activation of human platelets by a stimulatory monoclonal antibody*

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 17, Issue of June 15, pp. 1004%10046,199O Printed in U. 5’ A.

Activation of Human Platelets by a Stimulatory Monoclonal Antibody*

(Received for publication, October 6, 1989)

Elizabeth Korneckis, Bogdan Walkowiak, Ulhas P. Naik, and Yigal H. Ehrlichg From the Department of Anatomy and Cell Biology, State University of New York Health Science Center, Brooklyn, New York 11203, the §CSI/IBR Center for Developmental Neuroscience, College of Staten Island, City University of New York, and the New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10301

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed Fl 1. Interaction of mAb F 11 with human platelets resulted in dose-dependent granular secretion, measured by [‘4C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F 11 to platelets revealed a high affinity site with 8,067 2 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 f 0.9 x IO-’ M.

Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F 11. Incubation of 32Pi-labeled platelets with mAb Fll resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by de- phosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb Fll IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb Fll IgG blocked mAb Fll-induced platelet aggregation and the binding of “‘1-mAb Fll to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb GlO), which inhibits “‘I-fibrinogen binding and platelet aggregation, completely blocked mAb Fll-induced [‘“Clserotonin secretion and aggregation but not the binding of ‘261-mAb F 11 to platelets. mAb GlO also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb Fl 1, These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.

Platelets and platelet membrane glycoproteins play a significant role in immunologic reactions. Early studies have suggested that alloantibodies developed in patients following multiple transfusions activate platelets in vivo resulting in thrombocytopenia (1,2). Specific anti-platelet autoantibodies and alloantibodies to membrane glycoproteins (GP)’ such as GPIb, GPIIb, GPIIIa, and GPV now have been identified in patients with clinical disorders of drug-dependent thrombo-

* This work was supported by National Institutes of Health Grant HL 32914 (to E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence and reprint requests should be ad- dressed: Dept. of Anatomy and Cell Biology, SUNY Health Science Center, 450 Clarkson Ave., Box 5, Brooklyn, NY 11203.

’ The abbreviations used are: GP, glyciproteins; 5HT, serotonin; SDS, sodium dodecyl sulfate; PGE,, prostaglandin E1; AMP-PNP, 5’-adenylylimidodiphosphate; CHAPS, 3-[(3-cholamidopropyl)di- methylammoniol-1-propanesulfonic acid.

cytopenia purpura, posttransfusion purpura, neonatal isoim- mune thrombocytopenia, chronic immune thrombocytopenia purpura, and septicemia (3-16). AIDS patients with acute thrombocytopenia purpura were shown to have anti-platelet antibodies (17, 18). The study of the interaction of immunoglobulins with platelets has been enhanced by the develop- ment of monoclonal antibodies which induce platelet aggregation (19-26). The study of such monoclonal antibodies enables the identification of specific platelet membrane anti- gens involved in platelet activation by immunoglobulins in vivo, and in the elucidation of the molecular mechanisms resulting in this activation process.

In this paper we describe the properties and mechanism of action of a novel monoclonal antibody which acts as a potent inducer of aggregation and secretion in human platelets. This monoclonal antibody recognizes a unique receptor on the platelet surface which is involved in platelet activation, and we present data showing the association of the fibrinogen receptor in platelet secretion.

EXPERIMENTAL PROCEDURES

Collection of Blood and Plasma-Blood was obtained from normal individuals who were free of any medication for at least 1 week prior to experimentation. All volunteers signed an informed consent form approved by the State University of New York, Health Sciences Center, Brooklyn, New York Committee on Human Research.

Preparation and Washing of Platelets-Platelets were washed and isolated from blood freshly collected in acid citrate dextrose (27). Platelets were washed by djfferential centrifugation and resuspended in a Tvrode-albumin (0.35%) solution buffered with 11.9 mM sodium bicarbbnate (pH 7.4) containing heparin (2 units/ml) and 0.5 mg/ml potato apyrase (28). Platelets were- finally suspended in a final Ty- rode-albumin solution containing 2 mM CaCl*, 1 mM M&12, 11.9 mM NaHC03, NaH,P04 (0.36 mM)r glucose (0.1%) and govine serum albumin (0.35%). Gel-filtered platelets were prepared as described (29). Proteolytically treated platelets were prepared as described previously (30, 31). Platelets were counted microscopically using a phase contrast microscope and a hemocytometer.

Platelet Aggregation-The experiments were carried-out in a Chronolog Lumi-Aggregometer (Chronolog Corp., Havertown, PA). Platelet aggregation was initiated by the addition of monoclonal antibodies (10 ~1) at various concentrations to 0.45 ml of a platelet suspension containing 2-4 X 10’ platelets/ml. The extent of platelet aggregation was expressed in light transmission units and the initial velocity of aggregation was measured from the slope of platelet aggregation tracings (light transmission units/min). The aggregometer was calibrated with 0.45-ml suspensions of washed platelets for 10% light transmission.

Platelet ATP Release-The experiments were carried out in a Lumi-Aggregometer using the Chronolog luciferin/luciferase reagent.

Platelet Serotonin Release-Serotonin [side chain-2-“Cl5-hydroxy- tryptamine creatinine sulfate, 58 mCi/mmol) (Amersham Corp.) was added to washed platelets or platelet-rich plasma and incubated for 30 min at 22 “C. Imipramine (2 GM) was added to prevent reincorpor- ation of serotonin and incubation continued for 5 min. Formaldehyde (135 mM) was added to stop the release reaction and platelet suspensions were centrifuged for 1 min at 11,000 x g (32). The extent of release was calculated as the percentage of thrombin-releasable [‘“Cl

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Platelet Activation by a Monoclonal Antibody

5HT appearing in the supernatant fraction following stimulation as described previously (33).

Iodination of Antibodies and Antibody Binding to Platelets-Puri- fied monoclonal antibodies were radiolabeled by using the Iodo-Gen method (34) or by use of Iodo-Beads (Pierce Chemical Co.). The specific activities were approximately 7.4 X 10’ cpm/pg when 1 mg/ ml of monoclonal antibodies were radiolabeled by the Iodo-Gen method and 2 x lo7 cpm/pg when 50 pg/ml of monoclonal antibodies were radiolabeled by the Iodo-Bead method. Binding of radiolabeled antibody to platelet-rich plasma or to washed platelets was performed over 75 ~1 of silicon oil or 20% sucrose (35). The incubation mixture consisted of platelet aliquots (2-5 x lO’/ml) and radiolabeled monoclonal antibodies in total volumes of 220 or 300 ~1. Values for specific binding were analyzed according to the method of Scatchard (36).

Protein Phosphorylation in Platelets Labeled with 32P,-Washed platelets (lO’/ml), resuspended in phosphate-free Tyrode’s buffer containing albumin (pH 7.4), were incubated with 1 mCi/ml ‘*Pi for 30 min at 37 “C. The platelets were washed and resuspended in phosphate-containing, bovine serum albumin-free Tyrode’s solution. Aliquots (40 ~1) of the platelet suspension were incubated with agonists at 22 “C under nonstirring conditions. The reactions were stopped by the addition of SDS-Laemmli solution containing 2% p- mercaptoethanol and processed for SDS-gel electrophoresis followed by autoradiography.

Immunization and Hybridoma Production-Balb/c mice were injected with human platelet membranes emulsified with incomplete Freund’s adjuvant containing 4 mg/ml M. butyricum. Following weekly injections, mouse spleens were removed and fused with SP2/ O-Ag14 myeloma cells. Supernatants were tested for effects on platelet function. Selected hybridomas were cloned at least twice. Antibodies were characterized by enzyme-linked immunosorbent assay with the use of subclass-specific goat or rabbit anti-mouse antibodies. Mono- clonal antibody Fll is an IgG1, and monoclonal antibody GlO is an IgG2, isotype.

Preparation of ZgG and Fab Fragments-IgG was obtained from hybridoma supernatants by initial precipitation with 50% saturated ammonium sulfate followed by two washings with 40% ammonium sulfate (37). Following dialysis, the samples were chromatographed on a DEAE-cellulose column in 17 mM phosphate buffer (pH 7.0). The Fab fragments were prepared by papain digestion followed by carboxymethylcellulose column chromatography (38). IgG was also prepared from mouse ascites fluid by protein A-Sepharose column chromatography (39).

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting- SDS-polyacrylamide gel electrophoresis was performed in 4% stack- ing gels and in 7.5 or 10% separation polyacrylamide slab gels or 7- 14% polyacrylamide exponential gradient gels (40). The gels were stained for proteins with Coomassie Brilliant Blue, destained in 10% acetic acid, 50% methanol, dried in uocuo, and exposed to Kodak X- Omat AR film with DuPont-Cronex Lightning Plus intensifying screens for approximately 24 h at -70 to -85 “C and developed in a Kodak X-Omat developer. Reduced samples (reduced with 2% p- mercaptoethanol) or nonreduced samples were applied to SDS-polyacrylamide slab gels for electrophoresis. Molecular weight determi- nations were made by comparison to Bio-Rad reduced samples of myosin (200,000), Escherichia coli fl-galactosidase (116,000), phos- phorylase b (97,400), bovine serum albumin (66,200), ovalbumin (42,700), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and lysozyme (14,400). The completed nonstained gels were transferred also to nitrocellulose paper for staining with specific antibodies using the method of Towbin et al. (41).

RESULTS

Induction of Platelet Aggregation and Secretion by mAb Fll: Involvement of the Platelet Fll Antigen in Platelet Activa- tion-Fig. 1 shows the effects of addition of mAb Fll to intact, washed platelet suspensions. Following the addition of mAb Fll, there was an initial lag period followed by ATP release and platelet aggregation. We also measured serotonin release induced by mAb Fll. At concentrations of mAb Fll as low as 0.25 pg/ml, [“‘Clserotonin release was calculated to be 70.04 + 8.2% (mean f S.D.) of total uptake in seven separate experiments performed in triplicate. In comparison, thrombin (10 units/ml)-induced [Y]LiHT release was con- sistently lower, and thrombin induced 48.5 f 13.1% (mean +

E ol ‘i 5c P ‘3 z! ; 5

I 1c

90

10

I 1 F-11

1 min.

10043

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FIG. 1. Effect of mAb Fll on the aggregation and secretion of platelets. Aliquots (0.45 ml) of mustard-washed platelets were incubated for 30-60 s at 37 “C with 50 gl of luciferin/luciferase reagent in a Chronolog Lumi-Aggregometer. Aggregation (upper panel) and secretion (lower panel) were initiated by the addition of mAb Fll (1 gg/ml, final concentration).

S.D.) of [Y]5HT release in three separate experiments performed in triplicate. Similar results were obtained with mAb Fll added to platelet-rich plasma. Washed platelets, pretreated with chymotrypsin (30), also responded to mAb Fll by secreting ATP and aggregating in the absence of exoge- nously added fibrinogen. Similar secretion and aggregation were observed with platelets pretreated with human granulo- cyte and pancreatic elastase.

As shown in Fig. 1, mAb Fll-induced platelet aggregation (top panel) and ATP release (bottom panel) were not imme- diate events but were initiated after a latency period which was dependent on the concentration of mAb Fll. The latency observed for platelet activation following the addition of mAb Fll was shortened with increasing concentrations of mAb Fll. Approximately 5.8 pg/ml of mAb Fll-induced platelet activation within 3 min, whereas with higher concentrations of mAb Fll the latency period decreased to less than a minute.

Binding of ‘251-mAb Fll to Platelets-The binding of “‘I- mAb Fll to platelets increased rapidly with time and reached equilibrium within 15 min. The binding of mAb Fll to platelets was dependent on the concentration of radiolabeled li- gand. Fig. 2 shows the results obtained in 11 separate experiments using blood obtained from 11 different donors. This figure displays the exasperating but often present variability that can be observed in binding studies with human platelets from separate donors. In several experiments, the maximal amount of mAb Fll bound at saturation was approximately 0.35 pg/lO’ platelets, whereas in other experiments the binding of mAb Fll at saturation was approximately 0.1 pg/lO’ platelets. The two experiments which represent the maximal and minimal binding of mAb Fll to platelets, in our studies to date are highlighted in this figure. Scatchard analysis of


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0.3

0.2

0.1

0.0 10-5 10-4 10.3 10.2 10.’ 100

M.Ab. Fll ADDED (mghl)

FIG. 2. Binding of ‘““I-mAb Fll to platelets. Aliquots of platelet-rich plasma (2-5 x lO”/ml) (llO-~1 aliquots) were incubated at 22 “C for 15 min under nonstirring conditions with increasing concentrations of ‘“‘I-mAh Fll. Eleven separate experiments were performed and the nonspecific mAb Fll binding to platelets was sub- tracted from total binding. The results show the specific binding obtained in each experiment, and they are represented in this figure by different symbols. In order to emphasize the variability in binding among various donors, one donor who showed a high degree of mAb Fll binding is represented by open circles, and a donor who showed low amount of mAb Fll binding is shown by squares.

INTACT PLATELETS

I I ~1000 i

z 6 u

0 I,,,,,,,,,,,,.,,,

I2 3 4 5 6 7 8 9 101112131415 %Fd

TIME (min.)

FIG. 3. Time course of protein phosphorylation in platelets induced by mAb Fl 1. Washed intact platelets were incubated with “‘Pi for 30 min at 37 “C, washed, and resuspended in Tyrode’s buffer (pH 7.4). Aliquots (40 ~1) of the platelet suspension were incubated with mAb Fll (10 rg/ml) at 22 “C under nonstirring conditions for various periods of time. At the time points shown above, the reactions were stopped by the addition of SDS stop solution. Equal aliquots of each mixture were then applied to a 7-14% exponential gradient gel, followed by autoradiography. The autoradiographs were scanned with a laser microdensitometer and quantitated. The figure shows the phosphorylation of a 40-kDa dalton platelet protein (circles) and phosphorylation of a 20-kDa platelet protein (triangles). Each point represents the mean f S.E. of four separate experiments. Stars

indicate a significant difference @ < 0.05) from control values (platelets incubated without mAb Fll or with nonimmune IgG). The phosphorylated band indicated in this figure as “40K Dalton” is the same protein often referred to in the literature as the 47-kDa phos- phoprotein.

the data from all 11 experiments indicated a single class of binding sites with 8067 f 1307 binding sites/platelet with a dissociation constant of 2.7 + 0.9 X 10eR M. These values are the weighted means (x f SE.) which were calculated by using the correlation coefficient as a weighting factor.

Stimulation of Protein Phosphorylation in Platelets by mAb FII-The effect of mAb Fll on the phosphorylation of intracellular platelet proteins is shown in Fig. 3. Following incubation with mAb Fll, we found a selective and time-dependent increase in the phosphorylation of proteins with apparent molecular weights of 40,000 and 20,000. Such changes in phosphorylation pattern were found in intact platelets and in

chymotrypsin-pretreated platelets. The phosphorylation of the 40,000- and 20,000-dalton proteins increased significantly within seconds following the addition of mAb Fll. In intact platelets, the maximal increase in the phosphorylation of these proteins occurred following 5 min of incubation with mAb Fll. After longer incubations there was a decrease in the phosphorylation state of both the 40,000- and 20,000- dalton proteins. The changes in the phosphorylation state of the 40,000- and 20,000-dalton proteins induced by mAb Fll in platelets pretreated with chymotrypsin followed essentially the same time course shown for intact platelets in Fig. 3.

Platelet Proteins Recognized by mAb Fll-The platelet proteins recognized by mAb Fll in a Western immunoblotting procedure are shown in Fig. 4. mAb Fll recognized epitopes on two platelet-membrane proteins with molecular masses of 32 and 35 kDa. Both of these proteins were recognized by mAb Fll in 18 separate experiments conducted to date. Another monoclonal antibody, named mAb GlO, which is described below, as well as immunoglobulins obtained from SpB/O-injected mice (Fig. 4), showed no interaction with these proteins.

Involvement of GPIIb-IIIa in the Activation of Intact Plate- lets by mAb F-l I-We isolated a second monoclonal antibody, termed GlO, which is directed against the platelet GPIIIa molecule. Fig. 5 shows the results of Western blotting experiments using three different antibodies for comparison. mAb GlO is shown to immunoblot GPIIIa (lane A). As previously shown by us (30), glycoproteins IIb and IIIa were recognized by a polyclonal anti-human platelet membrane antibody (lane B). A polyclonal anti-human 66-kDa protein antibody (kind gift from Dr. S. Niewiarowski, Temple University, Philal*ll- phia, PA) also immunoblotted GPIIIa (lane C). This 66-kDa protein was shown by us previously to be a proteolytically derived fragment of the GPIIIa molecule (30). An antibody prepared against this protein immunoblotted GPIIIa in un- digested platelets (42). As control, immunoglobulins obtained from ascites of mice injected with Sp2/0 hybridoma cells did not immunoblot GPIIIa (lane D).

MW (I(d)

200.0 - 116.0 - 97.4 - 66.2-

42.7-

31.0- l *= 4-35 +32

2 1.5 -

14.4-

4B CD ABCD

FII SP2

FIG. 4. Immunoblots of platelet proteins recognized by mAb Fll. Western blots showing the recognition of 32- and 35.kDa proteins from platelet membranes (200 pg) (lane A); mAb Fll affinity column eluate of membrane proteins extracted with 1% CHAPS (20 fig) (lane B); mAb Fll affinity column eluate of membrane proteins extracted with 1% Triton X-100 (30 fig) (lane C); mAb Fll affinity column eluate of membrane proteins extracted with 1% octyl gluco- side (20 pg) (lane U). The left panel depicts immunoblots obtained using mAb Fll. The right panel depicts immunoblots obtained using nonspecific immunoglobulins from Sp2/0 hybridoma-injected mice. Goat anti-mouse IgG-conjugated horseradish peroxidase was used as second antibody.


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MW(Kd)

y”6.i - rib” . - 97.4 - -be + 66.2 -

42.7 -

31.0 -

21.5,

14.4 -

ABCD

FIG. 5. Immunoblots of platelet proteins recognized by mAb GlO. Western blots showing the recognition of GPIIIa by mAb GlO. Platelet membranes were extracted with 1% CHAPS. The detergent- extracted proteins were resolved on a 10% polyacrylamide gel, transferred onto nitrocellulose paper, incubated with antibodies, and de- tected using ““I-protein A. Lane A, mAb GlO; lane B, rabbit anti- human platelet membrane antibody; lane C, rabbit anti-66-kda antibody; lane D, immunoglobulins obtained from the ascites fluid of mice injected with Sp2/0 hybridoma cells.

The mAb Fll-induced platelet aggregation, ATP release, [‘C]5HT secretion and protein phosphorylation were tested in the presence of the anti-glycoprotein IIIa monoclonal antibody mAb GlO. mAb G10 completely inhibited mAb Fll- induced platelet aggregation. Fifty percent inhibition of platelet aggregation and ATP release occurred at concentrations of mAb GlO ranging from 0.35 to 0.45 pg/ml (Fig 6A). A slightly higher concentration of mAb GlO (1.8 pg/ml) inhibited 50% of the mAb Fll-induced [W]5HT release. Fig. 6B shows that the stimulation by mAb Fll of the phosphorylation of the intracellular 40,000- and 20,000-dalton proteins by mAb F-11 was completely inhibited by mAb GlO. Fig. 6B also shows that the mAb Fll-induced increase in phosphorylation starts before aggregation (compare time points marked d in upper and lower panels of Fig. 6B). These results were not due to blockade by mAb GlO of mAb Fll binding to platelets, as described below.

Inhibition of mAb Fl l-induced Platelet Aggregation by mAb Fll Fab Fragments-Fab fragments were prepared from pu- rified IgG of mAb Fll. Neither these monovalent molecules nor Fc fragments induced granular secretion or platelet aggregation. The effects of monovalent Fab fragments on mAb Fll-induced platelet aggregation are shown in Fig. 7A. The addition of increasing concentrations of Fab fragments pro- longed the latency of mAb Fll-induced platelet aggregation from 2 min to 1 h and longer. The mechanism responsible was found to be the inhibition by the Fab fragments of mAb Fll binding, as shown in Fig. 7B. The I&, of Fab fragment inhibition of mAb Fll binding is approximately 5 pg/ml. On the other hand, Fc fragments prepared from mAb Fll-IgG had no effect on mAb Fll binding to platelets, and did not inhibit mAb FIl-induced platelet aggregation even at a concentration as high as 435 rg/ml. In five separate experiments, mAb GlO-IgG did not inhibit the binding of ““I-mAb Fll to platelets. The binding data was similar to that seen in Fig. 7B using mAb Fll Fc fragments which did not inhibit mAb Fll binding.

Comparison of Fibrinogen-induced Platelet Aggregation and

FIG. 6. Effect of monoclonal antibody GlO (mAb GlO) on aggregation, ATP secretion, and phosphorylation of platelets induced by mAb Fll. A, dose-response curve of mAb GlO inhibition of aggregation and ATP release. Washed platelets (0.45 ml) were incubated at 37 “C in the presence of 50 ~1 of luciferin/luciferase reagent in a Chronolog Lumi-Aggregometer under stirring conditions. mAb GlO (O-O.9 pg/ml) was added and the incubation continued for an additional 1 min. mAb Fll(10 fig/ml) was added to initiate platelet aggregation and ATP release. B, inhibition of mAb Fll-induced platelet aggregation and phosphorylation by mAb GlO. Washed platelets (0.45 ml) (4 X lO*/ml) labeled with “PX were stirred at 37 “C in an aggregometer. Platelet aggregation was initiated by the addition of mAb Fll (1 rg/ml). At each time point (indicated by letters a through fl, 40-gl aliquots were removed and added to 10 ~1 of a 5 x concentrated SDS stop solution. The mixtures were applied to SDS- polyacrylamide gels and processed for autoradiography. The autora- diograms were scanned by a laser microdensitometer and quantitated. Upper panel, circles indicate phosphorylation of a 40-kDa protein, and triangles indicate phosphorylation of a PO-kDa protein. The basal level of phosphorylation in the absence of mAb Fll was set at 100%. Also shown above is the inhibition of mAb Fll-induced phosphorylation (squares) of 40- and 20-kDa proteins by mAb GlO (1 Kg/ml). Lower panel, the inhibition of mAb Fll-induced platelet aggregation by mAb GlO, simultaneously measured.

mAb Fl l-induced Aggregation in Chymotrypsin-pretreated Platelets-Fig. 8, A and B, shows the spontaneous aggregation of chymotrypsin-treated platelets upon the addition of fibrinogen in the presence and absence of PGEI. The result indicates that the elevation of cyclic AMP does not interfere with this type of aggregation. In contrast, the mAb Fll-induced aggregation of chymotrypsin-treated platelets is completely inhibited by PGE, as shown in Fig. 8 C, even though the fibrinogen-induced aggregation still occurs as shown in Fig. 8 D. This result indicates that mAb Fll-induced platelet aggregation is sensitive to raised levels of intracellular cyclic AMP.

Inhibition of mAb Fll-induced Secretion by ATP-mAb Fll-induced platelet aggregation can be inhibited by apyrase,


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A *rn


Fab Added kl&o FIG. 7. Inhibition of mAb Fll-induced platelet aggregation

and “‘1-mAb Fll IgG binding by mAb Fll Fab fragments. A, inhibition of platelet aggregation. mAb Fll (5 &g/ml)-induced platelet aggregation was performed in the presence of mAb Fll Fc fragments (90 rg/ml) or mAb Fll Fab fragments (90 @g/ml) as shown above. mAb Fll Fab and Fc fragments were incubated with platelets for 1 min at 37 “C prior to the addition of mAb Fll. mAb Fll Fc fragments did not inhibit mAb Fll-induced platelet aggregation. B, dose-dependent inhibition of ‘Y-mAb Fll binding to platelets by mAb Fll Fab fragments. mAb Fll Fab (circles) or mAb Fll Fc fragments (triangles) were incubated with platelets for 1 min prior to the addition of lz51-mAb Fll. The binding of ‘Z51-mAb Fll IgG to platelets was performed as described under “Experimental Procedures.” Each point is the mean of at least two separate experiments. mAb G10 IgG gave results in at least five separate experiments which were similar to those observed with mAb Fll Fc fragments (triangles).

ATP, and ATP analogues. Table I shows the I& values for such inhibition by ATP, 5’-p-fluorosulfonylbenzoyladenosine, and AMP-PNP. The simultaneous secretion of [14C]5HT from platelet-dense granules following the addition of mAb Fll was also measured (Fig. 9). We found that although ATP inhibited [14C]5HT secretion induced by mAb Fll (Fig. 9), maximal inhibition of secretion, even at high ATP concentra-

FIG. 8. Comparison of fibrinogen- induced aggregation and mAb Fll- induced aggregation of chymotrypsin-treated platelets: effect of PGE1. A, fibrinogen (200 pg/ml)-induced aggregation of chymotrypsin (500 pg/lO’ platelets/ml)-treated platelets; B, lack of inhibition of fibrinogen-induced aggregation of chymotrypsin-treated platelets by 10 pM PGI&; C, aggregation of chymotrypsin-treated platelets by mAb Fll and inhibition of aggregation by PGE,; and D, chymotrypsin-treated platelets aggregated by fibrinogen but not by mAb Fll in the presence of PGEI.

tions, was never greater than 70%. Thirty percent of the [‘“Cl 5HT release induced by mAb Fll could not be inhibited by ATP. Similar results of inhibition of [“‘C]5HT were found with apyrase. A high concentration of apyrase (0.9 mg/ml) inhibited 70% of [‘*C]5HT secretion induced by mAb Fll, and 29.3 f 2.6% of the secretion was not inhibited by apyrase. This would indicate that mAb Fll acts directly on the platelet surface to induce 30% granular secretion. Is the ADP receptor involved in this initial action? To test this possibility, platelets were made refractory to ADP by adding nanomolar concentrations of ADP as shown in the bottom panel of Fig. 10. Platelets which were made refractory to ADP still responded to mAb Fll with a shortened latency, even though maximal concentrations of ADP could not induce aggregation. This result, indicates that mAb Fll does not interact directly with the ADP receptor site.

DISCUSSION

We report here the characteristics and mechanisms of action of a monoclonal antibody named mAb Fll, a potent platelet agonist.. mAb Fll directly stimulates platelet secretion, measured as ATP and serotonin release, and fibrinogen- dependent platelet aggregation. By interacting with a unique receptor, termed Fll, mAb Fll induces rapid intracellular phosphorylation of two major proteins: a 40,000-dalton protein which is a known substrate for protein kinase C, and a 20,000-dalton protein, the light chain of myosin and the substrate for myosin light, chain kinase, a Ca2+-dependent enzyme. Thus, the cascade of intracellular biochemical events triggered by mAb Fll involves stimulation of protein kinase C and elevation of free calcium ion levels, in all likelihood through activation of the phosphoinositide cycle (43).

We have found that mAb Fll recognizes platelet surface membrane proteins of approximately 32,000 and 35,000 daltons, as determined by Western blotting and by analysis of the bound material eluted from a mAb Fll affinity column. By Scatchard analysis we have shown that there are approximately 8,000 high affinity Fll binding sites per platelet. The platelet Fll antigen appears to be resistant to surface prote- olysis since we observed that chymotrypsin- and elastase- pretreated platelets are stimulated by mAb Fll to secrete and aggregate. These proteolytically treated platelets show significant increases in intracellular phosphorylation of the 20,000-


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Platelet Activation by a Monoclonal Antibody 10047

TABLE I Inhibition of mAb Fll -induced platelet aggregation

Platelets were washed as described under “Experimental Proce- dures,” and aliquots (0.45 ml) were incubated in an aggregometer for 1 mm at 37 “C under stirring conditions with various concentrations of each compound. Platelet aggregation was initiated by the addition of 10 us/ml mAb Fll and the extents of aggregation were measured.

Compounds tested ICso values

ATP AMP-PNP

PM 220

83 5’-FSBA 85

FIG. 9. Inhibition of mAb Fl l-induced [‘%]5HT release by ATP. Platelet-rich plasma was incubated with [‘%]5HT for 30 min at 37 “C as described under “Experimental Procedures.” Aliquots (0.45 ml) of platelet-rich plasma were incubated for 1 min at 37 “C under stirring conditions with various concentrations of ATP. Aggre- gation was initiated by the addition of mAb Fll (5 rg/ml).

FE. 10. Stimulation of ADP refractory platelets by mAb Fl 1. Top panel, washed platelets were induced to aggregate by the addition of ADP (100 PM). The addition of mAb F11 (5 @g/ml) also induced platelet aggregation in separate aliquots of washed platelets. Bottom panel, platelets were made refractory to ADP by adding aliquots of suboptimal concentrations of ADP (0.5 PM). Finally, ADP at 100 FM (final concentration) was totally ineffective in inducing platelet aggregation. mAb Fll (5 pg/ml) was able to aggregate platelets made refractory to ADP.

and 40,000-dalton proteins following the addition of mAb Fll. The platelet receptor(s) recognized by mAb Fll consists of a protein duplex of molecular mass of 32,000 and 35,000 daltons. This appears to be a unique platelet antigen(s) previously not recognized by other stimulatory antibodies. De- tailed characterization of the structure of this unique receptor and its associated glycoproteins is in progress.

The fibrinongen receptor, consisting of glycoproteins IIb- IIIa, appears to play an important role in the action of mAb Fll. A monoclonal antibody developed in our laboratory

(named mAb GlO), which blocks aggregation and ‘*“I-fibrinogen binding to ADP-stimulated platelets, was found to be directed against GPIIIa. mAb GLO potently and completely inhibited mAb Fll-stimulated platelet aggregation. Moreover, mAb GlO blocks intracellular events induced by mAb Fll: these events include the increase in the phosphorylation of 40,000- and 26,600-dalton proteins and the initiation of [“Cl 5HT and ATP secretion. The complete inhibition by mAb GlO of intracellular protein phosphorylation events and the secretion induced by mAb Fll indicates that the GPIIIa molecule functions not only as a fibrinogen binding site required for fibrinogen-dependent platelet aggregation, but that GPIIIa also plays an important role in the transmission of signals that activate second messenger-generating systems leading to secretion. These results describe a new role for GPIIIa in platelet function. In a previous report we have described the platelets of a Friedreich’s ataxia patient with unique thrombopathy (44). mAb Fll induced secretion in platelets of this patient but not aggregation, due to a defect in the exposure of fibrinogen receptors. Interestingly, also in this patient mAb GlO inhibited mAb Fll-induced secretion, indicating that the role of GPIIIa in signals that lead to secretion can be separated from its role in exposure of fibrinogen binding sites.

In addition to mAb GlO, the Fab fragments of mAb Fll also inhibited the activation of platelets by mAb Fll. This inhibition was found to be due to direct interference of the Fab fragments with the binding of the mAb Fll IgG molecule to the platelet surface. Such interference is consistent with the possibility that mAb Fll-induced platelet activation involves receptor dimerization and microclustering (45) or the platelet Fc receptor (46). Agents which increase the level of cyclic AMP also inhibit mAb Fll-induced platelet aggregation, and this may be due to the inhibition of fibrinogen receptor exposure (47). The involvement of released ADP in mAb Fll-induced platelet aggregation is indicated by the finding that ATP and ATP analogues, which block the ADP receptor, and apyrase, which degrades the released ADP, completely inhibit aggregation. However, significant granular secretion (30% of uptake) still occurs in response to mAb Fll even in the presence of either ATP or apyrase, indicating that the direct interaction of mAb Fll with its receptor results, in part, in granular release.

In conclusion, mAb Fll interacts with specific protein components (32 and 35 kDa) at the platelet surface. This interaction leads to platelet granular secretion and aggregation. The biochemical pathways of platelet activation by mAb Fll involve stimulation of the activity of protein kinase C and of the Ca’+/calmodulin-dependent myosin light chain kinase, and is inhibited by elevating intracellular cyclic AMP. The mAb Fll-induced aggregation of platelets appears to be secondary to ADP release. The mAb Fll-induced secretion appears to involve action of glycoprotein IIIa. At least 30% of the granular secretion induced by mAb Fll is not mediated by ADP but by a specific Fll receptor. Detailed characterization of this unique receptor will provide novel information on the process of platelet activation.

Acknowledgments-We wish to thank Dr. Czeslaw Cierniewski for stimulating discussions and Monika Lange, Laurie DiCesare, Mark Fleming, David Harwick, Dan De Mars, and Nancy Harber for excellent technical assistance.

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E Kornecki, B Walkowiak, U P Naik and Y H EhrlichActivation of human platelets by a stimulatory monoclonal antibody.

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