Studies on the Dual Effects on Platelets of a Monoclonal Antibody to CD9, and on the Properties of Platelet CD9

Thrombosis Research 95 (1999) 215–227

REGULAR ARTICLE

Studies on the Dual Effects on Platelets of a Monoclonal Antibody to CD9, and on the Properties of Platelet CD9 Marit Inngjerdingen1,2, Karin Waterhouse1,2 and Nils Olav Solum 1 Research Institute for Internal Medicine, Rikshospitalet, and 2 Section of General Physiology, Department of Biology, University of Oslo, Oslo, Norway. (Received 27 September 1998 by B. Østerud; revised/accepted 1 February 1999)

Abstract The article describes effects on human platelets of a murine monoclonal antibody of the IgG2a subtype (clone FN99) directed against the membrane glycoprotein CD9. This antibody exerts a dual action on human platelets in plasma depending on whether the complement system can be activated or not, resulting either in membrane permeabilization or a true platelet aggregation. Secretion from the a-granules during permeabilisation was not observed in the sense that the granulelocated protein thrombospondin was retained in the platelets, as opposed to what was seen with platelets that had undergone an antibody-induced aggregation. Only a small fraction of P-selectin was found on the surface of the permeabilised platelets. The cytoskeletal protein actin-binding protein (filamin) was profoundly degraded during membrane permeabilisation, however, and scanning electron microscopy showed platelets that were swollen with only a few pseudopodia. Preincubation of platelets with three different antibodies to CD9 showed strong inhibition of a subsequent binding of FITC-labelled Fab fragment of FN99 indicating that antibodies tend to bind in the same area of Abbreviations: ABP, actin-binding protein; DTT, dithiothreitol; IAA, iodoacetamide; mAb, monoclonal antibody; PGE1, prostaglandin E1; c-PRP, citrated platelet-rich plasma; PPP, plateletpoor plasma; PBS, phosphate-buffered saline; TSP, thrombospondin. Corresponding author: N.O. Solum, Research Institute for Internal Medicine, Rikshospitalet, Pilestredet 32, 0027 Oslo, Norway. Tel: 147 (22) 86 82 26; Fax: 147 (22) 86 82 42; E-mail: ,n.o.solum @rh.uio.no..

the CD9 molecule. No association of CD9 to the platelet actin-based cytoskeleton was observed. CD9 was present on the surface of microvesicles derived from calcium ionophore-treated platelets.  1999 Elsevier Science Ltd. All rights reserved. Key Words: Platelets; CD9; Monoclonal antibodies; Cytoskeleton; Microvesicles

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D9 is a 24–28-kDa surface membrane glycoprotein present on platelets and a variety of different hematopoetic and nonhematopoetic cells [1]. The protein belongs to a recently defined family of cell surface molecules that spans the plasma membrane four times, called the tetraspan superfamily of proteins [2]. CD9 is constantly expressed on the platelet surface and is a major component of the platelet membrane proteins (,40000) in addition to GPIIb-IIIa [3]. CD9 may be involved in cellular activation and adhesion functions, but the true physiological role of the CD9 antigen in platelet function is as yet unknown. Practically all known monoclonal antibodies against the platelet CD9 have the property of inducing a sort of platelet response in platelet-rich plasma, which is a rather unique feature. The platelet response to mAbs against CD9 is either secretion and aggregation or complement activation. mAbs against CD9 of the IgG1-class (like ALB6) induce an aggregation of the platelets, a response mediated by signalling through FcgRII, which leads to secretion of material from storage granules [3–6]. mAbs of the IgM class (like FN52), on the

0049-3848/99 $–see front matter  1999 Elsevier Science Ltd. All rights reserved. PII S0049-3848(99)00035-3

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other hand, lead to a permeabilisation of the platelet after formation of complement complex C5b-9 [7,8]. By introducing leupeptin to c-PRP before stimulation with FN52, the membrane permeabilisation has been demonstrated to become abolished [8]. Platelets release small vesicles called microvesicles (or microparticles) when stimulated by agonists, such as thrombin, Ca-ionophores, or the introduction of the complement C5b-9 complex, due to the generation of a high concentration of calcium in cytosol [9–11]. The platelet glycoprotein GPIIbIIIa have been found to be present on the microvesicles [12]. The platelets contain a well-developed cytoskeleton consisting of actin filaments and microtubuli. The cytoskeleton contributes in keeping the discoid shape of the unstimulated platelet and is supposed to function in the shape change and secretion processes during platelet activation. There are two locations for the actin filament-based components in unstimulated platelets, (alternatively:, i.e.) throughout the cytoplasma and at the periphery of the cell where they coat the plasma membrane. The membrane skeleton consists of actin filaments that are connected to the cytoplasmic part of transmembrane glycoproteins in the plasma membrane by the actin binding protein (ABP, also known as filamin) [13,14]. One of these glycoproteins is GPIb [15]. ABP’s anchoring function is regulated by the calcium protease calpain [16]. The actin filaments are insoluble in nonionic detergents like Triton X-100. The largest actin filaments can be isolated by centrifugation at 130003g, while the short actin filaments require ultracentrifugation at 100,0003g for their isolation [17]. In the present study, we have examined the ability of the monoclonal antibody FN99 to induce platelet changes in the presence or absence of a functional complement system. We also studied whether the three mAbs FN99, FN52, and ALB6 have overlapping binding sites on CD9, whether CD9 is present on the microvesicles, and whether CD9 is anchored in the membrane skeleton.

1. Materials and Methods 1.1. Commercial Materials Apyrase (A 6535), 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIP)/p-nitro-blue-tetrazolium

chloride (NBT), Ca-ionophore A23187 (C 7522), Coomassie BBG (B 1131), ditiothreitol (DTT) (D 9760), hirudin (H 7016), iodoacetamide (IAA) (I 6125), leupeptin (acetyl-Leu-Leu-Arg-al) hemisulphate salt (L 2884), 2-mercaptoethanol (M 6250), papain (P 9886), prostaglandin E1 (PGE1) (P 1326), tris-hydroxymethyl-amino-methane (TRIS) (T 1378), and polyoxyethylene-sorbitan-monolaurate (Tween 20) (P 5927) were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Triton X-100 was from E. Merck (Darmstadt, Germany). Dimethyl sulphoxide (DMSO) was from Rathburn Chemicals Ltd. (Wallkerburn, Scotland). Fluoresceine isothiocyanate (FITC) was purchased from Molecular Probes, Inc., (Eugene, OR, USA). Alkaline phosphatase-conjugated goat-anti mouse IgG F(ab9)2 was from Jackson Immuno Research Laboratories (Avandale, PA, USA). Protein A-agarose columns (AffinityPak) were from Pierce Chemical Co., (Rockford, IL, USA). Sodium dodecyl sulphate (SDS) and nitrocellulose membrane were from BioRad Laboratories, (Richmond, CA, USA). Polyacrylamide tris-glycine (10–20%) gels were from Novex (San Diego, CA, USA). Inorganic salts for buffers were of p.a. (pro analyse) grade. The thrombin-mimicking peptide SFLLRN was synthesised at the Biotechnology Centre of Oslo, Norway. ReoPro (c7E3 Fab) was from Centocor, (Leiden, Holland).

1.2. Antibodies mAb FN99, belonging to the IgG2a subtype, and FN52 of the IgM class were produced by Dr. Steinar Funderud, Institute for Cancer Research, Radiumhospitalet, Oslo, Norway [8]. ALB6, a mAb belonging to the IgG1 subtype, was a generous gift from Dr. Boucheix and Dr. Rubinstein, Inserm Unite´ 268, Villejuif, France. FN99, ALB6, and FN52 are all mAbs against CD9. mAb PM6/81 directed towards an epitope in the glycocalicin region of the GPIbachain and mAb Ti10 against the 90-kDa end of the ABP subunit were used for immunoblotting after Western blotting. Both were gifts from Dr. J. M. Wilkinson, Royal College of Surgeons of England, London, UK. FITC-conjugated mAb Y2/51 (IgG1), against the platelet glycoprotein IIIa, and FITCconjugated IgG2a to be used as a negative control in flow cytometry, were purchased from DAKO A/S (Glostrup, Denmark). FITC-conjugated AK4

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against P-selectin (CD62P) was from Pharmingen (San Diego, CA, USA). Unless otherwise stated, FN99 and FN52 were used as dilutions of ascites in tris-buffered saline pH 7.4.

1.3. Buffers Medium A consisted of 148 mol/L NaCl, 0.02 mol/L tris-HCl, 5 mmol/L glucose, and 3.6 mmol/L EDTA Na2 (pH 7.4, 330 mosmol/L). Phosphate-buffered saline (PBS) were provided in the form of tablets giving 0.01 mol/L PBS (2.7 mmol/L KCl and 137 mmol/L NaCl), pH 7.4, when solubilised in H2O. Phillips suspension medium was composed of 138 mmol/L NaCl, 2.9 mmol/L KCl, 12 mmol/L NaHCO3, 5.5 mmol/L glucose, 0.36 mmol/L Na3PO4, and 1 mmol/L EDTA (pH 7.4). Phillips Triton X-100 solution consisted of 2% Triton X-100, 0.01 mol/L EGTA, and 0.1 mol/L tris-HCl (pH 7.4). Platelet washing solution (MPL) was composed of 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1 mmol/L NaH2PO4, 10 mmol/L glucose, 10 mmol/L sodium pyruvate, 5 mmol/L disodium malate, 10 mmol/L HEPES (acid), and 0.3 mmol/L bovine albumin (pH 7.4, 330 mosmol/L). SDS-solubilisation buffer for sample preparation consisted of (per mL) 500 mL sucrose, 100 mL 20% SDS, 19 mL 0.2 mol/L NaH2PO4, 81 mL 0.2 mol/L Na2HPO4, 20 mL bromphenol blue, and 280 mL H2O. Tris saline buffer (TS) was composed of 20 mmol/L trisHCl and 150 mmol/L NaCl (pH 7.4, 310 mosmol/L). Tris buffered saline (TBS) was composed of 20 mmol/L tris-HCl and 500 mmol/L NaCl (pH 7.5). TTBS was TBS with 0.05% Tween 20.

1.4. Platelets Venous blood from healthy volunteers was drawn into tubes containing one tenth volume of 0.129 mol/L sodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation at 3203g for 15 minutes. PRP was stored at 378C during the whole experiment to maintain the platelets in the discoid shape, seen as swirling, as well as distinct oscillations of the baseline in the aggregometer. When used to generate microvesicles, the platelets were isolated from ACD blood. Blood (34.4 mL) was drawn into 5.6 mL ACD consisting of 85 mmol/L Na3-citrate, 71.4 mmol/L citric acid, and 111 mmol/L glucose (pH 4.5). PRP was prepared as above. The

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platelets were sedimented by centrifugation for 10 minutes at 12003g, resuspended in 6.0 mL HAMPL (MPL solution supplemented with 5 U/mL hirudin and 0.02 U/mL apyrase) and left at 378C for 15 minutes. Immediately before recentrifugation, 2.0 mL ACD was added. The washing procedure was repeated once, this time without hirudin (AMPL). After another incubation and centrifugation, the platelet sediment was resuspended in AMPL. The washing solutions were kept at 378C during the whole procedure. A different procedure was used to isolate platelets to prepare Triton X-100 extracts. Forty milliliters of venous blood was drawn into one tenth volume of a solution of 45 mmol/L EDTA and 120 mmol/L NaCl. PRP was prepared and centrifuged for 15 minutes at 20003g. The platelet sediment was resuspended in 30 mL EDTA washing solution (0.148 mmol/L NaCl, 0.02 M tris-HCl, 5 mmol/L glucose, 0.6 mmol/L EDTA Na2) and centrifuged for 15 minutes at 20003g. The washing procedure was repeated twice, with 30 mL and 20 mL of EDTA washing solution, respectively. The resulting platelet sediment was resuspended in Phillips suspension medium to a platelet density of 1010 cells/mL. To obtain plateletpoor plasma (PPP) for calibration of the aggregometer, PRP was centrifuged for 5 minutes at 130003g.

1.5. Aggregometry Aggregometer experiments were done in a ChronoLog Dual Channel aggregometer model 440 (Chrono-Log Corporation, Havertown, PA, USA). The aggregometer was connected to a W1W linear recorder model 600 (W1W electronic Inc., Basel, Switzerland). For each series of aggregations, the aggregometer was calibrated using c-PPP and c-PRP as markers for light transmission. The difference in signal between PRP and PPP always represented 70 chart divisions on the recorder. In the aggregometer figures, light transmission through unstimulated and undiluted PRP is marked as corresponding to “100 % PRP”, and the light transmission through PPP is marked as corresponding to “0% PRP”. When washed platelets were used, a buffer was used as control instead of PPP.

1.6. Generation of Microvesicles ACD-washed platelets suspended in AMPL were stimulated to generate microvesicles in an aggrego-

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meter with 10 mmol/L of the calcium ionophore A23187 in the presence of 2.5 mmol/L CaCl2. Only initial stirring was used to avoid large aggregates. The platelets were incubated with the Ca-ionophore for 5 minutes and then processed for flow cytometry.

1.7. FITC Labelling of the CD9 MAb FN99 FITC conjugation of mAb FN99 was performed according to Godin [18]. Briefly, 5 mL FITC, solubilised in DMSO to a concentration of 10 mg/mL, was added to 1 mL of 0.5 mg/mL purified FN99. The solution, kept at pH 9.5 with 0.1 mol/L NaHCO3, was incubated for 2–3 hours in the dark at room temperature, and then dialysed for 18 hours at 48C in the dark against PBS, pH 7.4.

1.8. Flow Cytometry Labelled platelets were analysed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 15-mW air-cooled 488 nm argon laser. Forward and side scatter, as well as green fluorescence (FITC), were obtained with logarithmic amplification. Samples were transferred to polystyrene tubes containing PBS. The sample volumes were 100 mL after addition of the fluorescent probes (final concentrations in brackets): FITCY2/51 (5 mg/mL), FITC-IgG2a (5 mg/mL), or FITCFN99 (0.5 mg/mL). The tubes were incubated in the dark for 30 minutes. Thereafter PBS was added to a total volume of 600 mL. To study the amounts of different probes bound to the platelets, a gate based on the forward and side scatter properties of intact platelets was applied. Platelets and microvesicles were separated analytically on the basis of their difference in forward and light scatter. To discriminate between platelets and microvesicles, the lower limit of the platelet gate was set at the left-hand border of the forward scatter profile of unstimulated platelets with microvesicles representing GPIIIa positive particles below that size. The number of microvesicles were presented as percentage of the total number of fluorescent particles counted (i.e., platelets plus microvesicles). 10000 positive events were analysed each time and the data processing was conducted with the CellQuest software (Becton Dickinson) on an Apple Computer.

1.9. Preparation of Cytoskeleton Fractions EDTA-washed platelets resuspended in Phillips suspension medium was supplemented with an equal volume of Phillips Triton X-100 solution whereafter the platelets were solubilised by sucking and blowing using a Pasteur pipette. The insoluble material is known to represent most of the actinbased cytoskeleton [17]. A low-speed pellet was prepared by centrifugation of the suspension for 5 minutes at 130003g, and a high-speed pellet was prepared by a further centrifugation at 100,0003g for 2 hours.

1.10. SDS-PAGE and Immunostaining for CD9 SDS-PAGE was performed on 10–20%, or 4–20%, gradient tris-glycine polyacrylamide gels (Novex, San Diego, CA, USA). Samples were mixed with equal amounts of SDS-solubilisation buffer and reduced with 6% 2-mercaptoethanol in boiling water for 5 minutes before electrophoresis. The electrophoresis apparatus Xcell II (Novex, San Diego, CA, USA) was used, and 10–15 mL of samples were added in each lane. Prestained molecular weight standards were used (BioRad, Richmond, CA, USA). The proteins were transferred electrophoretically to a nitrocellulose membrane after SDS-PAGE. Subsequently, the membrane was blocked with 3% dried milk powder in TBS. The membrane was incubated with mAb FN99 against CD9. After washing with TBS and TTBS, the membrane was incubated with alkaline phosphatase conjugated goat-anti mouse IgG F(ab9)2 in TBS containing 0.3% dried milk powder. After washing, the proteins were visualised using the substrate solution NBT/BCIP in H2O.

1.11. Fragments of the CD9 MAb FN99 FN99 was purified on a Protein A agarose column and dialysed against PBS. FN99 was incubated with papain (0.25 mg/mL), DTT (500 mmol/L), and PBS for 1 hour at 378C, whereafter IAA (1.74 mmol/L) was added, followed by a further incubation for 30 minutes at 378C.

1.12. Scanning Electron Microscopy Platelets in citrated plasma were stimulated in the aggregometer with the mAb FN99. Samples were

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collected before stimulation, in the shape change phase, and at the lower part of the descending aggregometer curve. The samples were fixed (1 hour) in cacodylate-buffered glutaraldehyde (2%), and processed for scanning electron microscopy. Briefly, the samples were pelleted by centrifugation at 20003g for 10 minutes, washed in cacodylate buffer, fixed for 20 minutes with osmium tetroxide (1%), washed with cacodylate buffer, dehydrated in graded concentrations of ethanol, dried in a critical point drier, and sputtered with gold particles.

1.13. Secretion from Stimulated Platelets Platelets in citrated plasma were stimulated in an aggregometer with (1) FN99 alone, (2) FN99 in combination with leupeptin, or (3) ALB6. To reach maximal release, the samples were left for 5 minutes in the aggregometer after stimulation. The platelets were then sedimented by centrifugation for 5 minutes at 130003g, and resuspended in 500 mL EDTA washing solution. The procedure was repeated, but this time the platelets were resuspended in 200 mL of EDTA washing solution. The samples were centrifuged again under the same conditions and finally prepared for SDS-PAGE by resuspension in SDS solubilisation buffer and Medium A in a 1:1 relationship. Absence of the a-granula protein thrombospondin in platelet extracts was taken to mean that secretion had occurred. Secretion also was studied as exposure of P-selectin on the platelet surface after stimulation of platelets in c-PRP by using flow cytometry. For this, samples were withdrawn from the aggregometer cuvette and treated as described above.

2. Results 2.1. Effects of the IgG2a mAb FN99 in the Presence of Leupeptin ALB6 is a monoclonal antibody of the IgG1 subtype directed against the platelet CD9 antigen. Used as a control, it was added to citrated plateletrich plasma (c-PRP) and to a suspension of washed platelets, respectively (Figure 1). The response was a typical aggregation curve in both cases: A lag phase followed by shape change and an increase in light transmission associated with an oscillating, descending curve (Figures 1a and 1c). FN99 is a

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mAb against CD9 of the IgG2a subtype. Upon addition of FN99 to c-PRP, a membrane permeabilisation of the platelets occurred. Using the aggregometer, we observed an initial lag phase and shape change followed by an increase in the light transmission with practically no oscillations of the descending curve (Figure 1b), which is typical of complement-mediated membrane permeabilisation [8]. Stimulation of washed platelets with FN99, however, resulted in a platelet aggregation seen as distinct oscillations of the descending curve (Figure 1d). The protease inhibitor leupeptin has been shown to inhibit complement-mediated membrane permeabilisation when added to c-PRP before stimulation of platelets with CD9 mAbs of the IgM class [8]. Addition of leupeptin to c-PRP before stimulation with FN99 (Figure 1f) resulted in the same aggregometer pattern as seen for FN99 in washed platelets (Figure 1d) as well as for ALB6 in both PRP and washed platelet suspensions (Figures 1a and 1c). However, a long lag phase was always observed for FN99 in the presence of leupeptin. It therefore seems that the IgG2a mAb FN99 is able to stimulate the platelets in plasma via two mechanisms depending on the presence or absence of active complement factors. We therefore investigated whether the IgG2a antibody acted as a regular inducer of platelet secretion and aggregation in PRP when leupeptin was present. An FcgRII requirement for the observed aggregation with FN99 was demonstrated when antiFcgRII mAb IV.3 (0.04 mg/mL) was added to c-PRP along with leupeptin before the addition of FN99 (Figure 2). Then, no platelet activation could be observed. Only a stable baseline was seen (Figure 2b). This implies that when the complement cascade is inhibited, FN99 will activate the platelets via the Fc receptor and initiate responses like granule secretion and platelet aggregation via regular intracellular mechanisms. We further got an evidence for this idea by comparing the aggregation induced by FN99 in the presence of leupeptin with the aggregation induced by the thrombin mimicking peptide SFLLRN in the absence or presence of the aggregation inhibitors c7E3 Fab and PGE1. A sharp increase in light transmission occurred as soon as SFLLRN was added to the platelet suspension (Figure 3a). In contrast, there was a time delay before the onset of aggregation with FN99 in the presence of leupeptin (Figure 3d). When adding PGE1 (40 mmol/L) and leupeptin (2.42 mmol/L)

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Fig. 1. Effects of FN99 on platelets in citrated plasma and washed platelets. (a–d) 25 mL of ALB6 or FN99 were added to 475 mL c-PRP or washed platelet suspension in an aggregometer. (e and f) 25 mL leupeptin were added to an aggregometer cuvette containing 450 mL c-PRP before addition of 25 mL ALB6 or FN99. Aggregation is observed as an oscillating, descending aggregometer curve, while membrane permeabilisation is observed as a descending curve with practically no oscillations. A indicates ALB6 (diluted 1:3200 from stock solution); F, FN99 (diluted 1:200 from stock solution); L, leupeptin (2.42 mmol/L).

to c-PRP before stimulation with FN99, a total abolishment of the observed platelet aggregation was observed (Figure 3e). The same was seen after preincubation with PGE1, when platelets were stimulated with SFLLRN (100 mmol/L) instead of FN99 (Figure 3b). Adding c7E3 Fab (0.05 mg/mL) to the same system instead of PGE1 also lead to

Fig. 2. Effect of the mAb IV.3 on the FN99-mediated mechanisms in the presence of leupeptin in c-PRP. Due to the presence of 0.02% Na-azid in the IV.3 sample, this also was added to the control PRP before FN99. (a) Control, 20 mL Na-azid (0.02%), was incubated for 5 minutes in 430 mL c-PRP before addition of 25 mL leupeptin and 25 mL FN99. (b) 20 mL mAb IV.3 was preincubated with 430 mL c-PRP before the addition of 25 mL leupeptin and 25 mL FN99. N, Na-azid (0.02%); F, FN99 (diluted 1:200 from stock solution); L, Leupeptin (2.42 mmol/L).

an inhibition of the platelet aggregation in both cases (Figures 3c and 3f). Neither PGE1 nor c7E3 Fab inhibited the observed membrane permeabilisation in a system with c-PRP and FN99 alone (data not shown).

2.2. Platelet Response to Membrane Permeabilisation Platelet samples that were either stimulated to aggregate in c-PRP with FN99 in the presence of leupeptin, or stimulated to permeabilisation with FN99 alone, were studied by SDS-PAGE. Of interest was the finding that the band for the a-granule protein thrombospondin (TSP) at 150 kDa was still present in membrane-permeabilised platelet samples (Figure 4, lane 3), while TSP had disappeared from the aggregated platelets (Figure 4, lanes 1 and 2). This indicated that a regular secretion does not occur, or occurs only to a minor degree, during membrane permeabilisation via the antibody-activated complement system. Thus, flow cytometry experiments using a FITC-conjugated antibody to P-selectin showed that only a small fraction of the total P-selectin became exposed on the platelet surface after stimulation of platelets in c-PRP with FN99 in the absence of leupeptin at a concentration of FN99 inducing membrane permeabilisation. The mean fluorescence intensity obtained with the FITC-conjugated antibody to P-selectin after such stimulation with FN99 corre-

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Fig. 3. Effects of PGE1 (increases cyclic AMP) and c7E3 (blocker of activated GPIIb-IIIa) on platelet aggregation induced by FN99 in the presence of leupeptin, by using the thrombin mimicking peptide SFLLRN for comparisons. (a) 25 mL SFLLRN added to 475 mL c-PRP. (b) 5 mL PGE1 were preincubated for 2 minutes in 470 mL c-PRP before stimulation with 25 mL SFFLRN. (c) 12 mL c7E3 Fab were added to 463 mL c-PRP before addition of 25 mL SFLLRN. (d) 25 mL leupeptin added to 450 mL of c-PRP before addition of 25 mL FN99. (e) 5 mL PGE1 was preincubated for 2 minutes in 445 mL c-PRP before addition of 25 mL leupeptin and 25 mL FN99. (f) 12 mL c7E3 Fab, 25 mL leupeptin, and 25 mL FN99 were added to 438 mL c-PRP. S, SFLLRN (100 mmol/L); P, PGE1 (40 mmol/L); C, c7E3 Fab (0.05 mg/mL); L, leupeptin (2.42 mmol/L); F, FN99 (diluted 1:200 from stock solution).

sponded to 9.8% of that obtained either with an aggregating concentration of ALB 6 or with 100 mmol/L SFLLRN (mean of five experiments). The membrane-permeabilised platelets showed a profound degradation of the actin-binding protein (ABP) at 250 kDa and talin at 220 kDa as opposed to the aggregated platelets (Figure 4). An intensely

Fig. 4. Effect of platelet permeabilisation induced by FN99 on the thrombospondin (TSP) pool in the platelet a-granules. Platelets in c-PRP were stimulated in an aggregometer, sedimented by centrifugation, resuspended in medium A, solubilised in SDS-sample buffer, and subjected to SDS-PAGE under reducing conditions. Lane 1, platelets stimulated with FN99 in the presence of leupeptin (final concentration 2.42 mmol/L). Lane 2, platelets stimulated with ALB6. Lane 3, platelets stimulated with FN99 alone. Lane 4, unstimulated platelets sedimented directly from c-PRP. Lane 5, molecular weight markers. TSP, thrombospondin. ABP, actin-binding protein.

stained band at 90 kDa was observed with the permeabilised platelet sample (Figure 4, lane 3). Applying mAb Ti 10 directed towards ABP in an immunoblot study, this proved to represent a degradation product of ABP (Figure 5, lane 1). Thus, whereas membrane permeabilisation of the platelets does not involve a massive secretion of proteins

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Fig. 5. Effect on the ABP of platelet permeabilisation induced by FN99. The samples corresponding to those in Figure 4 were electrophoresed by SDS-PAGE, whereafter the proteins were transferred to a nitrocellulose membrane, and immunovisualised using the mAb Ti10 against the 90-kDa end of the ABP subunit. Lane 1, FN99permeabilised platelets. The 90-kDa band represents a degradation product from ABP. Lane 2, unstimulated control platelets. Lane 3, ALB6stimulated platelets. Lane 4, Platelets stimulated with FN99 in the presence of leupeptin.

located in the a-granules, a degradation of the actin binding protein does occur. Scanning electron microscopy of membrane permeabilised platelets clearly showed that the platelets had lost their regular, discoid shape (Figure 6). Only a few pseudopodia were observed.

2.3. Effect of Unlabelled FN99-Fab, FN52, and ALB6 on the Binding to CD9 of FITC-Labelled Fab Fragments of FN99 As would be expected for an antibody lacking an intact Fc part, purified FN99-Fab did not induce platelet activation (data not shown). However, when incubating FN99-Fab in c-PRP, it inhibited the induction of platelet permeabilisation by intact FN99 or FN52, as well as platelet aggregation by ALB6 (data not shown). With a flow cytometer, it is possible to measure the extent of binding of FN99-Fab to the platelet surface on a relative basis. When incubating platelets with either unlabelled Fab fragments of FN99, intact FN52, or ALB6 before addition of FITC-conjugated FN99-Fab, considerably lower mean fluorescence intensities were measured as compared with a control with only buffer and the FITC-labelled Fab fragment added to the platelets (Table 1). In the presence of the inhibitory components, the measured fluorescence intensities were comparable to that of a negative control containing an unspesific IgG2a antibody with no binding sites on the platelets. The competi-

tion indicates that the three antibodies have overlapping, or closely spaced, binding sites on the CD9 molecule.

2.4. Presence of CD9 on Microvesicles Washed platelets stimulated with the Ca-ionophore A23187 in the presence of extracellular Ca21-ions, release microvesicles. The mAb FITC-Y2/51 directed against an epitope on platelet GPIIIa (as well as the GPIIb-IIIa complex) was used as a positive control for registering microvesicles in the flow cytometer, as GPIIIa has been shown to be

Fig. 6. Scanning electron micrograph of permeabilised platelets.

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Table 1. Inhibition of binding of FITC-FN99-Fab to CD9 by anti-CD9 mAbs: FN99-Fab, intact FN52, and intact ALB6 Ligand (unlabelled)

Mean fluorescense intensity

Binding of FITC-FN99-Fab to CD9 (%)

PBS-buffer FN99-Fab FN52 ALB6

45.962.3 19.861.7 7.461.5 5.2261.2

100 43 16 11

Effect of preincubating platelets with unlabelled FN99-Fab, FN52, or ALB6 on the degree of binding of FITC-FN99-Fab to CD9. The results are expressed as the mean fluorescence per platelet 6SD in arbitrary units. PBS, Phosphate buffered saline.

present on the microvesicles [12]. Under our experimental conditions the percentage of microvesicles measured using FITC-Y2/51 was 0.760.7% in an unstimulated platelet sample and 2664.5% in a sample of platelets stimulated by Ca-ionophore in the presence of extracellular calcium ions (Figure 7). Under the same conditions, the percentage of microvesicles measured using FITC-FN99 was 1.060.8% for unstimulated platelets and 17.764.5% for stimulated platelets, respectively. Thus microvesicles display CD9 on their surface as detected by the mAb FITC-FN99.

2.5. Detection of CD9 in the Cytoskeleton Extraction of unstimulated platelets in 1% Triton X-100 is considered to result in an almost total

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solubilisation of the platelets except for the actin filament cytoskeleton and associated proteins [17]. The platelet cytoskeleton was isolated in two fractions; the low-speed pellet (130003g) and the highspeed pellet (100,0003g) as described in Materials and Methods. Figure 8 represents immunostaining for CD9 after SDS-PAGE and Western blotting. Bands for CD9 clearly can be seen around 28 kDa in the unstimulated platelet extract (Figure 8, lane 2), and in the supernatant after centrifugation at 100,0003g (Figure 8, lane 5). No bands for CD9 were seen with the cytoskeletal fractions (Figure 8, lanes 3 and 4). On the other hand, as used for comparisons, bands for GPIb and its degradation product, glycocalicin, were clearly present in all four samples.

3. Discussion Obviously, the platelet response to anti-CD9 mAbs are subtype dependent. All reported IgG1 antibodies against CD9 induce a relatively powerful platelet aggregation, such as that observed for ALB6 [3,6,7]. mAbs of the subclasses IgM, IgG2a, IgG2b, and IgG3 against CD9 are all reported to induce a platelet permeabilisation [7,8,19]. Two other groups have demonstrated an aggregation response with IgG2a antibodies when inactivating the complement factors by heating the plasma [7,20]. Thus, it may be typical for IgG2a mAbs that they induce aggregation whenever the complement

Fig. 7. CD9 on microvesicles released from platelets with the Ca-ionophore A23187 in the presence of extracellular calcium ions. The FITC-conjugated markers Y2/51 against GPIIIa and FN99 against CD9 were used for detecting their respective antigens on the surface of the microvesicles by using flow cytometry. The figure shows the number of microvesicles detected with the two different FITC-mAbs in percentage of the total fluorescent particles registered (microvesicles plus platelets). Mean6SD. For details, see Materials and Methods.

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Fig. 8. Experiment to see if CD9 is associated to the membrane skeleton. SDS-PAGE was conducted with unreduced platelet Triton X-100 extract and cytoskeletal fractions followed by immunovisualisation with a combination of the antibodies FN99 against CD9 and PM6/81 against GPIb after transfer to a nitrocellulose membrane. Lane 1, molecular weight markers. Lane 2, solubilised control platelets (Triton X-100 extract). Lane 3, 130003g low-speed cytoskeletal fraction. Lane 4, 100,0003g highspeed cytoskeletal fraction. Lane 5, Supernatant after ultracentrifugation. Band for CD9 at approximately 28 kDa. GC, Glycocalicin.

factors are inactivated. Upon addition of FN99 to platelets in citrated plasma, we observed an aggregometer curve typical for a platelet permeabilisation. This curve was clearly different from the aggregometer curve observed with platelet stimulation by the control mAb ALB6 that induces aggregation. When stimulating washed platelets with FN99, we demonstrated a platelet aggregation instead of a membrane permeabilisation. We demonstrated the same phenomenon in plasma when inhibiting the complement factors by using the protease inhibitor leupeptin. We then obtained an aggregometer curve directly comparable to those of ALB6-stimulated platelets and FN99-stimulated washed platelets, except for a longer lag phase. Thus, the IgG2a mAb FN99 induces platelet changes in plasma by two different mechanisms, dependent on the presence or absence of active complement factors, that is, membrane permeabilisation or platelet aggregation. We demonstrated that the platelet aggregation with FN99 in the presence of leupeptin was dependent on signalling through the Fc-receptor FcgRII and probably used a similar intracellular signalling pathway as the thrombin mimicking peptide SFLLRN as they were inhibited by the same inhibitors, that is, PGE1 and c7E3 Fab. The inhibitory effect of leupeptin in the complement system has been described previously [8,21,22]. After platelet permeabilisation stimulated with FN99, thrombospondin was still present in the permeabilised platelets indicating that a massive secre-

tion from the a-granules had not occurred. Thrombospondin was not detected in samples with aggregated platelets. A limited exposure of P-selectin on the platelet surface after stimulation by FN99 alone, indicates that a regular secretion may have occurred in a few platelets, however. The complement complex C5b-9 as produced from purified components has been reported to provoke secretion from the dense granules, as well as from a-granules [9]. A problem inherent in such studies, however, is that if the membrane permeabilisation is only minor, remaining intact platelets may become activated by ADP leaking from the affected platelets. We observed that the actin-binding protein (ABP) was degraded upon platelet permeabilisation. When leupeptin was added to this system prior to stimulation with FN99, only limited degradation of ABP was observed. In addition to its effects in the complement system, leupeptin is known for its ability to inhibit the calcium-activated protease calpain present in platelets, and calpain is known to degrade ABP upon its activation due to an increased cytosolic level of calcium-ions. Both of these effects may have contributed in the present situation. The abnormal platelet shape after membrane permeabilisation observed by the scanning electron microscopy may be explained by the idea that a degradation of ABP will lead to loss of contact between glycoproteins in the membrane and the membrane skeleton. This observation also confirms our previous observation with the IgM anti-

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body FN52 as far as degradation of ABP is concerned [8]. Three different mAbs against CD9 were available in our laboratory; ALB6, FN99, and FN52. To determine whether these three antibodies would compete for binding to CD9 or not, we used Fabfragmented FN99. Fab fragments bind to CD9 but will not elicit any of the responses described above as the fragments lack the reactive Fc fragment that reacts with the Fc receptor as well as with the complement system. We demonstrated a clear inhibition of binding of FITC-labelled FN99-Fab to platelets after preincubation with unlabelled ALB6, FN99, or FN52. A similar study was performed by Nomura et al. [20] by using the anti-CD9 mAbs ALB6, NNKY1-19, MALL13, and TP82. In flow cytometer experiments, an inhibition of binding of all the mAbs after preincubation with NNKY1-19 was observed [20]. As ALB6 was used both in Nomura’s and the present study, and the mAbs used in the surveys were of different subtypes, it may indicate that most, or all, mAbs against CD9 will bind in the same area of CD9. An increased concentration of calcium in the platelet cytosol induces a release of microvesicles from the platelet membrane [10,11]. This occurs after activation of the platelets with thrombin, collagen, Ca-ionophore A23187, or complement activation [9,10]. The microvesicles are rich in the platelet receptor GPIIb-IIIa involved in the aggregation process [12]. FITC-conjugated antibodies against GPIIb-IIIa often are used as markers for microvesicles in flow cytometry. Since CD9 is a membrane protein present on the platelets in a high copy number [3], we expected to register its presence also on the microvesicles. As expected, control experiments using FITC-labelled Y2/51 against GPIIb-IIIa demonstrated a significant amount of GPIIb-IIIa present on the microvesicles, and a significant binding of the anti-CD9 mAb FITC-FN99 to microvesicles was observed as well. Thus, microvesicles express CD9 on their surface. Nomura et al. [20] also demonstrated the presence of CD9 on microvesicles by using the IgG2a mAb MALL13. It has been suggested that CD9 and GPIIb-IIIa may associate physically when platelets are stimulated with mAbs against CD9 [23]. The mAbs might lead to a conformational change within the CD9 molecule that permits binding of GPIIb-IIIa to CD9 [24]. As this complex has not

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yet been identified with immunoblotting assays, it seems probable that the high number of GPIIbIIIa and CD9 on microvesicles simply reflects the high number of both of these molecules on the platelets, however. It is known that the platelet glycoprotein GPIb is connected to the membrane skeleton via ABP [14,15]. We used SDS-PAGE and immunoblotting to find out whether also CD9 is connected to the cytoskeleton, or not. We found no evidence for such a connection. In contrast to a high staining for GPIb after Western blotting, there was no staining for CD9 in samples with “low-speed” or “highspeed” cytoskeleton. CD9 has been reported to be closely associated with GPIIb-IIIa in platelets [23], probably by means of hydrophobic interactions [25], and to be colocalised with GPIIb-IIIa on the pseudopodia of activated platelets and in the a-granule membrane [26]. It also has been suggested that CD9 might be tightly associated with one or two small GTPbinding proteins [27], but we have found no evidence for this (unpublished observations). In summary, we demonstrate here that the IgG2a mAb FN99 induces platelet changes according to dual mechanisms in plasma, and this may be a reaction common to antibodies to CD9 of the IgG2a subtype [7]. The antibody normally will induce a complement-mediated platelet permeabilisation, but a regular secretion and platelet aggregation is induced when the complement system is inhibited by leupeptin. Secretion of the a-granule protein thrombospondin was not observed during platelet permeabilisation, but a limited amount P-selectin was exposed on the platelet surface. Furthermore, we demonstrated that the three mAbs against CD9, ALB6, FN99, and FN52 competed for binding to CD9, that microvesicles display CD9 on their surface, and that CD9 is not anchored to the platelet cytoskeleton. The present study has been supported by The Norwegian Council on Cardiovascular Diseases, Anders Jahres fund, and professor Paul A. Owrens fund.

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