non-apoptoticphosphatidylserineexternalizationinducedby ...proteins (gpi-aps). however, it is...

Non-apoptotic Phosphatidylserine Externalization Induced byEngagement of Glycosylphosphatidylinositol-anchored Proteins*

Received for publication, December 4, 2006, and in revised form, January 22, 2007 Published, JBC Papers in Press, February 5, 2007, DOI 10.1074/jbc.M611090200

Daniel Smrz, L’ubica Draberova, and Petr Draber1

From the Department of Signal Transduction, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,CZ 142 20 Prague 4, Czech Republic

The exposure of phosphatidylserine (PS) on the cell surface isa general marker of apoptotic cells. Non-apoptotic PS external-ization is induced by several activation stimuli, includingengagement of immunoreceptors. Immune cells can alsobe acti-vated by aggregation of glycosylphosphatidylinositol-anchoredproteins (GPI-APs). However, it is unknown whether cell trig-gering through these proteins, lacking transmembrane andcytoplasmic domains, also leads to PS externalization. Here weshow that engagement ofGPI-APs in rodentmast cells induces arapid and reversible externalization of PS by a non-apoptoticmechanism. PS externalization triggered by GPI-AP-specificmonoclonal antibodies was dependent on the activity ofH�-ATP synthase and several other enzymes involved in mastcell signaling but was independent of cell degranulation, freecytoplasmic calciumup-regulation, and a decrease in lipid pack-ing as determined by merocyanine 540 binding. Surprisingly,disruption of actin cytoskeleton by latrunculin B or plasmamembrane integrity by methyl-�-cyclodextrin had oppositeeffects on PS externalization triggered through GPI-AP or thehigh affinity IgE receptor.We further show that PS externaliza-tionmediated byGPI-APswas also observed in someother cells,and its extent varied with antibodies used. Interestingly, effectsof different antibodies onPS externalizationwere additive, indi-cating that independent stimuli converge onto a signaling path-ways leading to PS externalization.Our findings identify the cellsurface PS exposure induced through GPI-AP as a distinctmechanism of cell signaling. Such a mechanism could contrib-ute to “inside-out” signaling in response to pathogens and otherexternal activators and/or to initiation of other functions asso-ciated with PS externalization.

The plasma membrane exhibits a marked asymmetry intransbilayer distribution of phospholipids. Aminophospholip-

ids, including phosphatidylserine (PS),2 are usually restricted tothe inner leaflet of the membrane. This phospholipid asymme-try is maintained by activity of energy-dependent flippases andfloppases that mediate, respectively, inward-directed and out-ward-directed transfer of phospholipids (1–3). Furthermore,equilibration of phospholipids between the two plasma mem-brane leaflets seems to be regulated by lipid scramblase, whichfacilitates bi-directional migration of phospholipids across thebilayer (4, 5). In response to some stimuli, the phospholipidasymmetry is lost, and PS is translocated to the exoplasmicleaflet of plasma membrane (6). Externalized PS is observed inapoptotic, injured, infected, senescent, or necrotic cells andbecomes a target for recognition by phagocytes (7–11). PSexternalization is also detected at certain stages of cell develop-ment (12) and in the course of activation of immune cells bydifferent stimuli, including engagement of immunoreceptors(13–16).It has been described that PS externalization is triggered by

stimuli enhancing the concentration of free cytoplasmic cal-cium, which regulates the activities of lipid translocases andpromotes randomization of plasma membrane phospholipids(1, 17, 18). This notion was corroborated by experiments indi-cating that drugswhich enhance calcium influx also enhance PSexternalization (1). In mast cells, antigen or antibody-mediatedaggregation of the high affinity IgE receptor (Fc�RI) triggerssignaling pathways leading to increased tyrosine phosphoryla-tion of numerous proteins, enhanced concentration of freecytoplasmic calcium [Ca2�]i, and release of preformed granules(19). This process, called degranulation, is associatedwith rapidand transient externalization of PS (13, 14). A weak secretoryresponse can also be induced through aggregation of glyco-sylphosphatidylinositol-anchored proteins (GPI-APs) bymonoclonal antibodies (mAbs) (20, 21). However, the exactmechanism of cell activation through GPI-APs, which have notransmembrane and cytoplasmic domain, is unclear. Further-

* This work was supported in part by Center of Molecular and Cellular Immu-nology Project 1M6837805001 and Grant LC545 from Ministry of Educa-tion, Youth, and Sports of the Czech Republic, Grant Agency of the CzechRepublic Grant 301/06/0361, Grant Agency of the Academy of Sciencesof the Czech Republic Grant IAA5052310, and Institutional ProjectAVOZ50520514. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 Supported by an International Research Scholar award from HowardHughes Medical Institute. To whom correspondence should beaddressed: Dept. of Signal Transduction, Institute of Molecular Genet-ics, Academy of Sciences of the Czech Republic, Vıdenska 1083, CZ 14220 Prague 4, Czech Republic. Tel.: 420-241062468; Fax: 420-241062214;E-mail: [email protected].

2 The abbreviations used are: PS, phosphatidylserine; Fc�RI, high affinity IgEreceptor; [Ca2�]i, concentration of intracellular free calcium; GPI-AP, glyco-sylphosphatidylinositol-anchored protein; mAb, monoclonal antibody;TNP, trinitrophenyl; Ig, immunoglobulin; CEA, carcinoembryonic antigen;FITC, fluorescein isothiocyanate; zVAD-FMK, benzyloxycarbonyl-VAD-fluoromethyl ketone; PP2, 4-amino-5-(4-chlorophenyl)-7-(tbutyl)pyra-zolo[3,4-d]pyrimidine; �MM, methyl-�-D-mannopyranoside; PI-PLC,phosphatidylinositol-specific phospholipase C; DIDS, 4,4�-diisothiocya-natostilbene-2,2�-disulfonic acid disodium salt hydrate; RBL, rat baso-philic leukemia; FCS, fetal calf serum; BMMC, bone marrow mast cell;LAT, linker for activation of T cell; RPMC, rat peritoneal mast cell; PBS,phosphate-buffered saline; BSS, buffered saline solution; BSA, bovineserum albumin; PI, propidium iodide; MC540, merocyanine 540; Con A,concanavalin A; M�CD, methyl-�-cyclodextrin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 14, pp. 10487–10497, April 6, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

APRIL 6, 2007 • VOLUME 282 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 10487

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more, it is not known whether engagement of GPI-AP couldalso lead to PS externalization. In this study we have analyzedexternalization of PS in mast cells and some other cell typesafter GPI-AP triggering. Our data indicate for the first time thatengagement of GPI-AP can induce PS externalization by a dis-tinct mechanism of intracellular signaling.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—The following mAbs were used:MRCOX7 (OX7), specific for Thy-1.1 (22); 1aG4/C5, recogniz-ing Thy-1.2 (23); anti-Fc�RI � subunit, clone 5.14 (24); trinitro-phenyl (TNP)-specific immunoglobulin (Ig) E (IGEL b4 1) (25);TEC-21, recognizing a GPI-AP TEC-21 (21); D6.17.7, specificfor carcinoembryonic antigen (CEA; CD66e) (26). Phosphoty-rosine-specific mAb (PY-20) conjugated to horseradish perox-idase was purchased from BD Biosciences. Rat CD48-specificmAb (CD48) was obtained as hybridoma supernatant fromSerotec (Oxford, UK). Anti-mouse IgG-cyanine 3 conjugatewas bought from Jackson ImmunoResearch Laboratories, Inc.(West Grove, PA). F(ab) fragment of OX7 mAb was preparedusing ImmunoPure Fab preparation kit (Pierce). The followingreagents were used: annexin V-fluorescein isothiocyanate(FITC; BD Biosciences), benzyloxycarbonyl-VAD-fluoro-methyl ketone (zVAD-FMK) (QBIOGENE-Alexis, Grunberg,Germany), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor, methyl-�-D-mannopyranoside(�MM), latrunculin B (Merck-Calbiochem), and Fura-2-AM(Molecular Probes, Eugene, OR). Phosphatidylinositol-specificphospholipase C (PI-PLC) from Bacillus cereus, 4,4�-diisothio-cyanatostilbene-2,2�-disulfonic acid disodium salt dihydrate(DIDS) and all other chemicals were from Sigma-Aldrich.Cells—The origin of rat basophilic leukemia (RBL) cells,

clone 2H3, and their transfectant, RBL-gT1.2/1, expressingboth the endogenousThy-1.1 and the transfectedThy-1.2 gene,have been described (27). The cells were grown in completeculture medium consisting of a 1:1 mixture of RPMI 1640 andminimal essential medium supplemented with 10% (v/v) fetalcalf serum (FCS), extra D-glucose (2.5 mg/ml), and antibiotics(100 units/ml penicillin and 100 �g/ml streptomycin). RBL-2H3 cells expressing human CEA, clone RBL-CEA/2D1, wereprepared by co-transfection of human CEA cDNA (28), sub-cloned into the p91023B expression vector (courtesy of R. Kauf-man, Genetics Institute, Boston), and psTneo B vector (29),conferring resistance to the neomycin analogue Geneticin(G418). Permanent transfectants were isolated by growing thecells in selective medium containing G-418 (Invitrogen, 400�g/ml estimated pure G418). Mouse myelomonocyte WEHI-3cells were cultured inDulbecco’smodified Eagle’smedium sup-plemented with 10% (v/v) FCS, antibiotics, 1% minimal essen-tialmediumnonessential amino acids, and 10mMsodiumpyru-vate. Mouse 3T3 fibroblasts were cultured in complete culturemedium. Human colon carcinoma cells, SW1417, wereobtained from the American Type Culture Collection and cul-tured in RPMI 1640 with 10% (v/v) FCS and antibiotics. Isola-tion and culturing of bone marrowmast cells (BMMC) derivedfromwild type or linker for activation of T cells (LAT)-deficientmice have been previously described (30). Rat peritoneal mastcells (RPMC) were recovered by peritoneal cavity lavage with

phosphate-buffered saline (PBS; 135 mM NaCl, 1.7 mMNa2HPO4�2 H2O, and 5 mM KH2PO4, pH 7.4) followed by den-sity gradient centrifugation over Ficoll gradient (31). The sus-pensions contained �95%mast cells as determined by stainingwith 0.1% toluidine blue. Rat peritoneal exudate cells were elic-ited by intraperitoneal administration of 8 ml of 3% (w/v) thio-glycolate in PBS intomale rats (Wistar). Elicited peritoneal exu-date cells were harvested 5 days later by peritoneal lavage withsterile RMPI 1640, 10% FCS, added to glass Petri dishes, andincubated at 37 °C in 5% CO2. After 6 h the dishes were flushedto remove non-adherent cells, and those adherent (macrophages)were further incubated in fresh RPMI 1640, 10% FCS. Two dayslater the cells were collected by trypsinization. Peripheral bloodfrom C57/BL6 mice was obtained by tail bleeding into 3.8% (w/v)sodium citrate. Mononuclear blood cells were obtained usingHISTOPAQUE� 1119 and 1077 (Sigma-Aldrich) according tothe manufacturer’s instructions. A fraction of mononuclearcells was collected, resuspended in RPMI 1640, 2% FCS, andincubated with Leuko-Pak� leukocyte filter (Fenwal Laborato-ries, Deerfield, IL) for 40 min at room temperature. The non-adherent cells, �85% T lymphocytes as inferred from theexpression of Thy-1.2 glycoprotein (32), were collected andsubjected to further analysis. Escherichia coli (strain M15) wasgrown in LB medium (33) at 37 °C for 12–16 h (A600 � 3). Thecollected cells were washed, adjusted to �1010/ml, and imme-diately used for activation studies.Cell Activation and PI-PLC Treatment—Cells were washed

with buffered saline solution (BSS; 20 mM HEPES, pH 7.4,135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5.6 mM glucose)supplemented with 1.8 mM CaCl2 and 0.1% bovine serumalbumin (BSS/BSA). The cells were resuspended in BSS/BSAat a concentration 20 � 106/ml. The suspension was mixedwith twice-concentrated activators in BSS/BSA at a ratio 1:1,incubated at 37 °C for 20 min, and centrifuged at 150 � g for5 min. The effect of inhibitory drugs was studied on cellspretreated with particular inhibitors at 37 °C as indicatedunder “Results.” The drugs were also present during cell acti-vation and annexin V-FITC labeling. To remove GPI-AP, thecells (15 � 106/ml) in BSS/BSA without calcium were incu-bated without (control) or with PI-PLC (1.5 units/ml) for 50min at 37 °C.Determination of Cell Degranulation and Intracellular Free

Calcium Concentration—Degranulation of the cells wasassessed by the amount of�-glucuronidase released into super-natant. 20-�l aliquots of the supernatant weremixedwith 60�lof 40 �M 4-methylumbelliferyl �-D-glucuronide. After 60 minof incubation at 37 °C, the reaction was stopped by adding 200�l of ice-cold 0.2 M glycine buffer, pH 10.0, and fluorescencewas determined in a microtiter plate reader Fluorostar (SLTLabinstruments GmbH, Grodig, Austria) with 365-nm excita-tion and 460-nm emission filters. Total cell content of theenzyme was evaluated in supernatants from cells lysed in 0.1%Triton X-100. Concentrations of intracellular free calcium[Ca2�]i was measured using Fura-2 as a probe as previouslydescribed (34). Briefly, the cells were incubated at 37 °C for 30min with 2 �M Fura-2-AM and 2.5 mM probenecid. Cells werethen washed in BSS/BSA/probenecid and immediately beforeassessment washed once more in BSS/BSA. The samples were

Phosphatidylserine Externalization via GPI-anchored Proteins

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transferred into cuvettes and analyzed on luminescence spec-trometer LS 50B (PerkinElmer Life Sciences) with excitationwavelengths at 340 and 380 nm and constant emission at 510nm. Calcium concentrations were calculated bymeans ofWin-lab software (PerkinElmer).PS Externalization and Flow Cytometry Analysis—Exposure

of PS on the cell surface was detected by FITC-labeled annexinV (35, 36) using a modified manufacturer protocol (BD Bio-sciences). Briefly, cells were spun down and resuspended inannexinV binding buffer (10mMHEPES, pH 7.4, 140mMNaCl,2.5 mM CaCl2) at a concentration of 2 � 106 cells/ml. 38 �l ofthe cell suspension wasmixed with 1�l of annexin V-FITC and2.5 �l of propidium iodide (PI, 50 �g/ml) and incubated for 15min at room temperature in the dark. The reactionwas stoppedby adding 200�l of annexin V binding buffer, and samples wereimmediately analyzed by flow cytometry on FACSCalibur withCellQuest software (BD Biosciences). Only PI-negative cellswere further analyzed. In experiments using cells activated byco-culturing with E. coli or under conditions lacking extracel-lular calcium, the sampleswerewashedwith ice-cold annexinVbinding buffer followed by incubation with annexin V-FITCand PI for additional 30 min on ice. Aliquots of 200 �l of ice-cold annexin V binding buffer were then added, and the sam-ples were immediately subjected to flow cytometry; to getreproducible results, keeping the cells under ice-cold condi-tions during the whole procedure was essential. In experimentsevaluating the kinetics of PS externalization, cells were incu-batedwith orwithout priming antibodies (5�g/ml) in BSS/BSAfor 30min on ice, washed twicewith ice-cold annexinVbindingbuffer, and incubated with annexin V-FITC and PI on ice. After30 min the cells were transferred to 37 °C, and the reaction wasstopped by adding 200 �l of ice-cold annexin V binding bufferat various time intervals. The amount of annexin V-FITCbound was immediately quantified by flow cytometry. Todetermine antibody binding, cells were washed with ice-coldPBS supplemented with 1% BSA (PBS/BSA) and stained on icefor 30 min with anti-mouse IgG-FITC conjugate (10 �g/ml inPBS/BSA). The cells werewashed 3 timeswith ice-cold PBS andanalyzed by flow cytometry. To determine merocyanine 540(MC540) binding, cells were washed and resuspended at a con-centration of 5 � 106/ml in BSS/BSA supplemented with 0.05�M MC540 followed by incubation at 37 °C for 3 min beforeanalysis by real time flow cytometry carried out at 37 °C.Confocal Microscopy—Cells were spun down and resus-

pended in annexin V binding buffer at a concentration of 6 �106 cells/ml. Fifty �l of the cell suspension was mixed with 3 �lof annexin V-FITC and incubated at room temperature in thedark for 15 min. Then the cells were transferred on poly-L-lysine-coated coverslips (12 mm in diameter) placed in wells ofa 24-well plate. Fivemin later 400�l of 5% paraformaldehyde inannexin V binding buffer was added, and the samples wereincubated at room temperature for 15min followed by washingwith PBS and blocking with PBS/BSA for 10 min at room tem-perature. The cells were then stained on ice for 20 min withanti-mouse IgG-cyanine 3 conjugate (10 �g/ml in PBS/BSA)and washed again with PBS. Images were acquired with a LeicaTCS NT/SP confocal system in conjunction with a Leica DMR

microscope (Leica Microsystems GmbH, Wetzlar, Germany)equipped with oil objective100�/1.4 numerical aperture.Evaluation of the Results—Means � S.D. were calculated

from at least three independent experiments. Statistical signif-icance of differences was calculated using Student’s t test.

RESULTS

PS Externalization Induced in Activated Mast Cells in theAbsence of the Secretory Response—The study was initiated byanalyzing the correlation between cell surface exposure ofPS, assessed by annexin V-FITC binding, and secretoryresponse of mast cells exposed to various stimuli. Experi-ments using TNP-specific IgE and antigen (TNP-BSA) con-firmed previous reports (13, 14) showing that the engage-ment of Fc�RI in BMMC induced PS externalization whichcorrelated with the secretory response determined by therelease of �-glucuronidase (Fig. 1A). Interestingly, when thecells were exposed to lectin concanavalin A (Con A), PSexternalization was comparable with that found in cells acti-vated through Fc�RI even though Con A induced onlyminute degranulation. Similarly, exposure of the cells to bac-terial strain E. coli induced PS externalization in the absenceof secretory response. As expected, in BMMC from micedefective in LAT, an important transmembrane adaptor pro-tein required for Fc�RI signaling (37), both the antigen-in-duced PS externalization and the secretory response werereduced. Surprisingly, however, PS exposure induced by ConA or E. coli in LAT�/� cells was not diminished (Fig. 1A). PSexternalization concomitant with weak or no degranulationwas also observed in RBL-2H3 cells or in freshly isolatedRPMC exposed to Con A or E. coli (Fig. 1, B and C). In thelatter cells strong secretory response was observed after pre-treatment of the cells with thapsigargin, an inhibitor ofendoplasmic reticulum Ca2� ATPase. Enhanced secretionfrom RPMC in response to thapsigargin was, however, notassociated with any stronger increase in PS externalization.Thus, the extent of PS externalization in mast cells does notalways correlate with the extent of degranulation.PS externalization induced by Con A was completely

inhibited in cells pretreated with �MM, a specific competi-tive inhibitor of Con A binding (Fig. 1D; �MM/Activ). Partialinhibition of PS externalization by �MM was also observedin cells stimulated with E. coli. Even when �MM was addedafter stimulation with Con A or E. coli (Activ/�MM), theamount of surface PS was still reduced, suggesting that theprocesses are reversible and depend on constant engage-ment of mannose-containing plasma membrane glycoconju-gates. Pretreatment of the cells with PI-PLC, which cleavesGPI linkage, significantly (p � 0.005) reduced the PS exter-nalization induced by both Con A and E. coli (Fig. 1D),implying that GPI-anchored proteins are involved in theprocess of PS externalization.PS Externalization Induced by Triggering through GPI-AP—

Next we tested whether PS externalization could be induced bymAb specific for GPI-AP. For these experiments we selectedseveral antibodies recognizing GPI-AP expressed on mastcells: Thy-1.1 (recognized by OX7 mAb) (20, 27), CD48 (38,39), and TEC-21 (21). The antibodies were used at such con-




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centrations (5 �g/ml for OX7 andanti-TEC-21 and 1:10-dilutedhybridoma supernatant for anti-CD48) that induce dimerization ofthe target antigens but no degran-ulation (see below). As shown inFig. 2, A and B, all antibodiesbound to RBL-2H3 cells andinduced PS externalization. PSexternalization was also observedin freshly isolated RPMC exposedto OX7 or anti-CD48 (Fig. 2, C andD). As expected, anti-TEC-21induced no PS externalization inRMPC because TEC-21 is notexpressed in these cells (not shown).These data indicate that the engage-ment of GPI-AP leads to PSexternalization.PS Externalization Induced

through GPI-AP Is Rapid andReversible—To further characterizethe mechanism leading to PS exter-nalization through engagement ofGPI-AP, we used RBL-2H3 cells andThy-1.1-specific mAb OX7. Wefound that PS externalizationinduced through Thy-1.1 is a rapidprocess reaching a plateau at �20min after triggering (Fig. 3A). PSexternalization was not absent incells incubated without antibody orwith 1aG4/C5, an antibody that isspecific for the Thy-1.2 isoform(Fig. 3A) and does not bind to RBL-2H3 cells (27). These findings con-firmed the specificity of the reactionand proved that the binding ofannexin V-FITC to nonactivatedcells is not enhanced during thewhole (40min) incubation period at37 °C. Pretreatment with PI-PLCabolished the binding of OX7 to thecells and PS externalization (Fig. 3B;PI-PLC/OX7). IncubationwithOX7to allow externalization of PS fol-lowed by PI-PLC treatment reducednot only the amount of OX7bound but also PS exposure (Fig.3B; OX7/PI-PLC); this indicatesthat the process of Thy-1.1-in-duced PS externalization is revers-ible and depends on a continuousThy-1.1 engagement. When intactOX7 mAb or its F(ab) fragmentwas used, they bound to the RBL-2H3 cells at comparable levels, butonly the intact (divalent) antibody

FIGURE 1. Various stimuli induce PS externalization and degranulation in different mast cell types. A, BMMCwere obtained from wild type (WT) or LAT deficient (LAT �/�) mice, sensitized with TNP-specific IgE (1 �g/ml), andstimulated for 30 min with antigen (TNP-BSA; 0.5 �g/ml). Alternatively, non-sensitized BMMC were activated for 30min with Con A (20 �g/ml) or E. coli (�1010/ml). B–C, RBL-2H3 cells (B) or freshly isolated RPMC (C) were exposed tothapsigargin (Th, 4 �M), Con A (20 �g/ml), or E. coli (�1010/ml) for 20 min. PS externalization was determined by flowcytometry after staining of the cells with annexin V-FITC. Thin and thick lines indicate annexin V-FITC binding tononactivated (Control) and activated cells, respectively. Degranulation was determined by measurement of �-glu-curonidase released into supernatant. D, RBL-2H3 cells were activated by adding Con A (50 �g/ml) or E. coli (�1010/ml) after (�MM/Activ) or before (Activ/�MM) treatment with �MM (50 mM, 30 min). Alternatively, the cells were pre-treated with PI-PLC before activation. Data were normalized to Con A- or E. coli-activated cells treated with vehicle alone(BSS/BSA, Control). In A--C, typical flow cytometry profiles from at least three independent experiments are shown.




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induced PS externalization (Fig. 3C). Thus, the minimumrequirement for induction of PS externalization is Thy-1.1dimerization.Distinct Mechanisms of PS Externalization in Cells Activated

through GPI-AP—To find out whether Thy-1.1-induced PSexternalization is mediated by the same mechanism as the oneeffective in Fc�RI-activated cells, we compared levels ofannexin V-FITC binding in cells activated by the OX7 mAb(which dimerizes Thy-1.1) and cells activated by the 5.14 mAb(which dimerizes Fc�RI) (40, 41). Data presented in Fig. 4Ashow that performance of OX7 was comparable with 5.14. As acontrol we also used calcium ionophore (ionomycin)-activatedcells and confirmed previous data (13, 17, 42) that ionomycin

also induces PS externalization. Recently, Elliott et al. (42) sug-gested a model in which calcium ionophore-induced PS redis-tribution is preceded by and dependent on a decreased lipidpacking,whichwas detected by a fluorescent probeMC540 thatbinds preferentially to the outer leaflet of the plasmamembranewith relatively loosely packed lipids (43, 44). To determinewhether engagement of Thy-1.1 is also accompanied withdecreased lipid packing, we studied the kinetics of MC540binding to control and activated RBL-2H3 cells. Data in Fig. 4Bshow a dramatic increase in MC540 binding in ionomycin-ac-tivated cells but no enhanced binding in cells activated by OX7or 5.14. These data indicate that PS exposure in Thy-1.1- andFc�RI-activated cells is mediated by a mechanism independentof a decrease in lipid packing. Further experiments showed thatdimerized Thy-1.1, unlike dimerized Fc�RI, was ineffective ininducing any degranulation (Fig. 4C) or calcium response (Fig.

FIGURE 2. PS externalization is induced by mAb against different GPI-AP.RBL-2H3 cells (A and B) or RPMC (C and D) were exposed for 20 min toanti-Thy-1.1 (OX7, 5 �g/ml), anti-CD48 (CD48, 1:10 diluted hybridomasupernatant), or anti-TEC-21 (TEC-21, 5 �g/ml; RBL-2H3 cells only).Annexin V-FITC binding (A and C) was determined as described in Fig. 1.Antibody binding (B and D) was determined by binding of FITC-labeledsecondary anti-IgG followed by flow cytometry analysis; thin lines indicatebinding of FITC-labeled secondary antibody alone. Typical flow cytometryprofiles from at least three independent experiments are shown.

FIGURE 3. PS externalization induced by Thy-1.1-specific mAb is rapid,reversible, and dependent on Thy-1.1 aggregation. A, kinetics of PS exter-nalization induced by engagement of Thy-1.1 glycoprotein with OX7 mAb(filled circles) in RBL-2H3 cells. Untreated (filled triangles, Control) or anti-Thy-1.2 mAb (open circles, 1aG4/C5)-treated cells were used as negative controls.The data were normalized to responses obtained at 0 min for untreated cells(0%) and at 20 min for OX7 mAb-treated cells (100%). B, RBL-2H3 cells weretreated with PI-PLC before (PI-PLC/OX7) or after (OX7/PI-PLC) exposure to OX7.Annexin V-FITC (black bars) and OX7 mAb (empty bars) binding was deter-mined and normalized to values in control cells (OX7-activated and PI-PLC-untreated). C, role of Thy-1.1 dimerization. The cells were exposed to wholeOX7 IgG or its F(ab) fragments, and annexin V-FITC and antibody binding wasdetermined as described in Figs. 1 and 2. In C, a typical result from at leastthree independent experiments performed is shown.




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4D); tyrosine phosphorylation of numerous cellular proteinswas also substantially lower in Thy-1.1-activated cells as com-pared with Fc�RI-triggered cells (Fig. 4E).

Next, we compared the sensitivity of Thy-1.1- and Fc�RI-mediated PS externalization to various pharmacological inhib-itors. When cells were pretreated with zVAD-FMK, an inhibi-tor of caspases, no inhibition of PS externalizationwas observed(Fig. 4F), suggesting a non-apoptotic origin of PS externaliza-tions induced by both Thy-1.1 and Fc�RI. In contrast, severalother drugs that inhibit phospholipid translocases (DIDS andglybenclamide), phosphatidylinositol-3 kinase (wortmannin),Src-family kinases (PP2), and Syk family kinases (piceatannol)reduced both types of PS externalization. Importantly, cells cul-tured in the absence of glucose and treated with oligomycin oraurovertin B, H�-ATP-synthase inhibitors, also exhibitedreduced PS externalization, indicating that ATP-dependentmechanisms are involved. Impaired PS externalizations werealso observed after exposure of cells to the mAb in the absenceof extracellular Ca2� followed by transfer of the cells on ice andthe addition of annexin V binding buffer with Ca2�. A dramaticdifference between Thy-1- and Fc�RI-mediated PS exposurewas observed in cells pretreated with methyl-�-cyclodextrin(M�CD), which does not bind to the cells but effectivelyremoves cellular cholesterol (45, 46). In OX7-activated cells PSexternalization was inhibited by M�CD, whereas it wasenhanced in 5.14-activated cells (Fig. 4G). Similarly, if the cellswere pretreated with latrunculin B, an inhibitor of actin polym-erization (47, 48), PS externalization was inhibited in OX7-ac-tivated but potentiated in cells treated with 5.14 (Fig. 4G).Data on spatial distribution of externalized PS obtained by

confocal microscopy show that externalized PS in cells acti-vated through OX7-dimerized Thy-1.1 is distributed in spotsand patches, whereas Thy-1.1 is distributed mostly homoge-neously (Fig. 4H). A similar distribution pattern was observedin cells activated through 5.14-dimerized Fc�RI; externalizedPS was present in spots and patches. Whereas Fc�RI showed ahomogeneous distribution. Thus, the dramatic difference indegranulation between Thy-1- and Fc�RI-activated cells doesnot have its counterpart in a similarly different distribution ofexternalized PS.Additive Effects of Different GPI-AP on PS Externalization—

In an attempt to better understand themechanisms of PS exter-nalization induced through various GPI-AP and/or Fc�RI, westudied the effect of triggering with various combinations ofantibodies. We found that stimulation of RBL-2H3 cells withincreasing concentrations of priming antibodies reached a pla-teau in PS externalization at 2 �g/ml of OX7 and 0.5 �g/ml of5.14 (Fig. 5, A–B). Nevertheless, after simultaneous activation

FIGURE 4. Molecular mechanisms leading to PS externalization are differ-ent in Thy-1.1- and Fc�RI-activated cells. A, PS externalization induced inRBL-2H3 cells stimulated with OX7 mAb (5 �g/ml; dashed red line), 5.14 mAb(5 �g/ml; dashed and dotted blue line), or ionomycin (4 �M; dashed and double-dotted green line) for 20 min and determined as described in Fig. 1. Control(black line) represents stimulation with vehicle alone (BSS/BSA). B, real timeflow cytometric analysis of cells labeled with MC540 after stimulation (arrow)with OX7, 5.14, or ionomycin. For further explanation, see the legend to Fig.4A. C, secretory response of the mAb-stimulated cells as determined by meas-uring the concentration of�-glucuronidase in supernatants. D, calcium responsein mAb-stimulated cells. The cells were labeled with Fura-2 and stimulated byOX7 mAb (5 �g/ml; dashed red line), 5.14 mAb (5 �g/ml; dashed and dottedblue line), or vehicle alone (black line) added at the time point indicated by anarrow. E, tyrosine phosphorylation of the mAb-stimulated cells as determinedby immunoblotting (IB) using lysates from the corresponding cells. Positionsof molecular mass standards in kDa are shown on the left. F, PS externalizationinduced in mAb-stimulated cells pretreated for 10 min with the caspaseinhibitor (zVAD-FMK, 200 �M), inhibitors of PS translocation (DIDS and gly-benclamide, both at 200 �M), inhibitor of phosphatidylinositol 3-kinase (wort-mannin, 1 �M), Src- and Syk-family kinase inhibitors (PP2 (20 �M) and piceat-annol (200 �M)), H�-ATP synthase inhibitors (oligomycin (Oligo/-Glc, 2 �M)

and aurovertin B (Auro/-Glc, 2 �M)) used in the absence of glucose, or stimu-lated in the absence of extracellular calcium (�Ca2�). G, PS externalizationinduced in the mAb (black bars, OX7; white bars, 5.14)-stimulated cells pre-treated for 15 min with M�CD (20 mM) or latrunculin B (La, 0.5 �M). H, confocalmicroscopy. PS on the surface of the mAb (OX7 or 5.14)-stimulated cells wasdetected with annexin V-FITC (left). Antibody binding was detected with anti-mouse IgG-cyanine 3 (middle). The merge of these two labels is shown on theright (Overlay). The bar represents 10 �m. In A and F–G, PS externalization wasdetermined as described in Fig. 1. Data in F–G were normalized to the mAb-stimulated cells pretreated with vehicle alone. In A–B, D–E, and H a typicalexperiment from at least three performed is shown.




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of the cells by OX7 and 5.14 (both at 5 �g/ml; Fig. 5C), anadditive effect on PS externalizations was observed. Interest-ingly, a similar additive effect on PS externalizations was alsoobserved after simultaneous engagement of the target anti-gens with the following pairs of mAb: OX7 and TEC-21 (Fig.5D), OX7 and CD48 (Fig. 5E), and CD48 and TEC-21 (Fig.5F). These data suggest that different plasma membranemolecules, even those anchored to the plasma membrane vialipid tail, could utilize different signaling pathways leading toPS externalization.Prolonged Engagement of Thy-1.1 or Fc�RI Has a Different

Effect on PS Externalization but a Comparable Effect on CellProliferation—PS externalization induced by engagement ofGPI-AP or Fc�RI depends on the enzymatic activity of severalsignaling molecules. In all experiments described so far cellswere exposed to the activatingmAb for a short time interval, 40min or less. Next, we studied PS externalization and antibodybinding in cells exposed to anti-Thy-1.1 or anti-Fc�RI for lon-ger time intervals.When cells were cultured withOX7mAb for0.5, 24, or 72 h and then analyzed for PS externalization andantibody binding, no dramatic difference in the levels of PSexternalizationwas noticed.However, with 5.14mAb, PS exter-nalization reached its maximum 0.5 h after triggering anddecreased 24 h later. In cells incubated with 5.14 for 72 h, no PSexternalization was observed even though binding of 5.14 mAbwas still detectable (Fig. 6,A–B). Thus, Fc�RI triggering inducesonly a transient PS externalization, whereas engagement ofThy-1.1 leads to a sustained PS exposure. It should be notedthat incubation of cells for up to 72 h with OX7 or 5.14 did not

impair the cell viability, as determined by PI staining (notshown), and had little effect on cell proliferation (Fig. 6C).PSExternalization Induced throughGPI-AP Is Cell Type- and

Antibody-dependent—To determine whether PS externaliza-tion induced throughGPI-AP is confined tomast cells and anti-bodies actually used, we extended the studies to several othercell types and mAb. When RBL-gT1.2/1 cells, expressing bothendogenous Thy-1.1 and the transfected mouse Thy-1.2 gene,were exposed toOX7 (Thy-1.1 specific) or 1aG4/C5mAb (Thy-1.2 specific), PS externalization was evident after triggeringwith both mAb (Fig. 7, top row, two left panels). CEA is anotherGPI-AP expressed in a wide variety of epithelial malignancies,including colon cancer and colon cancer-derived cell lines such

FIGURE 5. Additive effects of different stimuli on PS externalization.RBL-2H3 cells were stimulated via increasing concentrations of anti-Thy-1.1 (A, OX7) or anti-Fc�RI (B, 5.14), and PS externalization and mAb bindingwere determined as described in Figs. 1 and 2. Alternatively, the cells wereexposed independently or simultaneously to antibody (Ab) doses giving sat-uration of PS exposure by OX7 and/or 5.14 (C), OX7 and/or anti-TEC-21 (D),OX7 and/or anti-CD48 (E), and anti-CD48 and/or anti-TEC-21 (F); all mAb wereused at a concentration 5 �g/ml, except for anti-CD48, which was used as a1:10-diluted hybridoma supernatant. PS externalization was determined asdescribed in Fig. 1, and the data were normalized to responses obtained atthe highest mAb concentration used (A–B) or to the maximum responsesobtained in each experiment (C–F).

FIGURE 6. Prolonged incubation with Thy-1.1- or Fc�RI-specific mAb hasdifferent impact on PS exposure but a negligible effect on cell prolifera-tion rate. A, RBL-2H3 cells were grown in the absence (Control) or presence ofOX7 or 5.14 mAb (both at 2 �g/ml) in complete culture medium. After 0.5, 24,or 72 h the cells were harvested, and annexin V-FITC and antibody bindingwere determined as described in Figs. 1 and 2. Typical flow cytometry profilesfrom at least three independent experiments are shown. B, binding dataobtained as above were normalized to maximal response in each experiment.C, cell numbers were determined at the indicated time interval in culturesincubated with (OX7 or 5.14) or without (Control) mAb.




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as SW1417. Antibody-mediated aggregation of CEA inSW1417 induced no PS externalization (not shown), suggestingthat either the cells are unresponsive or themAb is inefficient ininducing PS exposure in these cells. To get more details onCEA-mediated PS externalization in responsive cells, we trans-fected CEA cDNA into RBL-2H3 cells and analyzed their prop-erties. As shown in Fig. 7 (top row, two right panels) CEA-spe-cificmAb bound to the RBL-CEA/2D1 cells but failed to inducePS externalization. The same CEA-transfected cells were stillcapable of responding toOX7 triggering; this suggests that anti-body-mediated dimerization of CEA is not sufficient to inducePS externalization even in RBL-derived cells.Mouse thymocytes and peripheral T cells express large

amount of Thy-1.2 detectable by 1aG4/C5 mAb, and this anti-body induced PS externalization in both those cell types (Fig. 7,second row). Engagement of Thy-1.1 in rat thymocytes withOX7 mAb induced PS externalization as well (Fig. 7, thirdrow, two left panels). Weak PS externalization was alsoobserved in rat peritoneal macrophages exposed to anti-

CD48 mAb (Fig. 7, third row, tworight panels). Thy-1.2 glycopro-tein is also expressed in mousemyelomonocytic cell line WEHI-3and mouse fibroblasts, 3T3.Although these two cell linesbound comparable amounts of Thy-1.2-specific mAb 1aG4/C5, PSexternalizationwas only observed inWEHI-3 cells (Fig. 7, bottom pan-els). These data indicate that PSexternalization induced throughengagement of GPI-AP is cell type-and antibody-specific.

DISCUSSION

Here we show that the engage-ment of GPI-AP induces external-ization of PS in living cells by a dis-tinct mechanism of cell signaling.Most of the experiments were per-formed with rat mast cell line RBL-2H3 expressing large amounts ofGPI-AP Thy-1.1 (27), CD48 (38),and TEC-21 (21). However, PSexternalization was not confined tothose cells but was also evident insome other cell types triggeredthrough different GPI-AP. It shouldbe noted that PS externalizationafter exposure to a limited panel ofmAbs specific for GPI-APs wasabsent in several cell lines such asSW1417 and 3T3 fibroblast, indi-cating that it depends on cells andon antibodies actually used.PS externalization is a character-

istic feature of early apoptotic cells(1, 49) and seems to be phylogeneti-

cally conserved (50). Data presented in this study indicate, how-ever, that in Thy-1.1-activated mast cells PS externalizationoccurs in the absence of apoptosis. Incubation of cells for pro-longed time intervals with antibodies specific for GPI-AP hadno effect on cell viability, and the proliferation rate was almostthe same as that in control cells. Furthermore, PS externaliza-tion was dependent on steady dimerization of Thy-1.1 glyco-protein; treatment with PI-PLC of Thy-1.1-activated cells,already externalizing PS, resulted not only in removal of Thy-1.1 but also in decreased PS exposure. Finally, PS externaliza-tion could be reduced by several compounds known to specifi-cally inhibit either the enzymes involved in lipid transfer (DIDSand glybenclamide), in the early stages of mast cell signaling(PP2, piceatannol and wortmannin), and/or in the productionofATP (oligomycin, aurovertinB).On the other hand, PS exter-nalization was not inhibited by zVAD-FMK, an inhibitor ofcaspases, enzymes that play an important role in apoptosis.Thus, although non-apoptotic PS externalization has recentlybeen described after activation of several cell types including B

FIGURE 7. PS externalization induced by engagement of GPI-AP is dependent on the cell type and anti-body used. Cells of different origin were exposed to anti-Thy-1.1 (OX7), anti-Thy-1.2 (1aG4/C5), anti-CEA (CEA),or anti-CD48 (CD48) mAb for 30 min, and annexin V-FITC and antibody binding were determined as describedin Figs. 1 and 2. Typical flow cytometry profiles from at least three independent experiments are shown.




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and T lymphocytes, granulocytes, andmast cells (13, 14, 16, 42,51–53), our data are the first showing that non-apoptotic PSexternalization can be induced through engagement ofGPI-AP.PS externalization inmast cells has previously been observed

after Fc�RI triggering (13, 14). Because activation through thisimmunoreceptor leads to degranulation, it has been proposedthat PS externalization could be a useful marker of degranula-tion in individual cells (13, 14, 54). Degranulation of mast cellscan also be induced by an extensive aggregation of GPI-AP (20,21, 27), and therefore, GPI-AP-induced PS externalizationmight also be expected to reflect GPI-AP-induced degranula-tion, utilizing similar signaling pathways as those used in Fc�RI-triggered cells. However, several lines of evidence presented inthis study and summarized in Table 1 indicate that the mecha-nisms leading to the PS externalization induced in Thy-1.1- orFc�RI-activated cells differ and that PS can be externalized inthe absence of degranulation. First, the extent of PS external-ization inThy-1.1-activated cells was at least the same or higheras the one in Fc�RI-activated cells, although only the lattershowed degranulation. For cell activation experiments we usedIgG class antibodies recognizing Fc�RI or GPI-AP and dimer-izing them. Dimerization of Fc�RI is sufficient to induce secre-tion (40, 41), but dimerization of Thy.1.1 is not (Fig. 4C). Sec-ond, a dramatic difference between cells activated throughdimerized Thy-1.1 and Fc�RI was also observed at the level ofcalcium response; rapid and strong in Fc�RI-activated cells butundetectable in Thy-1.1-activated cells. Data obtained withThy-1.1-activated cells corroborate the previous observations,suggesting that enhanced [Ca2�]i is not necessarily a prerequi-site for PS externalization (53, 55). It should be noted, however,that extracellular calcium was required for full PS exposureinduced by both Thy-1.1 and Fc�RI triggering. The combineddata suggest that the role of Ca2� in PS externalization is con-fined to the plasma membrane; in Thy-1.1-activated cells thelocal increase of Ca2� may be sufficient to induce PS exposurebut insufficient to induce further signal propagation anddegranulation. Third, a dramatic difference between Thy-1.1-and Fc�RI-activated cells was also observed at the level of tyro-sine phosphorylation. In Thy-1.1-activated cells, the spectrumof phosphorylated proteins was very limited yet still sufficientfor PS externalization. An important role of tyrosine-phospho-

rylated proteins could be deduced from the fact that inhibitorsof Src- and Syk-family kinases inhibited the PS externalizationin both Thy-1.1 and Fc�RI-activated cells. Fourth, the processof Thy-1.1- and Fc�RI-induced PS externalization exhibiteddifferent sensitivity to several pharmacological inhibitors. Themost dramatic difference was observed in cells pretreated withlatrunculin B, which enhanced PS externalization in Fc�RI-ac-tivated cells but inhibited this process in Thy-1.1-activatedcells. Latrunculin B inhibits actin polymerization by sequester-ing themonomeric actin (47) and has been reported to enhancedegranulation in mast cells activated through both Fc�RI andextensively aggregated Thy-1.1 (48, 56). The observed differ-ences in the sensitivity of PS externalization to latrunculin sug-gest that actin serves as a negative regulator in Fc�RI-mediatedPS externalization but a positive regulator in Thy-1.1-mediatedPS externalization. Effects ofM�CDonPS externalizationwerealso opposite; M�CD pretreatment caused an enhanced PSexposure in Fc�RI-activated cells and a reduced PS exposure inThy-1.1-activated cells. M�CD is known to deplete cellularcholesterol and enhances in this way the degranulation in mastcells activated through both Fc�RI and extensively aggregatedThy-1.1 (34, 57). Inhibition of PS externalization in M�CD-pretreated and GPI-AP-activated cells could hypothetically berelated to localization of GPI-anchored proteins in putativelipid rafts (58–61) and their involvement in PS externalizationthrough GPI-AP proteins (see below). Our data with Thy-1.1-activated cells are in accordance with previous studies showingthat PS externalization is inhibited by actin cytoskeleton inhib-itor, cytochalasin D, and M�CD (17, 62). However, in Fc�RI-activated cells the same treatment led to opposite results, indi-cating that actin and cholesterol requirements for PSexternalization depend on the activation pathway utilized.Fifth, the additive effect of simultaneous engagement ofGPI-AP and Fc�RI on PS externalization suggests that signalingpathways induced through these different plasma membranemolecules initiate signals independently and converge on acommon signaling pathway leading to PS externalization.Interestingly, this additive effect was also observed when twodifferent GPI-anchored proteins were engaged, supporting theconcept that GPI-AP could inhabit different membranemicrodomains and could, thus, trigger different signaling path-ways (63). Finally, prolonged incubation of cells with anti-Fc�RIor anti-Thy-1.1 mAb induced only a transient or sustained PSexternalization, respectively. The sustained exposure of PS incells cultured in the presence of anti-Thy-1.1mAbhadno effecton proliferation; cells cultured in the presence anti-Thy-1.1 oranti-Fc�RI mAb proliferated almost at the same rate as did thecontrol cells.The exact molecular mechanism of PS externalization in

cells activated through GPI-AP remains enigmatic. GPI-APshave been putatively localized in lipid rafts (58–61). Engage-ment of GPI-AP could be sensed by those signaling moleculeslocated in the same raft, and in this way the signal could betransduced (60). Thismodel is supported by studies document-ing the formation of complexes of GPI-APs with Src familykinases (58, 59). Alternatively, GPI-APs might form complexeswith transmembrane proteins, which in turn could create abridge between GPI-APs and cytoplasmic signaling molecules

TABLE 1Summary of differences between Thy-1- and Fc�RI-mediated PSexternalization in RBL-2H3 cells�, response; �, no response; �, partial response; 2, decreased activity; 1 ,increased activity.

Parameter

Activationmediated throughdimerization of

Thy-1 Fc�RIPS externalization � �Degranulation � �Ca2� response � �Tyrosine phosphorylation � �PS externalization after treatment of the

cells withLatrunculin 2 1M�CD 2 1

PS externalization after prolonged � �stimulation




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or could themselves have signaling properties as suggested byliterature data (63–65). Our data are compatible with both ofthese models.Recently, Elliott et al. (42) proposed a model assuming that

PS externalization may occur by a translocase-independentmechanism at energetically favorable sites of membrane per-turbations where lipid packing is decreased. These data werebased in part on the finding that fluorescent dyeMC540, whichbinds preferentially to the plasma membranes with relativelyloosely packed lipids, showed enhanced binding to calciumionophore-stimulated lymphocytes with enhanced PS expo-sure (42). Our finding that Thy-1.1- and Fc�RI-activated cellsexhibited MC540 binding comparable with nonactivated cellssuggests, however, that other mechanisms are more likely to beinvolved.The asymmetric distribution of PS in the plasma membrane

of nonactivated cells could be due to the activity of ATP-de-pendent flippases that hydrolyze ATP to flip PS against a con-centration gradient (1–3). Export of phospholipids from theinner to theouterplasmamembrane leaflet ismediatedbyATP-dependent floppases. In addition, Ca2�-dependent activationof ATP-independent scramblase rapidly randomizes the phos-pholipid distribution across the membrane bilayers. Our find-ing that GPI-AP-induced PS externalization is not accompa-nied by enhanced levels of Ca2� and is reduced in cellspretreated with H�-ATP-synthase inhibitors oligomycin andaurovertin B (see Fig. 4F) suggests that scramblase is notinvolved in this process, and therefore, floppases/flippasescould be the major players.What might be the physiological role of PS externalization

induced by mAb against GPI-AP? In this study we used themAbs as highly specific probes recognizing individual GPI-APson the plasma membrane. Although it is unlikely that suchmAbs act under in vivo conditions, it is possible that their bind-ing mimics events induced by physiological ligands and patho-gens. It is known that GPI-APs serve as receptors for variouspathogens (38, 66, 67), which could infect cells and induce PSexternalization. Externalized PS may serve as an early markerfor phagocytes to remove such infected cells (6, 38, 68). BesidesGPI-APs, there are other molecules involved in pathogen-hostinteraction that could also contribute to PS externalization,enhancing, thus, the probability of infected cells being rapidlyrecognized by phagocytes even in the absence of apoptosis. Thiscould form a physiological basis for the observed additiveeffects of engagement of distinct membrane molecules on PSexternalization. In addition, GPI-APs are involved in numerouscell-cell and cell-matrix interactions, and exposure of PS couldmodulate such important biological processes as cellular adhe-sion, migration, neurite outgrowth, and cell death (12, 69, 70).

Acknowledgments—We thank Anna Koffer for critical reading ofthe manuscript and HanaMrazova, Dana Lorencıkova, and SarkaSilhankova for expert technical assistance.

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Daniel Smrz, L'ubica Dráberová and Petr DráberGlycosylphosphatidylinositol-anchored Proteins

Non-apoptotic Phosphatidylserine Externalization Induced by Engagement of

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non-apoptoticphosphatidylserineexternalizationinducedby ...proteins (gpi-aps). however, it is...

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