated in Tyrode’s buffer (control) or in Tyrode’s buffer containing 10pM ADP with and without fibrinogen. Incubations were performed at room temperature for 15 min without stirring and the binding of ‘251-IgGtrimer was then assessedin the presence of 1 mMEDTA.
for efficient platelet binding of oligomeric IgG. The biological activity of the cytochalasin B was confirmed by its ability to inhibit ADP and thrombin-induced platelet aggregation. Modulation of Platelet Fey Binding Sites We next sought to determine what factors might modulate expression of the platelet Fey receptor. We first investigated whether platelet activation induced by the interaction of physiologic agonists, such as thrombin and ADP, alters the binding of IgG ligand to its platelet Fey-binding site. Platelets were incubated with 0.01-10.0 units/ml of thrombin for 30 min at 37°C without stirring. The ability of these concentrations of thrombin to cause platelet aggregation in titrated PRP confirmed that the preparations were biologically active. We observed that preincubation with thrombin had no substantial effect on Fey receptor number or affinity (Table 1). The effect of ADP on the binding of IgG trimer by platelets was examined under two conditions: (i) in the presence of 2 mM EDTA and (ii) in Tyrode’s buffer in the presence of 2 mM Ca‘+ , 2 mM Mg2+ and fibrinogen ( 100 pg/ml), but without stirring to avoid aggregation. In the latter case,the platelets were isolated from blood collected in 0.38% citrate, washed, and resuspended in Tyrode’s buffer. Following preincubation with ADP, EDTA was added to the platelets to a final concentration of 2 mM for the IgG ligand binding studies. We found that incubation of platelets with 10 N ADP did not substantially affect their ability to bind IgG trimer (Table 1). The activity of the ADP employed in these studies was confirmed by observing its ability to aggregate platelets which were incubated under identical conditions as our experimental preparations, but with stirring. Eflect of Dexamethasone Glucocorticoids have been observed to modulate Fey receptor expression in some cells and cell lines. Therefore, we studied the effect of the glucocorticoid hormone
Fey RECEPTOR
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TABLE 2 Effect of Dexamethasone
on Fey Receptor Expression”
Fey receptors/cell b Donor 1 2 3 4 5 6
Control 8,824 10,679 9,072 13,561 5,312 f 322’ 8,074 f 443d
Dexamethasone 4846 3805 7910 2709 3096 f 248 4910+619
% Inhibition 45.1 64.4 12.8 80.0 42.4 39.2
’ Platelets were incubated in PRP with either PBS (control) or 5 & dexamethasone for 18-24 hr at room temperature and then isolated and washed. b Represents the maximum number of binding sites for IgG trimer as determined by Scatchard analysis. ’ Mean + 2 SEM for five separate determinations on the platelets from patient 5. d Mean f 2 SEM for two separate determinations on the platelets from patient 6.
dexamethasone (Sigma Chemical Co., St. Louis, MO) on platelet Fey-receptor expression. Platelets were incubated with 5 PM dexamethasone or an equal volume of PBS in PRP with ACD for 18-48 hr at room temperature under sterile conditions. The ability of these cells to bind IgG trimer and aggregatein response to heat-aggregated IgG was then assessed.Platelet yield was >95% in both the dexamethasone- and buffer-treated preparations. In 7 of 12 donors tested, we found that dexamethasone treatment decreasedthe binding of IgG trimer to platelets by as much as 80% (Table 2). The extent of the effect varied from donor to donor. In parallel, we examined the effect of dexamethasone on IgG-dependent platelet aggregation. Dexamethasone-treated platelets used in these studies were isolated from PRP by gel filtration. We observed in each of three donors whose dexamethasonetreated platelets expressed a decreasednumber of binding sites for IgG trimer, that dexamethasone also inhibited platelet aggregation by heat-aggregatedIgG (Table 3). The ability of these dexamethasone-treated platelets to aggregatein response to the platelet agonists ADP and thrombin was unaltered. Platelets from several individuals had no alteration in the expression of their binding sites for IgG following preincubation with dexamethasone. Dexamethasone-treated platelets from these donors also aggregated normally in response to heat-aggregated IgG. Dexamethasone had no effect on anti-FcyRII monoclonal antibody binding to platelets, as assessedby flow cytometry, in any of the donors tested. Binding of IgG to Platelets from Patients with Glanzmannk Thrombasthenia and Bernard Soulier Syndrome We were able to study the binding of IgG trimer to platelets from patients with two disorders characterized by the lack of specific glycoproteins on the platelet surface, Glanzmann’s thrombasthenia and Bernard Soulier syndrome. In each experiment, equilibrium binding studies were compared to a concurrent normal control. Equilibrium binding experiments showed that the platelets from all the patients expresseda number of binding sites and affinity of binding similar to that of the platelets from
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TABLE 3 Effect of Dexamethasone on Fey Receptor-Dependent Platelet Aggregation’ Donor
Platelet agonist b
% Inhibition of aggregation’ by dexamethasone
1
Heat-aggregated IgG ADP Thrombin Heat-aggregated IgG ADP Thrombin Heat-aggregated IgG ADP Thrombin
86.7 0 3.4 58.8 0 0 64.4 0 1
2 3
’ Platelets were preincubated in PRP with 5 pkfdexamethasone for 18-24 hr at room temperature and then isolated by gel filtration. ’ 50 pl platelet agonist (5 mg/ml heat-aggregatedIgG, 50 &f ADP, or0.2 units/ml thrombin) were added to 450-p] gel-filtered platelets in Tyrode’s buffer. ’ Determined by comparison to control platelets incubated with PBS.
our population of 25 normal controls. Platelets from the patients with Glanzmann’s thrombocytopenia expressed 14,172 and 13,320 binding sites/platelet. The platelets from the patient with Bernard Soulier syndrome expressed 5865 sites/platelet. In studying the Bernard Soulier platelets, extensive care was taken to remove contaminating leukocytes from the platelet preparations. Concurrent controls, which enumerated the number and affinity of binding sites for IgG trimer on patient leukocytes, confirmed that the results with the patient preparations were indeed due to platelets and not to neutrophils.
Efect ofAnti-Fey ReceptorMonoclonal Antibodies We examined the effect of two anti-Fey receptor monoclonal antibodies on platelet binding of IgG trimer. Monoclonal antibody 3G8, which is directed at an Fcy-binding site (FcyRIII) on tissue macrophages and polymorphonuclear leukocytes (16), did not inhibit the binding of IgG trimer to platelets or interefere with the affinity of binding. A second monoclonal antibody (IV.3, isotype IgG 2b) which binds to a 40,000 M, platelet membrane protein and interferes with platelet activation caused by IgG aggregates(17) did inhibit IgG trimer binding to both normal (Fig. 4a) and Bernard Soulier platelets. No inhibition was observed with a control IgG2b monoclonal antibody at identical protein concentrations.
Binding ofAnti-FcyRII Monoclonal Antibody to Platelets Binding studies with ‘251-radiolabeledanti-FcyRII were also performed in order to further define the number of Fey receptors expressed by normal human platelets. These studies were conducted under conditions identical to those previously described for the IgG trimer binding assay, namely at p = 0.07 ionic strength and 4°C. The binding of IgG trimer to these cells was concurrently studied in order to effectively compare the number of Fey receptors measured by the two assay systems. In
Fe-r RECEPTOR ON HUMAN
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4,
b
3
6
14
18 B (molecules/cell)
125 I - I gG ADDED (pg)
FIG. 4. (a) Equilibrium binding of IgG trimer to platelets preincubated with anti-Fey receptor monoclonal antibody. Platelets were preincubated with anti-Fey receptor monoclonal antibody or buffer for 30 min at 37°C and washed. Results with platelets preincubated with monoclonal antibody (A) or buffer (0) are shown. Buffer = 3 149 sites/cell, Kd = 7.8 X 10e8M; platelets + Mab = 775 sites/cell, Kd = 16.2 X 10e8 M. (b) Binding of IV.3 IgG and IV.3 Fab to normal human platelets. Washed platelets from a single donor were concurrently incubated with either “‘I-IV.3 IgG or “‘I-IV.3 Fab for I hr at 4°C p = 0.07. Platelets were incubated in both the presenceand absenceof a 50-fold molar excessof unlabeled protein in order to assessthe amount of nonspecific binding. Scatchard plots of the data are shown and indicate 2.2 times more binding sites for Fab as compared to the intact IgG. Binding of 12’I-IV.3 Fab (Cl) yielded 4729 binding sites per platelet and a Kd of 2.2 X 10e9M while only 2 172 sites per platelet were detected with ‘251-IV.3 IgG @) with a Kdof 2.98 X lo-” M.
five such studies performed, the mean number of Fey receptors was 5044 as determined by ‘251-anti-FcyRII binding compared to 523 1 obtained with IgG trimer. The individual results obtained with platelets from the five donors examined are presented in Table 4. The binding of ‘*‘I-Fab, generated by papain digestion of anti-FcyRII, TABLE 4 Comparison of Fey Receptor Expression as Determined by 1251-anti-FcyRII and ‘2SI-IgGTrimer Binding Fcr receptors/cell ’ Donor
Anti-FcyRII
IgG trimer
I 2 3 4 5
2695 5099 4745 4437 8243
3220 5793 3813 4962 8366
LINumber of &S-binding sites/cell expressedon the platelets obtained from each of five normal donors.
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SCHREIBER
was also investigated. Because of the possibility that the divalent IgG antibody may bind to two rather than to one receptor, we compared the number of Fcr receptors determined with ‘251-Fabto that measured with the intact radiolabeled IgG molecule. We detected approximately twice as many receptor sites with the Fab fragment than with the whole molecule (Fig. 4b). In 10 normal donors studied, we observed a mean ratio of Fab-detected sites to sites detected with the intact IgG molecule of 1.86 + 0.20 (SEM).
Isolation of Platelet Fey-Binding Protein We also isolated the platelet Fey-binding protein from intact whole platelets. Approximately 58 mg of protein extract, solubilized from 4 X 10” washed human platelets with 1% NP-40, were incubated with Fey-Sepharose 4B in a total volume of 50 ml. Following overnight incubation and extensive washing, as described under Methods, the protein bound to the gel was eluted with 0.5 N acetic acid and immediately neutralized with 2 M Tris base. An increase in OD at 280 nm was observed in five fractions eluting off the column after the void volume. These fractions were immediately dialyzed against 0.01 M Tris-HCl (pH 6.8) or PBS with 0.05% NP40. Total protein determinations performed on the material eluted off the column indicated a yield of 0.7 mg which corresponds to 1.2%of the protein extract originally applied to the column. A control experiment which involved incubation of the NP40-solubilized platelet extract with Sepharose 4B coupled to bovine serum albumin yielded no protein recovery following elution of the gel with acetic acid.
Characteristicsof the Fey-Binding Protein SDS-polyacrylamide gel electrophoresis was employed to determine the molecular weight of the protein recovered from the Fey-Sepharose 4B column. Upon reduction with 2-P-mercaptoethanol, a major band with apparent M, = 50,000 was observed on a 7.5% polyacrylamide gel (Fig. 5). In the unreduced state, the protein readily entered the stacking gel (3% acrylamide, pH 6.8) but did not enter the separating gel (7.5% acrylamide, pH 8.8). Molecular weight determination ofthe unreduced protein required the use of an agarose-acrylamide gel or a 4.5% SDS-polyacrylamide gel in a pH 7 phosphate-buffer system and yielded a band with M, = 220,000 upon Western blot analysis (Fig. 6). Identification of the platelet protein isolated from the Fey-Sepharose 4B column also was accomplished by assessingits ability to bind ‘251-IgGtrimer. First, when the Fey-binding protein was incubated with radiolabeled IgG trimer in an aqueous detergent-free EDTA buffer (1 mM EDTA/PBS), following extraction of the NP-40 with Extracti-Gel D (Pierce Chemical), the two coprecipitated. No such precipitation occurred when radiolabeled IgM was employed. Secondly, Western blot analysis further confirmed that the isolated protein was an IgG-binding protein (Fig. 6). When the protein was electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose, and then overlayed with I*?-IgG trimer, autoradiography revealed single bands of molecular weight 50,000 for the reduced form and 220,000 for the unreduced protein on 4.5% SDS-PAGE. DISCUSSION Much of the evidence supporting the existence of Fe-yreceptors on human platelets has come from studies of the effect of IgG on platelet function. Recently, more direct
Fey RECEPTOR ON HUMAN
ABCDEF
MW
92,500
-
66,200
-
31,000
-
21,500
-
PLATELETS
475
GHIJK
FIG. 5. SDS-PAGE of isolated Fey-binding protein. The material eked off the Fey-Sepharose 4B column was run on a 10% polyacrylamide gel under nonreducing (lanes A-F) and reducing (lanes G-K) conditions and then stained with Coomassie blue: lane A, molecular weight standards; lanes B and G, human IgG; lanes C and H, Fcr fragment; lanes D and I, platelet lysate; lanes E and J, platelet lysate that did not bind to column; lanes F and K, Fey-binding protein.
evidence has been provided by binding studies utilizing IgG ligand ( 18-20). We employed a similar approach to further characterize the interaction of IgG ligand with this platelet receptor. The determination of binding sites for IgG trimer on human platelets and the Kd for binding assumed that all three Fc subunits of a single trimer molecule interact with Fey receptors and that each IgG subunit binds to a single receptor. Evidence to support such an assumption has been provided in studies describing IgG oligomer binding to other cell types ( 17,2 1) and the results of our anti-FcyRII binding studies are consistent with this concept. When Fey receptors were enumerated on platelets from individual donors, similar results were determined using either anti-FcyRII antibody or IgG trimer (Table 4). However, it is not known whether each IgG antiFcyRII molecule binds to a single Fey receptor or whether the receptors are in close proximity such that a single molecule can bind to two different receptors. To further investigate this issue, binding studies with anti-FcyRII Fab fragments were performed concurrently. The observed approximate 2: 1 ratio of sites detected with the Fab compared with the intact molecule has one of two possible interpretations: (i) there are two epitopes recognized by the anti-FcyRII antibody per receptor, or (ii) each intact anti-FcyRII molecule is binding to two separatereceptors. The consistent correlation of binding sites obtained with IgG anti-FcyRII and IgG trimer supports the former possibility. In establishing the conditions under which IgG trimer binds optimally to human platelets, we examined the effect of ionic strength. Ionic strength dependence has been
476
ICING, MCDERMOTT,
AND SCHREIBER
b
a
A
MW
A6
MW
C
D
6 82,500-
200,000
-
116,250 92,50066,200
-
66,200 -
45,000 -
31,000
-
21,500
-
45,000-
FIG.6. (a) Western blot of Fey-binding protein. The material eluted off the Fey-Sepharose 48 column was electrophoresed on a 4.5% SDS-polyacrylamide phosphate gel and then transferred to nitrocellulose. The nitrocellulose was then overlayed with ‘2sI-IgG trimer: lane A, unreduced protein; lane B, reduced protein. (b) Western blot of Fey-binding protein. Neither the unreduced Fey-binding protein (lane B) nor corresponding band present in the whole platelet lysate (lane A) entered a 7.5% SDS-polyacrylamide Trisbuffered gel. Both remained at the interface between the stacking and separating gel. The reduced samples of each, which appear in lanes D and C, respectively, readily entered the separating gel and yielded 50,000 molecular weight bands.
reported for ligand-receptor interactions in other cell systems (22). In our studies, decreasing the ionic strength from P = 0.15 to 0.07 resulted in an increase in the number of IgG binding sites expressed per platelet without a change in affinity of binding. Although the mechanism of this effect is uncertain, it appears that lowering the ionic strength results in the expression of previously unexposed Fey binding sites. Alternatively, the observed increase in IgG trimer binding at low ionic strength could reflect changes induced in the ligand itself. Lowering the ionic strength may cause a conformational change in the Fey subunits resulting in greater accessibility for interaction with Fcr receptors. In addition, a decreasein ionic strength may induce further IgG trimer aggregation. However, the unchanged affinity and linearity of the Scatchard plots at or.= 0.7 argue against these latter possibilities. It has recently been reported that lowering the ionic strength resulted in enhancement of binding of aggregated murine IgG2b to U937 cells, presumably to FcyRII (23). Since it appearsthat platelet FcrRII is similar to FcyRII on U937 cells, FcyRII may react preferentially with oligomeric IgG at low ionic strength in several cell types. One focus was to examine the modulation of the platelet Fcr receptor. Since the platelet Fey receptor is likely important in the pathogenesis of platelet destruction in
Fe-y RECEPTOR
ON HUMAN
PLATELETS
477
some individuals with immune complex disorders, it would be helpful to identify those factors capable of modulating its expression. We first examined whether activation of the platelet by receptor-mediated agonists, such as thrombin and ADP, altered the binding of oligomeric IgG to the Fey receptor. The data indicate that perturbation of the platelet membrane by these agonists does not influence Fey receptor expression (Table 1) and suggestthat the platelet Fey receptor is distinct from those sites on the cell surface which interact with thrombin and ADP. Furthermore, the fact that ADP in the presence of fibrinogen also does not alter Fey receptor number or affinity suggeststhat interaction of fibrinogen with its GPIIb/GPIIIa binding site does not influence Fey receptor expression. These data, however, do not exclude an effect of these platelet agonists on a select population of individual platelets. Glucocorticoids are among the most commonly used pharmacologic agents in the treatment of immune platelet disorders (24, 25). The decreasein platelet-associated IgG observed in some patients with immune thrombocytopenic purpura (ITP) following glucocorticoid therapy may represent a direct effect on the platelet Fey receptor. Glucocorticoids also have been noted to alter Fey receptor expression on other cells (26,27). Our results with dexamethasone are consistent with this thesis (Table 2). Dexamethasone treatment of platelets from selectindividuals resulted in a decreasein the amount of IgG trimer bound by these cells and in the ability of these same cells to aggregatein response to heat-aggregatedIgG (Table 3). The mechanism of dexamethasone’s action is uncertain, but may reflect a direct effect on the platelet membrane (26, 28). Alternatively, but less likely, due to the minimal amount of platelet mRNA, dexamethasone may induce translational changes similar to that attributed to glucocorticoids in other cell systems(29). Anti-FcyRII binding to dexamethasonetreated platelets was unaltered suggesting that the effect of dexamethasone on the platelet is not on Fcr receptor protein expression per se. Binding of IgG ligand to platelets may involve interactions with Fc-,Jreceptor sites other than the epitope recognized by the IV.3 anti-FcyRII monoclonal antibody. Dexamethasone may influence such sites or alter the platelet membrane in a manner which perturbs Fey receptor function. Although the minimal concentration of dexamethasone necessary to decreasebinding of IgG ligand to platelets is uncertain, our preliminary data suggest that concentrations below 5 &4 are effective in some instances. Further studies will be necessary to delineate whether the platelet Fcr receptor is influenced by other pharmacologic agents and whether these agents in vivo alter the clinical course of immune platelet disorders. An additional focus of this study was to further establish the biochemical identity of the platelet Fe-yreceptor. Several investigators have proposed that the platelet Fey receptor may be one of several previously identified platelet glycoproteins. We studied the binding of IgG to platelets from patients with Glanzmann’s thrombasthenia and Bernard Soulier syndrome and did not observe any decrease or alteration in affinity of Fey-binding sites. Since thrombasthenic platelets are deficient in the GPIIb/IIIa complex (30), and Bernard Soulier platelets lack GPIb, GPV, and GPIX (3 l-34), the results suggestthat neither GPIIb/IIIa, GPIb, GPV nor GPIX represent the physiologic platelet surface Fcr receptor. Furthermore, we directly isolated an Fey-binding protein from normal human platelets by affinity chromatography with immobilized Fey fragments. A 220-kDa protein was recovered which upon reduction dissociated into a single major subunit with a molecular weight of 50,000 as determined by PAGE. These results are similar to those obtained by Cheng and Haw-
478
KING, MCDERMOTT,
AND SCHREIBER
iger (35) who isolated an Fey-binding protein from platelets which had apparent molecular weights of 255,000 and 50,000 under nonreducing and reducing conditions, respectively. These results are also consistent with observations made by Beardsley et al. (36), who studied the binding of radiolabeled anti-platelet antibodies to platelet lysates which were electrophoretically separated. They employed aggregated IgG as a control and observed that it bound to a 200,000 molecular weight protein on a nonreduced gel and to a 45,000 molecular weight band on a reduced gel. Recently, Vancura and Steiner (37) reported that derivatized Fey fragments bind to a 200,000 molecular weight platelet protein which appears as a 50-kDa band after reduction. On the basis of the above studies, we believe the platelet Fey receptor is a multicomponent protein with an apparent molecular weight of 200-250 kDa, with subunits of approximately 40-50 kDa. Recently, we have demonstrated this protein on human megakaryocytes and in megakaryocyte mRNA (38). The platelet Fey receptor has been described as a 40,000 molecular weight membrane protein by two independent groups of investigators (17, 39). Rosenfeld et al. (17) used the IV.3 anti-FcyRII antibody to affinity purify a 40,000 molecular weight protein from surface-radioiodinated human platelets. Kelton et al. (39) reported similar results using immobilized IgG for affinity purification. In neither casewas a large M, protein recovered. There are several possibilities for this apparent discrepancy. First, the 200-kDa protein was isolated from intact platelets which were not surface radiolabeled and, thus, may represent an intracellular structure. In addition, the methods employed in the isolation of the protein may be responsible for the molecular weight difference. IgG-Sepharose immunoabsorbants were used to isolate the 40kDa proteins which were then separated from the affinity gel by boiling in an SDSTris buffer ( 17,40). In contrast, the 200-kDa protein was isolated using an Fcy-fragment-Sepharose column and was recovered by washing the gel with either an acid or chaotropic agent (35). These differences in the isolation conditions may have favored one structure or the other. Perhaps the methods employed in the isolation of the 40kDa receptor disrupted the structure of a larger complex into 40-50 kDa subunits. Alternatively, the 200-kDa structure recovered may represent an aggregate of the smaller receptor. However, this seems less likely since radiolabeled aggregated IgG readily binds to a high M, band on polyacrylamide gels of lysed human platelets both in our studies and those of others (Fig. 6) (36). The fact that the isolated 200-kDa protein forms an in vitro complex with polyvalent IgG satisfies an important requirement for an Fey receptor, its binding of IgG ligand, and further supports the multicomponent high M, nature of the platelet Fey receptor. ACKNOWLEDGMENTS We thank Drs. C. M. Ingerman-Wojenski and Dr. Scott Murphy of Jefferson Medical College and Dr. Margaret Johnson of Wilmington, Delaware for permitting us to study their patients. We also thank Dr. Jay Unkeless of Mt. Sinai School of Medicine and Dr. Clark Anderson of Ohio State University for the gracious use of their monoclonal antibodies to Fey receptors. We appreciate the help of Ruth Rowan and Diane Meredith in preparing this manuscript for publication.
REFERENCES 1. Humphrey, J. H., and Jaques, R., J. Physiol. 128,9, 1955. 2. Mustard, J. F., Movat, H. Z., Macmorine, D. R. L., and Senji, A., Proc. Sot. Exp. Biol. Med. 119,988, 1965.
Fey RECEPTOR ON HUMAN
PLATELETS
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3. Mueller-Eckhardt, C. L., and Luscher, E. F., Thromb. Diath. Haemorrh. 20, 155, 1968. 4. Pfueller, S. L., and Luscher, E. F., J. Immunol. 109,5 17, 1972. 5. Henson, P. M., and Spiegelberg, H. L., J. Clin. Invest. 52, 1281, 1973. 6. Israels, E. D., Nisli, G., Paraskevas,F., and Israels, L. G. Thromb. Diath. Haemorrh. 29,434, 1973. 7. Trent, R. J., Clancy, R. L., Davis, V., and Basten, A., Brit. J. Haematol. 44,645, 1980. 8. Lurhama, A. G., Roccomi, H., and Masson, P. L., Clin. Exp. Immunol. 28,49, 1977. 9. Walsh, C. M., Nardi, M. A., and Karpatkin, S., N. Eng. J. Med. 311,635, 1984. 10. Kunicki, T. J., Johnson, M. M., and Aster, R. H., J. Clin. Invest. 62,716, 1978. 11. Segal, D. M., and Titus, J. A., J. Immunol. 120, 1395, 1978. 12. Porter, R. R., Biochem. J. 73,119, 1959. 13. McConahey, P. J., and Dixon, F. J., Int. Arch. Allergy Appl. Immunol. 29, 185, 1966. 14. Laemmli, U. K., Nature(London) 227,680, 1970. 15. Segal, D. M., and Hurwitz, E., J. Immunol. 118,1338, 1977. 16. Fleit, H. B., Wright, S. D., and Unkeless, J. C., Proc. Nat. Acad. Sci. USA 79,3275, 1982. 17. Rosenfeld, S. L., Looney, R. J., Leddy, J. P., Phipps, D. C., Abraham, J. N., and Anderson, C. L., J. Clin. Invest. 76,23 17, 1985. 18. Pfueller, S. L., Weber, S., and Luscher, E. F., J. Immunol. 118,5 14, 1977. 19. Moore, A., and Nachman, R. L., J. Clin. Invest. 67, 1064, 1981. 20. Karas, S. P., Rosse,W. F., and Kurlander, R. J., Blood60, 1272, 1982. 2 1. Kurlander, R. J., and Batker, J., J. Clin. Invest. 69, 1, 1982. 22. Fries, L. F., Prince, G. M., Gaither, T. A., and Frank, M. M., J. Immunol. 135,2673, 1985. 23. Jones, D. H., Looney, R. J., and Anderson, C. L., J. Immunol. 135,3348,1985. 24. Cines, D. B., and Schreiber, A. D., New Engl. J. Med. 300, 106, 1974. 25. Cines, D. B., Dusak, B., Tomaski, A., Mennuti, M., and Schreiber, A. D., New Engl. J. Med. 306,826, 1982. 26. Schreiber, A. D., Parson, J., McDermott, P., and Cooper, R. A., J. Clin. Invest. 56, 1189, 1978. 27. Crabtree, G. R., Munck, A., and Smith, K. A., Nature (London) 274,338, 1979. 28. Naray-Fejes-Toth, A., Cornwell, G. G., and Guyre, P. M., Immunology56,359, 1985. 29. Kern, J. A., Reed, J. C., Daniele, R. P., and Nowell, P. C., Clin. Res. 34,498A, 1986. 30. Bennett, J. S., Vilaire, G., and Cines, D. B. J. Biol. Chem. 257,8049, 1982. 3 1. Nurden, A. T., and Caen, J. P., Nature (London) 25,720, 1975. 32. Clemetson, K. J., McGregor, J. L., and James, E., J. Clin. Invest. 70, 304, 1982. 33. Hagen, I., Nurden, A., Bjerrum, 0. J., Solum, N. O., and Caen, J. P., J. Clin. Invest. 65,722, 1980. 34. Berndt, M. C., Gregory, C., Chong, B. H., Zola, H., and Costaldi, P. A., Blood62,800, 1983. 35. Cheng, C. M., and Hawiger, J., J. Biol. Chem. 254,2165, 1987. 36. Beardsley, D. S., Spiegel, J. E., Jacobs, M. M., Hardin, R. I., and Lux, S. E., J. Clin. Invest. 74, 1701, 1984. 37. Vancura, S., and Steiner, M., Proc. Natl. Acad. Sci. USA 84,3575, 1987. 38. Gewirtz, A., Rappaport, E., King, M., and Schreiber, A. D., Blood 72,332A, 1988. 39. Kelton, J. G., Smith, J. W., Santos, A. N., Murphy, W. G., and Horsewood, P., Amer. J. Hem. 25, 299, 1987.
IMMUNOLOGY
128,462-479 (1990)
Characterization MARIANNE
of the Fey Receptor on Human Platelets’T2
KING, PATRICIA MCDERMOTT,
AND ALAN
D. SCHREIBER
University of Pennsylvania Graduate Group in Immunology and Cancer Center, University of Pennsylvania School ofMedicine, Philadelphia, Pennsylvania 19104 Received October 4,1989; accepted January 13, 1990 IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fey receptor on the platelet surface. We studied the platelet Fey receptor and characterized its interaction with IgG ligand and anti-Fey receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. L&id-binding studies indicated that normal human platelets express a single Fcr receptor (FcrRII) with 8559 + 852 sites per cell, Kd = 12.5 f 1.7 X lo-* M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-FcyRII were consistent with each Fey receptor expressing two epitopes recognized by the antibody. The number of Fey binding sites and affinity of binding were unchanged by the presence of 2.0 mMMg*+ or 10 pg/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fe-r binding sites or the affinity of binding. However, platelets preincubated with 5 &dexamethasone expressed a decreasednumber of Fcr binding sites as well as decreasedIgG-dependent platelet aggregation. Platelets from patients with Glanzmann’s thrombasthenia and from patients with the Bernard Soulier syndrome expresseda normal number and affinity of Fcr binding sites. The data suggest that platelet FcyRII binding of trimeric IgG occurs independent of actin filament interaction, Mg*+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fey-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 M, subunits. 0 1990 Academic Press, Inc.
INTRODUCTION Platelets contain a number of factors that may be involved in inflammation (l-2). In immune complex disorders, platelets are thought to interact with IgG-containing immune complexes through an Fey binding site on the platelet surface (l-6). Only immunoglobulin aggregatesof the y isotype have the ability to stimulate platelets, suggesting that the critical site(s) on the immunoglobulin molecule responsible for platelet activation resideson the heavy chain ($6). These data have led to the concept of a platelet Fey receptor. i Supported by NIH Grants HL-28207 and HL-40387. ZPresented in part at the National Meetings of the American Association of Immunologists and the American Society of Hematology. 462 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
Fcr RECEPTOR
ON HUMAN
PLATELETS
463
The association of thrombocytopenia with diseasestates in which circulating immune complexes occur further suggeststhat immune complex-platelet interaction may represent a mechanism by which platelet destruction is induced. There is evidence that some patients with chronic immune thrombocytopenic purpura (ITP) have circulating immune complexes which correlate with the degreeof thrombocytopenia (7-9). Thus, in some immune platelet disorders platelet clearance may be induced by immune complexes which bind to the platelet Fey receptor in such a manner that Fey domains remain available for macrophage detection within the reticuloendothelial system. In this study we further characterized the interaction of the platelet Fey receptor with IgG ligand and anti-Fey receptor monoclonal antibody. In addition, we examined the effectsof several possible modulatory signals on the expression of this receptor, since the regulation of this Fey receptor may have pathophysiologic significance. Finally, we attempted to ascertain the biochemical identity of the platelet Fcr receptor through both ligand binding studies with glycoprotein-deficient platelets and through direct isolation of the platelet Fey binding protein. The data suggest that human platelets have a single surface Fe-r receptor which is distinct from platelet glycoproteins IIb/IIIa, and Ib, V, and IX and which migrates on PAGE at M,
= 220,000. METHODS
Patients We studied 25 normal platelet donors ranging in age from 25-50 years who were not on any medications and who were aspirin-free for at least 2 weeks. We studied in a single experiment two individuals with type II Glanzmann’s thrombasthenia known to be deficient in GPIIb/IIIa, who were kindly provided by Dr. Margaret Johnson, Wilmington, Delaware. We also studied three patients from a family with Bernard Soulier syndrome kindly provided by Drs. Scott Murphy and Ingerman Wojensky of Jefferson Medical College. The platelets from these patients have been previously reported and have been demonstrated to be deficient in GPIb (10).
Preparation of Platelets Platelets were isolated daily from the blood of normal human donors and were used in experiments immediately following their isolation. Briefly, blood was drawn into 0.3 ml of 5% disodium ethylene diaminetetraacetate (EDTA) per 10 ml blood, unless otherwise noted, and centrifuged for 15 min at 18Ogat room temperature in a Sorvall centrifuge (Beckman Instruments, New York). The platelet-rich plasma (PRP) was aspirated and centrifuged a second time at 18Ogto remove any remaining red cells or leukocytes. Leukocyte contamination was lessthan 0.0 1%.The platelets were then centrifuged at 1OOOgfor 13 min and washed three times with phosphate-buffered saline containing 12.5 mM EDTA and 1.0 mM adenosine. The washed cells were suspendedin the appropriate assaybuffer, quantitated and adjusted to a concentration of l-2 X 109/ml.
Preparation of Human IgG Human IgG was isolated from fresh human serum by ammonium sulfate precipitation followed by ion exchange chromatography (QAE-Sephadex, Pharmacia, Piscata-
464
KING,
MCDERMOTT,
AND
SCHREIBER
way, NJ) and/or gel filtration chromatography (Sephacryl S-300, Pharmacia, Piscataway, NJ). Oligomeric IgG was prepared according to the method of Segal and Titus (11). Briefly, purified IgG (70 mg/ml in 0.2 MTris-HCl buffer, pH 8.4) was incubated with a 16-fold molar excessof dimethyl suberimidate (Sigma Chemical Co., St. Louis, MO) for 1 hr at 30°C. The reaction was then quenched by the addition of a fivefold molar excessof glycine (with respect to dimethyl suberimidate) and the mixture immediately applied to a tandem column of Sephadex G-200 (Pharmacia, Piscataway, NJ) and Ultrogel AcA 22 (LKB Instruments, Inc., Gaithersburg, MD) and fractionated into oligomers of defined molecular weight. Pools of each oligomer were made and concentrated to 5- 10 mg/ml. Fey and Fab fragments of IgG were prepared by papain digestion of IgG monomer according to the method of Porter ( 12). Heat aggregated IgG was prepared each day by incubating IgG monomer for 20 min at 63°C. IgG was radiolabeled with ‘251-Na(New England Nuclear, Boston, MA) to a specific activity of l-2 X lo5 cpm/pg protein (13). Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis (PAGE) was performed using a modification of the Laemmli system (14). The stacking gel contained 3.0% acrylamide in 0.125 A4 Tris-HCl buffer, pH 6.8, and the separating gel 5.0-10.0% acrylamide in 0.375 M Tris-HCl buffer, pH 8.8. Samples were dissolved in 0.01 M Tris-HCl, pH 6.8, containing 0.1% SDS and incubated for 10 min in a boiling water bath before application to the gels. Reduced samples also contained 0.1% 2+mercaptoethanol. Gels (16 X 20 cm) were run in a Bio-Rad Protein II vertical gel apparatus (Bio-Rad, Richmond, CA) at 4°C overnight at 65 V. After electrophoresis, gels were fixed and stained in methanol-water-acetic acid (5:5: 1) containing 0.25% Coomassie blue for l-2 hr. Gels were destained in the same solution in the absenceof Coomassie blue. IgG Binding to Platelets The ability of oligomeric IgG to aggregate platelets necessitated that all binding studies be performed in the presence of EDTA. Assayswere performed in Dulbecco’s calcium and magnesium-free PBS (GIBCO, Grand Island, NY) containing 0.2% human serum albumin (Sigma Chemical, St. Louis, MO), 2 mM EDTA and 15 mA4 sodium azide. Washed platelets were suspended in this buffer at a concentration of l-2 X lo9 cells/ml and 1 X 10’ platelets were incubated with increasing concentrations of radioiodinated IgG oligomer in a total volume of 0.5 ml in the presence or absence of a loo-fold excessof unlabeled heat-aggregated IgG. Incubations were performed at either 37°C for 1 hr or 4°C for 3 hr at which time equilibrium of binding was achieved. The reaction mixture was layered over silicone oil (15 ml silicone + 80 ml biphenyl silicone; William F. Nye, Inc., New Bedford, MA), the platelets sedimented and the amount of cell-associated ‘251-IgGquantitated in a Beckman gamma counter (Beckman Instruments, New York). The concentration of free ‘25I-IgG was defined as the amount of radioactivity which remained unbound. Specific binding was defined as the cell-associated radioactivity not inhibited by the presence of a 1OOfold excessof unlabeled IgG and was analyzed by Scatchard plot. For these calculations a molecular weight of 150,000 for IgG was employed assuming that each IgG subunit binds to a single receptor (15). The number of IgG-binding sites and the
Fey RECEPTOR
ON HUMAN
PLATELETS
465
affinity of specific binding to platelets was determined by a linear regression program using the least squares method. Preparation of Purified Anti-FcTRII Monoclonal Antibody Anti-FcyRII monoclonal antibody IV.3 was isolated from ascites fluid using protein A (Affi-Gel Protein A Maps II, Bio-Rad, Richmond, CA). Briefly, 1.5 ml ascites fluid (kindly provided by Dr. Clark L. Anderson, Ohio State University, Columbus, OH) was applied to a 5-ml protein A-agarose column. The column was washed with 15 vol of buffer and the IgG eluted with pH 2.8 buffer. The eluted protein was dialyzed against PBS, concentrated to 7 mg/ml and radiolabeled with “‘1 (New England Nuclear, Boston, MA) using chloramine T ( 13) to a specific activity of 200,000 cpm/ pg. Experiments of equilibrium binding to platelets were performed using ‘251-antiFcyRII and unlabeled anti-FcyRII in a manner similar to the human IgG ligandbinding studies above. Fab fragments of the monoclonal were prepared by incubating purified IV.3 antibody with immobilized papain (Pierce, Rockford, IL) for 4 hr at 37°C. The Fab fragments were separated from the Fc fragments and undigested IgG on a protein A column as described above. Purity of the Fab fragment was confirmed by Ouchterlony analysis and SDS-PAGE. Protein concentration was estimated by measuring the absorbance at 280 nm (E f’?&= 14.0) and by the Lowry method. Platelet Aggregation Studies Platelets used in aggregation studies were isolated from PRP containing ACD by gel filtration. Approximately 4 ml PRP were applied to a 50-ml Sepharose4B (Pharmacia, Piscataway, NJ) column and the platelets eluted with Tyrodes buffer containing 0.1% glucose and 0.35% BSA. The platelets were adjusted to 2 X 108/ml and used immediately. Since IgG-dependent platelet aggregation requires the presence of extracellular Ca*‘, CaC12was added to the cell suspension to a final concentration of 2 mM. The platelet suspension (450 ~1) was pipetted into an aggregometer cuvette and incubated in a dual channel recording aggregometer(Chrono-Log Corp., Havertown, PA) at 37°C with stirring for 1 min. Fifty microliters of platelet-aggregating agent (heat-aggregatedhuman IgG, thrombin, or ADP + fibrinogen) were then added and the response was recorded for 10 min. Results were expressed as percentage of aggregation and were determined graphically from the aggregometercurve. Preparation of Fey-Sepharose 4B Column Fifteen grams of freeze-dried cyanogen bromide-activated Sepharose 4B (Pharmacia, Piscataway, NJ) were swollen and washed with 2 mM HCl and then resuspended in a coupling buffer consisting of 0.1 M NaHC03 + 0.5 M NaCl, pH 8.3. Isolated Fey fragment (10 mg/ml), solubilized in the same buffer, was immediately added to the gel in the proportion of 20 ml Fcr fragment solution/55 ml CNBractivated Sepharose 4B. Following a 2-hr incubation at room temperature, the gel was washed once with coupling buffer and then incubated an additional 1.5 hr at room temperature with 0.2 A4 glycine, pH 8.0. The gel was then washed with four cycles of alternating pH with each cycle consisting of one wash with 0.1 M acetate buffer, pH 4.0, and one wash with coupling buffer, pH 8.3. The gel was then sus-
466
KING,
MCDERMOTT,
AND
SCHREIBER
pended in PBS with 0.02% sodium azide and stored at 4°C until use. The residual protein present in the coupling buffer supernatant following the initial incubation and in the subsequent wash solutions was determined and indicated that the coupling efficiency for the Fcr fragment was 98%.
Isolation of Platelet Fey-Binding Protein Platelets were isolated from 3-4 units of blood obtained from normal donors as described above. The cells were washed 5X with PBS containing 12.5 mM EDTA and 1.O mM adenosine and pooled to yield approximately l-2 X 10” cells. The washed cells were pelleted, resuspended with 7.5 ml PBS containing 1%NP-40, 1 mA4 EDTA, 0.05 mMN-carbobenzoxy-L-glutamyl+tyrosine, l/3 units/ml aprotinin and 2 mMphenylmethyl-sulfonyl fluoride (all from Sigma Chemical Co., St. Louis, MO), and incubated on ice for 30 min. The detergent-solubilized cells were then centrifuged in an ultra centrifuge (Beckman Instruments, Inc., Palo Alto, CA) for 1 hr @ 100,OOOg. The supematant was removed and incubated with 42.5 ml ofgel consisting of Fey fragments (3.8 mg Fey fragment per milliliter Sepharose4B) coupled to cyanogen bromide-activated Sepharose4B (Pharmacia, Piscataway, N.J.). The incubation was performed overnight in a sterile flask with shaking at 4°C. The gel was then poured into a 50-ml column and washed with at least 25 vol of PBS containing 1 r&4 EDTA and 0.5% NP-40. Bound protein was eluted from the column by washing with 2 vol of 0.5 N acetic acid containing 0.5% NP-40. One-milliliter fractions were collected into 0.2 ml 2 MTris baseand examined at OD 280 nm. Protein-containing fractions were immediately dialized against 0.01 A4 Tris-HCl, pH 6.8, or PBS with 0.05% NP-40, pH 7.0.
WesternBlot Analysis Western blots were employed to identify platelet Fey binding proteins isolated from intact platelets. Proteins electrophoresed on polyacrylamide or agarose/polyacrylamide gels were transferred to nitrocellulose (Schleicher and Schuell, Inc., Keene, NH) using Bio-Rad’s Trans-Blot electrophoretic transfer cell (Bio-Rad, Richmond, CA). Gels were first preequilibrated in transfer buffer (25 mA4 Tris, 192 mM glycine, 20% methanol) for 20-60 mitt, overlayed with nitrocellulose and sandwiched between Whatman 3-mm filter paper and gel pads as described in the Trans-Blot protocol. The transfer was performed at 4°C overnight. The efficacy of the transfer procedure was assessedby staining the polyacrylamide gel slab and a portion of the nitrocellulose with Coomassie blue. Following the transfer, the nitrocellulose blots were incubated at room temperature with 3.0% bovine serum albumin in Denhardt’s borate-buffered saline (166.5 mM H3B03, 27.5 mM NaOH, 150 mM NaCl with 6 mM NaNa,, 1 g/liter Ficoll, 1 g/liter polyvinylpyrrolidone, 15 g/liter BSA, and 0.1% NP-40) for at least 3 hr to minimize nonspecific binding of proteins to the paper. The blots were then incubated an additional 2 hr with ‘*‘I-IgG trimer (200,000 cpm/lO ml Denhardt’s). Unbound radioactive protein was removed by washing the blots five times in Denhardt’s buffer or until any background radioactivity was eliminated. The blots were air-dried and exposed to Kodak X-Omat AR film for 3-7 days for development of the autoradiograph.
Fey RECEPTOR ON HUMAN
PLATELETS
467
125 I - IgG Added (pg)
FIG. 1. Binding of ‘251-IgGto human platelets. Isolated ‘251-IgGmonomer, dimer, and trimer were added in increasing concentrations to 1 X 10’ platelets and incubated in the presence and absence of a IOO-fold excessof unlabeled heat-aggregatedIgG at 4°C for 3 hr. A representative experiment illustrating the number of molecules of ‘*%IgG specifically bound is shown.
RESULTS Characteristics of IgG Binding to Normal Platelets We first examined the ability of normal human platelets to bind both radiolabeled monomeric and oligomeric IgG. We observed no significant specific binding of IgG monomer to platelets (less than 150 molecules IgG/platelet) over a wide range of monomeric IgG input. We then compared isolated IgG oligomers of varying molecular weight in their binding to platelets and we observed that the amount bound increasedas the degree of IgG polymerization increased (Fig. 1). Oligomer binding was inhibited by isolated Fcr but not by Fab fragments. We employed IgG of M, = 450,000 (IgG trimer) in all subsequent binding studies to avoid inconsistencies which might result with the use of larger, more heterogenous oligomer preparations. When platelets were incubated with radiolabeled IgG trimer at 4°C binding of lz51IgG ligand reached equilibrium within 3 hr. Furthermore, platelet-bound IgG trimer was displaced by the addition of heat-aggregatedIgG. Aggregated IgG was five times more effective than monomer in displacing the bound trimer. After 3 hr at 4°C platelet binding of IgG trimer was saturable and Scatchard plots of the data were linear, consistent with a single class of binding sites for IgG trimer on platelets (Fig. 2). Efect of Ionic Strength We further characterized the optimal conditions for IgG trimer binding by studying the effect of changesin ionic strength. We varied the ionic strength of the assaybuffer, while maintaining constant osmolarity, by the addition of either dextrose, D-mannose, or D-mannitol. The results were similar for each of these three sugars. Specific binding of IgG trimer under equilibrium conditions was saturable at ionic strengths
468
RING, MCDERMOTT,
AND SCHREIBER
125 I - I gG Added (pg) FIG. 2. Equilibrium binding of IgG trimer to platelets. A representative experiment illustrates the equilibrium binding of I25I- I gG trimer to platelets. Washed platelets were incubated with increasing concentrations of IgG trimer in a final volume of 0.5 ml for 3 hr at 4°C. The Scatchard plot of the data (inset) suggests that only one class of binding sites is present.
ranging from P= 0.07 to CL= 0.15 and Scatchard plots of the data were linear. When the ionic strength was progressively lowered from p = 0.15 to p = 0.07, we observed an increase in the total number of IgG binding sites expressedper platelet (Fig. 3). At ionic strength P = 0.07, 10,200 f 2741 (mean + SD) IgG binding sites/platelet were expressed (N = 5), while at P = 0.15, 4045 f 1195 (mean f SD) binding sites were expressed per platelet (N = 5). The affinity of IgG trimer binding to platelets was similar at p = 0.07 and 0.15. We also observed that this effect of ionic strength was reversible.
Normal Platelets We next studied the platelets obtained from 25 consecutive normal adults for the number of binding sites and affinity of binding for IgG trimer. The platelets from these normal donors expressed 8559 + 852 (mean +- SEM); range = 2825 - 18,290) binding sites for IgG trimer at ionic strength P= 0.07. The affinity of binding was Kd = 12.5 + 1.7 X 1O-’ M (mean + SEM) and did not differ substantially for the platelets expressing high or low numbers of Fcr binding sites.
Fcr RECEPTOR ON HUMAN
PLATELETS
469
I23
2
4
6
8
IO
12
14
1251- IgG Added (pg) FIG. 3. Effect of ionic strength. Platelets were incubated with %IgG at ionic strength of rr = 0.15 (A) or p = 0.07 (B). At r.~= 0.07, more ‘*51-IgGspecifically bound to platelets at equilibrium. Scatchard plots of the data (insets) demonstrate an increase in the number of IgG-binding sites expressed per platelet at low ionic strength. In this representative experiment at p = 0.15 (A), 2700 binding sites/platelet were expressed; at Jo= 0.07 (B), 5200 siteswere expressed.The affinity ofbinding was unaffected by change in ionic strength.
Efect of Divalent Cations We explored the divalent cation requirement for the binding of IgG to platelets. Since the above studies were performed in the presence of EDTA and in the absence of free Ca*+ and Mg*+, we determined whether the binding of IgG trimer by platelets was altered in the presence of Mg *+. We examined the effect of Mg*+ by substituting EGTA for EDTA at equimolar concentrations. We compared the effect of 2 mM EDTA, 2 m1J4EGTA, and 2 mM EGTA containing 2 mit4 Mg*+. Equal numbers of IgG-binding sites and affinities of binding were observed under each of these three conditions (Table 1). Efect of Cytochalasin B The presentation of a relatively fixed array of IgG to the platelet surface appears to be important in platelet binding and stimulation by IgG and suggeststhat the bridging of some cell surface molecules, e.g., the Fcr receptor, may be necessaryfor effective interaction of platelets with IgG oligomer. Such an interaction may require an intact platelet membrane cytoskeleton. We examined the effect of cytochalasin B, an inhibitor of cytoskeleton function, on the ability of platelets to bind IgG trimer. Platelets were incubated with lo-100 &ml of cytochalasin B for 30 min at 37°C and the number and affinity of IgG binding sites were determined in the presence of cytochalasin B (Table 1). Cytochalasin B did not alter the number or affinity of plateletbinding sites for IgG trimer. Thus, an intact cytoskeleton does not appear necessary
470
RING, MCDERMOTT,
AND SCHREIBER
TABLE 1 Effect of Mg’+, Thrombin, and ADP on Platelet Fey Receptor Expression Number of binding sites/cell’
Kd
Experiment
Control-EDTA (2 mA4) EGTA (2 mM) EGTA (2 mA4) + Mg*+ (2 mM)
6153 5872 6012
6.6 x lOmEM 7.6 X 1O-8M 6.6 X 1O-8 M
Control’ Thrombin (1 unit/ml)
5798 5522
9.1 x 10m8M 8.3 x 1O-8 M
Control’ ADP(lO&) ADP ( 10 PM) + fibrinogen (100 &ml)
2825 2326 2551
9.5 x IO-* M 6.9 X 1O-8M 11.1 X IO-*M
’ Represents the maximum number of IgG molecules bound per platelet at saturation (B max). ’ Platelets were preincubated in either 2 mM EDTA/PBS (control) or 2 m&f EDTA/PBS containing 1 unit/ml thrombin for 30 min at 37°C. Similar results were observed with 10 units/ml thrombin. ’ Platelets were incubated in Tyrode’s buffer (control) or in Tyrode’s buffer containing 10pM ADP with and without fibrinogen. Incubations were performed at room temperature for 15 min without stirring and the binding of ‘251-IgGtrimer was then assessedin the presence of 1 mMEDTA.
for efficient platelet binding of oligomeric IgG. The biological activity of the cytochalasin B was confirmed by its ability to inhibit ADP and thrombin-induced platelet aggregation. Modulation of Platelet Fey Binding Sites We next sought to determine what factors might modulate expression of the platelet Fey receptor. We first investigated whether platelet activation induced by the interaction of physiologic agonists, such as thrombin and ADP, alters the binding of IgG ligand to its platelet Fey-binding site. Platelets were incubated with 0.01-10.0 units/ml of thrombin for 30 min at 37°C without stirring. The ability of these concentrations of thrombin to cause platelet aggregation in titrated PRP confirmed that the preparations were biologically active. We observed that preincubation with thrombin had no substantial effect on Fey receptor number or affinity (Table 1). The effect of ADP on the binding of IgG trimer by platelets was examined under two conditions: (i) in the presence of 2 mM EDTA and (ii) in Tyrode’s buffer in the presence of 2 mM Ca‘+ , 2 mM Mg2+ and fibrinogen ( 100 pg/ml), but without stirring to avoid aggregation. In the latter case,the platelets were isolated from blood collected in 0.38% citrate, washed, and resuspended in Tyrode’s buffer. Following preincubation with ADP, EDTA was added to the platelets to a final concentration of 2 mM for the IgG ligand binding studies. We found that incubation of platelets with 10 N ADP did not substantially affect their ability to bind IgG trimer (Table 1). The activity of the ADP employed in these studies was confirmed by observing its ability to aggregate platelets which were incubated under identical conditions as our experimental preparations, but with stirring. Eflect of Dexamethasone Glucocorticoids have been observed to modulate Fey receptor expression in some cells and cell lines. Therefore, we studied the effect of the glucocorticoid hormone
Fey RECEPTOR
ON HUMAN
471
PLATELETS
TABLE 2 Effect of Dexamethasone
on Fey Receptor Expression”
Fey receptors/cell b Donor 1 2 3 4 5 6
Control 8,824 10,679 9,072 13,561 5,312 f 322’ 8,074 f 443d
Dexamethasone 4846 3805 7910 2709 3096 f 248 4910+619
% Inhibition 45.1 64.4 12.8 80.0 42.4 39.2
’ Platelets were incubated in PRP with either PBS (control) or 5 & dexamethasone for 18-24 hr at room temperature and then isolated and washed. b Represents the maximum number of binding sites for IgG trimer as determined by Scatchard analysis. ’ Mean + 2 SEM for five separate determinations on the platelets from patient 5. d Mean f 2 SEM for two separate determinations on the platelets from patient 6.
dexamethasone (Sigma Chemical Co., St. Louis, MO) on platelet Fey-receptor expression. Platelets were incubated with 5 PM dexamethasone or an equal volume of PBS in PRP with ACD for 18-48 hr at room temperature under sterile conditions. The ability of these cells to bind IgG trimer and aggregatein response to heat-aggregated IgG was then assessed.Platelet yield was >95% in both the dexamethasone- and buffer-treated preparations. In 7 of 12 donors tested, we found that dexamethasone treatment decreasedthe binding of IgG trimer to platelets by as much as 80% (Table 2). The extent of the effect varied from donor to donor. In parallel, we examined the effect of dexamethasone on IgG-dependent platelet aggregation. Dexamethasone-treated platelets used in these studies were isolated from PRP by gel filtration. We observed in each of three donors whose dexamethasonetreated platelets expressed a decreasednumber of binding sites for IgG trimer, that dexamethasone also inhibited platelet aggregation by heat-aggregatedIgG (Table 3). The ability of these dexamethasone-treated platelets to aggregatein response to the platelet agonists ADP and thrombin was unaltered. Platelets from several individuals had no alteration in the expression of their binding sites for IgG following preincubation with dexamethasone. Dexamethasone-treated platelets from these donors also aggregated normally in response to heat-aggregated IgG. Dexamethasone had no effect on anti-FcyRII monoclonal antibody binding to platelets, as assessedby flow cytometry, in any of the donors tested. Binding of IgG to Platelets from Patients with Glanzmannk Thrombasthenia and Bernard Soulier Syndrome We were able to study the binding of IgG trimer to platelets from patients with two disorders characterized by the lack of specific glycoproteins on the platelet surface, Glanzmann’s thrombasthenia and Bernard Soulier syndrome. In each experiment, equilibrium binding studies were compared to a concurrent normal control. Equilibrium binding experiments showed that the platelets from all the patients expresseda number of binding sites and affinity of binding similar to that of the platelets from
472
RING, MCDERMOTT,
AND SCHREIBER
TABLE 3 Effect of Dexamethasone on Fey Receptor-Dependent Platelet Aggregation’ Donor
Platelet agonist b
% Inhibition of aggregation’ by dexamethasone
1
Heat-aggregated IgG ADP Thrombin Heat-aggregated IgG ADP Thrombin Heat-aggregated IgG ADP Thrombin
86.7 0 3.4 58.8 0 0 64.4 0 1
2 3
’ Platelets were preincubated in PRP with 5 pkfdexamethasone for 18-24 hr at room temperature and then isolated by gel filtration. ’ 50 pl platelet agonist (5 mg/ml heat-aggregatedIgG, 50 &f ADP, or0.2 units/ml thrombin) were added to 450-p] gel-filtered platelets in Tyrode’s buffer. ’ Determined by comparison to control platelets incubated with PBS.
our population of 25 normal controls. Platelets from the patients with Glanzmann’s thrombocytopenia expressed 14,172 and 13,320 binding sites/platelet. The platelets from the patient with Bernard Soulier syndrome expressed 5865 sites/platelet. In studying the Bernard Soulier platelets, extensive care was taken to remove contaminating leukocytes from the platelet preparations. Concurrent controls, which enumerated the number and affinity of binding sites for IgG trimer on patient leukocytes, confirmed that the results with the patient preparations were indeed due to platelets and not to neutrophils.
Efect ofAnti-Fey ReceptorMonoclonal Antibodies We examined the effect of two anti-Fey receptor monoclonal antibodies on platelet binding of IgG trimer. Monoclonal antibody 3G8, which is directed at an Fcy-binding site (FcyRIII) on tissue macrophages and polymorphonuclear leukocytes (16), did not inhibit the binding of IgG trimer to platelets or interefere with the affinity of binding. A second monoclonal antibody (IV.3, isotype IgG 2b) which binds to a 40,000 M, platelet membrane protein and interferes with platelet activation caused by IgG aggregates(17) did inhibit IgG trimer binding to both normal (Fig. 4a) and Bernard Soulier platelets. No inhibition was observed with a control IgG2b monoclonal antibody at identical protein concentrations.
Binding ofAnti-FcyRII Monoclonal Antibody to Platelets Binding studies with ‘251-radiolabeledanti-FcyRII were also performed in order to further define the number of Fey receptors expressed by normal human platelets. These studies were conducted under conditions identical to those previously described for the IgG trimer binding assay, namely at p = 0.07 ionic strength and 4°C. The binding of IgG trimer to these cells was concurrently studied in order to effectively compare the number of Fey receptors measured by the two assay systems. In
Fe-r RECEPTOR ON HUMAN
473
PLATELETS
4,
b
3
6
14
18 B (molecules/cell)
125 I - I gG ADDED (pg)
FIG. 4. (a) Equilibrium binding of IgG trimer to platelets preincubated with anti-Fey receptor monoclonal antibody. Platelets were preincubated with anti-Fey receptor monoclonal antibody or buffer for 30 min at 37°C and washed. Results with platelets preincubated with monoclonal antibody (A) or buffer (0) are shown. Buffer = 3 149 sites/cell, Kd = 7.8 X 10e8M; platelets + Mab = 775 sites/cell, Kd = 16.2 X 10e8 M. (b) Binding of IV.3 IgG and IV.3 Fab to normal human platelets. Washed platelets from a single donor were concurrently incubated with either “‘I-IV.3 IgG or “‘I-IV.3 Fab for I hr at 4°C p = 0.07. Platelets were incubated in both the presenceand absenceof a 50-fold molar excessof unlabeled protein in order to assessthe amount of nonspecific binding. Scatchard plots of the data are shown and indicate 2.2 times more binding sites for Fab as compared to the intact IgG. Binding of 12’I-IV.3 Fab (Cl) yielded 4729 binding sites per platelet and a Kd of 2.2 X 10e9M while only 2 172 sites per platelet were detected with ‘251-IV.3 IgG @) with a Kdof 2.98 X lo-” M.
five such studies performed, the mean number of Fey receptors was 5044 as determined by ‘251-anti-FcyRII binding compared to 523 1 obtained with IgG trimer. The individual results obtained with platelets from the five donors examined are presented in Table 4. The binding of ‘*‘I-Fab, generated by papain digestion of anti-FcyRII, TABLE 4 Comparison of Fey Receptor Expression as Determined by 1251-anti-FcyRII and ‘2SI-IgGTrimer Binding Fcr receptors/cell ’ Donor
Anti-FcyRII
IgG trimer
I 2 3 4 5
2695 5099 4745 4437 8243
3220 5793 3813 4962 8366
LINumber of &S-binding sites/cell expressedon the platelets obtained from each of five normal donors.
474
KING,
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SCHREIBER
was also investigated. Because of the possibility that the divalent IgG antibody may bind to two rather than to one receptor, we compared the number of Fcr receptors determined with ‘251-Fabto that measured with the intact radiolabeled IgG molecule. We detected approximately twice as many receptor sites with the Fab fragment than with the whole molecule (Fig. 4b). In 10 normal donors studied, we observed a mean ratio of Fab-detected sites to sites detected with the intact IgG molecule of 1.86 + 0.20 (SEM).
Isolation of Platelet Fey-Binding Protein We also isolated the platelet Fey-binding protein from intact whole platelets. Approximately 58 mg of protein extract, solubilized from 4 X 10” washed human platelets with 1% NP-40, were incubated with Fey-Sepharose 4B in a total volume of 50 ml. Following overnight incubation and extensive washing, as described under Methods, the protein bound to the gel was eluted with 0.5 N acetic acid and immediately neutralized with 2 M Tris base. An increase in OD at 280 nm was observed in five fractions eluting off the column after the void volume. These fractions were immediately dialyzed against 0.01 M Tris-HCl (pH 6.8) or PBS with 0.05% NP40. Total protein determinations performed on the material eluted off the column indicated a yield of 0.7 mg which corresponds to 1.2%of the protein extract originally applied to the column. A control experiment which involved incubation of the NP40-solubilized platelet extract with Sepharose 4B coupled to bovine serum albumin yielded no protein recovery following elution of the gel with acetic acid.
Characteristicsof the Fey-Binding Protein SDS-polyacrylamide gel electrophoresis was employed to determine the molecular weight of the protein recovered from the Fey-Sepharose 4B column. Upon reduction with 2-P-mercaptoethanol, a major band with apparent M, = 50,000 was observed on a 7.5% polyacrylamide gel (Fig. 5). In the unreduced state, the protein readily entered the stacking gel (3% acrylamide, pH 6.8) but did not enter the separating gel (7.5% acrylamide, pH 8.8). Molecular weight determination ofthe unreduced protein required the use of an agarose-acrylamide gel or a 4.5% SDS-polyacrylamide gel in a pH 7 phosphate-buffer system and yielded a band with M, = 220,000 upon Western blot analysis (Fig. 6). Identification of the platelet protein isolated from the Fey-Sepharose 4B column also was accomplished by assessingits ability to bind ‘251-IgGtrimer. First, when the Fey-binding protein was incubated with radiolabeled IgG trimer in an aqueous detergent-free EDTA buffer (1 mM EDTA/PBS), following extraction of the NP-40 with Extracti-Gel D (Pierce Chemical), the two coprecipitated. No such precipitation occurred when radiolabeled IgM was employed. Secondly, Western blot analysis further confirmed that the isolated protein was an IgG-binding protein (Fig. 6). When the protein was electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose, and then overlayed with I*?-IgG trimer, autoradiography revealed single bands of molecular weight 50,000 for the reduced form and 220,000 for the unreduced protein on 4.5% SDS-PAGE. DISCUSSION Much of the evidence supporting the existence of Fe-yreceptors on human platelets has come from studies of the effect of IgG on platelet function. Recently, more direct
Fey RECEPTOR ON HUMAN
ABCDEF
MW
92,500
-
66,200
-
31,000
-
21,500
-
PLATELETS
475
GHIJK
FIG. 5. SDS-PAGE of isolated Fey-binding protein. The material eked off the Fey-Sepharose 4B column was run on a 10% polyacrylamide gel under nonreducing (lanes A-F) and reducing (lanes G-K) conditions and then stained with Coomassie blue: lane A, molecular weight standards; lanes B and G, human IgG; lanes C and H, Fcr fragment; lanes D and I, platelet lysate; lanes E and J, platelet lysate that did not bind to column; lanes F and K, Fey-binding protein.
evidence has been provided by binding studies utilizing IgG ligand ( 18-20). We employed a similar approach to further characterize the interaction of IgG ligand with this platelet receptor. The determination of binding sites for IgG trimer on human platelets and the Kd for binding assumed that all three Fc subunits of a single trimer molecule interact with Fey receptors and that each IgG subunit binds to a single receptor. Evidence to support such an assumption has been provided in studies describing IgG oligomer binding to other cell types ( 17,2 1) and the results of our anti-FcyRII binding studies are consistent with this concept. When Fey receptors were enumerated on platelets from individual donors, similar results were determined using either anti-FcyRII antibody or IgG trimer (Table 4). However, it is not known whether each IgG antiFcyRII molecule binds to a single Fey receptor or whether the receptors are in close proximity such that a single molecule can bind to two different receptors. To further investigate this issue, binding studies with anti-FcyRII Fab fragments were performed concurrently. The observed approximate 2: 1 ratio of sites detected with the Fab compared with the intact molecule has one of two possible interpretations: (i) there are two epitopes recognized by the anti-FcyRII antibody per receptor, or (ii) each intact anti-FcyRII molecule is binding to two separatereceptors. The consistent correlation of binding sites obtained with IgG anti-FcyRII and IgG trimer supports the former possibility. In establishing the conditions under which IgG trimer binds optimally to human platelets, we examined the effect of ionic strength. Ionic strength dependence has been
476
ICING, MCDERMOTT,
AND SCHREIBER
b
a
A
MW
A6
MW
C
D
6 82,500-
200,000
-
116,250 92,50066,200
-
66,200 -
45,000 -
31,000
-
21,500
-
45,000-
FIG.6. (a) Western blot of Fey-binding protein. The material eluted off the Fey-Sepharose 48 column was electrophoresed on a 4.5% SDS-polyacrylamide phosphate gel and then transferred to nitrocellulose. The nitrocellulose was then overlayed with ‘2sI-IgG trimer: lane A, unreduced protein; lane B, reduced protein. (b) Western blot of Fey-binding protein. Neither the unreduced Fey-binding protein (lane B) nor corresponding band present in the whole platelet lysate (lane A) entered a 7.5% SDS-polyacrylamide Trisbuffered gel. Both remained at the interface between the stacking and separating gel. The reduced samples of each, which appear in lanes D and C, respectively, readily entered the separating gel and yielded 50,000 molecular weight bands.
reported for ligand-receptor interactions in other cell systems (22). In our studies, decreasing the ionic strength from P = 0.15 to 0.07 resulted in an increase in the number of IgG binding sites expressed per platelet without a change in affinity of binding. Although the mechanism of this effect is uncertain, it appears that lowering the ionic strength results in the expression of previously unexposed Fey binding sites. Alternatively, the observed increase in IgG trimer binding at low ionic strength could reflect changes induced in the ligand itself. Lowering the ionic strength may cause a conformational change in the Fey subunits resulting in greater accessibility for interaction with Fcr receptors. In addition, a decreasein ionic strength may induce further IgG trimer aggregation. However, the unchanged affinity and linearity of the Scatchard plots at or.= 0.7 argue against these latter possibilities. It has recently been reported that lowering the ionic strength resulted in enhancement of binding of aggregated murine IgG2b to U937 cells, presumably to FcyRII (23). Since it appearsthat platelet FcrRII is similar to FcyRII on U937 cells, FcyRII may react preferentially with oligomeric IgG at low ionic strength in several cell types. One focus was to examine the modulation of the platelet Fcr receptor. Since the platelet Fey receptor is likely important in the pathogenesis of platelet destruction in
Fe-y RECEPTOR
ON HUMAN
PLATELETS
477
some individuals with immune complex disorders, it would be helpful to identify those factors capable of modulating its expression. We first examined whether activation of the platelet by receptor-mediated agonists, such as thrombin and ADP, altered the binding of oligomeric IgG to the Fey receptor. The data indicate that perturbation of the platelet membrane by these agonists does not influence Fey receptor expression (Table 1) and suggestthat the platelet Fey receptor is distinct from those sites on the cell surface which interact with thrombin and ADP. Furthermore, the fact that ADP in the presence of fibrinogen also does not alter Fey receptor number or affinity suggeststhat interaction of fibrinogen with its GPIIb/GPIIIa binding site does not influence Fey receptor expression. These data, however, do not exclude an effect of these platelet agonists on a select population of individual platelets. Glucocorticoids are among the most commonly used pharmacologic agents in the treatment of immune platelet disorders (24, 25). The decreasein platelet-associated IgG observed in some patients with immune thrombocytopenic purpura (ITP) following glucocorticoid therapy may represent a direct effect on the platelet Fey receptor. Glucocorticoids also have been noted to alter Fey receptor expression on other cells (26,27). Our results with dexamethasone are consistent with this thesis (Table 2). Dexamethasone treatment of platelets from selectindividuals resulted in a decreasein the amount of IgG trimer bound by these cells and in the ability of these same cells to aggregatein response to heat-aggregatedIgG (Table 3). The mechanism of dexamethasone’s action is uncertain, but may reflect a direct effect on the platelet membrane (26, 28). Alternatively, but less likely, due to the minimal amount of platelet mRNA, dexamethasone may induce translational changes similar to that attributed to glucocorticoids in other cell systems(29). Anti-FcyRII binding to dexamethasonetreated platelets was unaltered suggesting that the effect of dexamethasone on the platelet is not on Fcr receptor protein expression per se. Binding of IgG ligand to platelets may involve interactions with Fc-,Jreceptor sites other than the epitope recognized by the IV.3 anti-FcyRII monoclonal antibody. Dexamethasone may influence such sites or alter the platelet membrane in a manner which perturbs Fey receptor function. Although the minimal concentration of dexamethasone necessary to decreasebinding of IgG ligand to platelets is uncertain, our preliminary data suggest that concentrations below 5 &4 are effective in some instances. Further studies will be necessary to delineate whether the platelet Fcr receptor is influenced by other pharmacologic agents and whether these agents in vivo alter the clinical course of immune platelet disorders. An additional focus of this study was to further establish the biochemical identity of the platelet Fe-yreceptor. Several investigators have proposed that the platelet Fey receptor may be one of several previously identified platelet glycoproteins. We studied the binding of IgG to platelets from patients with Glanzmann’s thrombasthenia and Bernard Soulier syndrome and did not observe any decrease or alteration in affinity of Fey-binding sites. Since thrombasthenic platelets are deficient in the GPIIb/IIIa complex (30), and Bernard Soulier platelets lack GPIb, GPV, and GPIX (3 l-34), the results suggestthat neither GPIIb/IIIa, GPIb, GPV nor GPIX represent the physiologic platelet surface Fcr receptor. Furthermore, we directly isolated an Fey-binding protein from normal human platelets by affinity chromatography with immobilized Fey fragments. A 220-kDa protein was recovered which upon reduction dissociated into a single major subunit with a molecular weight of 50,000 as determined by PAGE. These results are similar to those obtained by Cheng and Haw-
478
KING, MCDERMOTT,
AND SCHREIBER
iger (35) who isolated an Fey-binding protein from platelets which had apparent molecular weights of 255,000 and 50,000 under nonreducing and reducing conditions, respectively. These results are also consistent with observations made by Beardsley et al. (36), who studied the binding of radiolabeled anti-platelet antibodies to platelet lysates which were electrophoretically separated. They employed aggregated IgG as a control and observed that it bound to a 200,000 molecular weight protein on a nonreduced gel and to a 45,000 molecular weight band on a reduced gel. Recently, Vancura and Steiner (37) reported that derivatized Fey fragments bind to a 200,000 molecular weight platelet protein which appears as a 50-kDa band after reduction. On the basis of the above studies, we believe the platelet Fey receptor is a multicomponent protein with an apparent molecular weight of 200-250 kDa, with subunits of approximately 40-50 kDa. Recently, we have demonstrated this protein on human megakaryocytes and in megakaryocyte mRNA (38). The platelet Fey receptor has been described as a 40,000 molecular weight membrane protein by two independent groups of investigators (17, 39). Rosenfeld et al. (17) used the IV.3 anti-FcyRII antibody to affinity purify a 40,000 molecular weight protein from surface-radioiodinated human platelets. Kelton et al. (39) reported similar results using immobilized IgG for affinity purification. In neither casewas a large M, protein recovered. There are several possibilities for this apparent discrepancy. First, the 200-kDa protein was isolated from intact platelets which were not surface radiolabeled and, thus, may represent an intracellular structure. In addition, the methods employed in the isolation of the protein may be responsible for the molecular weight difference. IgG-Sepharose immunoabsorbants were used to isolate the 40kDa proteins which were then separated from the affinity gel by boiling in an SDSTris buffer ( 17,40). In contrast, the 200-kDa protein was isolated using an Fcy-fragment-Sepharose column and was recovered by washing the gel with either an acid or chaotropic agent (35). These differences in the isolation conditions may have favored one structure or the other. Perhaps the methods employed in the isolation of the 40kDa receptor disrupted the structure of a larger complex into 40-50 kDa subunits. Alternatively, the 200-kDa structure recovered may represent an aggregate of the smaller receptor. However, this seems less likely since radiolabeled aggregated IgG readily binds to a high M, band on polyacrylamide gels of lysed human platelets both in our studies and those of others (Fig. 6) (36). The fact that the isolated 200-kDa protein forms an in vitro complex with polyvalent IgG satisfies an important requirement for an Fey receptor, its binding of IgG ligand, and further supports the multicomponent high M, nature of the platelet Fey receptor. ACKNOWLEDGMENTS We thank Drs. C. M. Ingerman-Wojenski and Dr. Scott Murphy of Jefferson Medical College and Dr. Margaret Johnson of Wilmington, Delaware for permitting us to study their patients. We also thank Dr. Jay Unkeless of Mt. Sinai School of Medicine and Dr. Clark Anderson of Ohio State University for the gracious use of their monoclonal antibodies to Fey receptors. We appreciate the help of Ruth Rowan and Diane Meredith in preparing this manuscript for publication.
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