Merocyanine 540 as a Fluorescent Probe of AlteredMembrane Phospholipid Asymmetry in Activated

Whole Blood PlateletsCezary Watala,1* Iveta Waczulikova,2 Boguslawa Wieclawska,1 Marcin Rozalski,1 Peter Gresner,2

Krzysztof Gwozdzinski,3 Anton Mateasik,4 and Libusa Sikurova2

1Laboratory of Haemostatic Disorders, Medical University of Łodz, Łodz, Poland2Department of Biophysics and Chemical Physics, Faculty of Mathematics, Physics, and Informatics, Comenius University,

Bratislava, Slovak Republic3Department of Molecular Biophysics, Institute of Biophysics, University of Łodz, Łodz, Poland

4International Laser Centre, Bratislava, Slovakia

Received 3 April 2002; Revision Received 24 June 2002; Accepted 30 July 2002

Background: Platelet activation leads to the loss of anatural asymmetry of membrane phospholipids (PL) andthe subsequent exposure of negatively charged PL inplatelets with procoagulant activity that can be monitoredroutinely with annexin V (AN-V).Methods: Flow cytometric analysis of merocyanine 540(MC540) binding may be the alternate choice for themonitoring of platelet procoagulant activity. Due to theincreased partition of negatively charged phosphatidylser-ine (PS) in the membrane outer leaflet of activated plate-lets, the interaction with MC540 is reduced.Results: Collagen, which facilitated platelet PL bilayersymmetrization, vastly reduced MC540 fluorescence andaugmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in thepresence of thrombin receptor-activating peptide (TRAP,SFLLRNPNDKYEPF). In the presence of VO4

(�3) (the in-hibitor of aminophospholipid translocase), the rebuilt ofmembrane asymmetry was attenuated, which resulted infurther reduced MC540 fluorescence and enhanced AN-Vbinding in activated cells. In platelets incubated with

thapsigargin, the inhibitor of platelet tubular system Ca2�

ATP-ase, which elevates intraplatelet Ca2� concentration,TRAP increased AN-V and reduced MC540 binding. Thechelating of Ca2� with EGTA outside of activated plateletsreduced AN-V binding, but did not affect MC540-positiveplatelets. The fluctuations in reduced staining with MC540paralleled enhanced AN-V binding (r � �0.481, P �0.01), especially for strong “procoagulant” activatingagents.Conclusions: (1) MC540 may be used in whole bloodflow cytometry for the monitoring of platelet membranesymmetrization as an alternate or compounding methodto AN-V. (2) Platelet staining with MC540 is sensitive tothe fluctuations in the intraplatelet [Ca2�] during plateletactivation. (3) Use of MC540 is characterized by improveddiagnostic precision and reliability compared with AN-V.Cytometry 49:119–133, 2002. © 2002 Wiley-Liss, Inc.

Key terms: platelet procoagulant activity; merocyanine540 (MC540); annexin V; blood platelet activation; flowcytometry

The response of blood platelets to agonists results in thesecretion of intraplatelet granules, platelet degranulation,and formation of the procoagulant surface. The latter iscrucial for the activation of the prothrombinase complexon the platelet membrane surface and underlies thrombingeneration. A mutual interaction exists between the for-mation of the procoagulant surface on activated plateletsand thrombin generation. Prothrombinase complex acti-vation is enhanced strongly upon the exposure of theprocoagulant phospholipids (PL) at the platelet surface,and is referred to as platelet procoagulant response (1). Itis linked intimately to the loss of natural membrane PLasymmetry, which is an intimate feature of the membranelipid bilayer in the majority of cells, including red blood

cells (RBC) and blood platelets. PL asymmetry is associ-ated with the lower exposure of the negatively charged PLin the outer leaflet of platelet plasma membranes and is

Presented at the 16th International Symposium on Bioelectrochemistryand Bioenergetics, June 1-6, 2001, Bratislava, Slovakia (Abstract 86).

Grant sponsor: Medical University of Łodz; Grant number: 503-130-2;Grant number: 500-130-2; Grant sponsor: Slovak Grant Agency; Grantnumber: 1/7673/20.

*Correspondence to: Dr. Cezary Watala, Laboratory of Haemostasis andHaemostatic Disorders, Medical University of Łodz, ul. Narutowicza 96,90-141 Łodz, Poland.

E-mail: cwatala@csk.am.lodz.plPublished online in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/cyto.10152

© 2002 Wiley-Liss, Inc. Cytometry 49:119–133 (2002)

actively maintained due to membrane PL translocase (1–3). In blood platelets, the redistribution of membrane PLand the translocation of phosphatidylserine (PS), which isobligatory for the formation of procoagulant activity, ispreceded by Ca2� mobilization — an initial stage of plate-let activation (4,5). It is believed that the release andmobilization of Ca2� are crucial for the chemicophysicaltransformations in the PL bilayer because blocking of sucha mobilization attenuates redistribution of the membranePL (6,7). In resting nonactivated platelets, negativelycharged PL dominate in the cytoplasmic inner bilayerleaflet. This natural membrane asymmetry vanishes uponplatelet activation and mobilization of calcium ions. Bothnatural platelet agonists (collagen) and the modulators ofCa2� mobilization (thapsigargin, ionophore A23187)strongly influence platelet membrane PL asymmetry, andmay affect the procoagulant properties of activated plate-lets. The redistribution of some PL of the membranebilayer inner leaflet, including PS and phosphatidylethano-lamine (PE), mainly contributes to an altered plateletmembrane PL asymmetry in activated blood platelets.Shifted PL asymmetry (PS/PL) across the bilayer may resultin altered surface membrane potential (surface chargedensity) and in altered lipid packing (membrane lipidfluidity) (8–10). The redistribution of membrane PL dur-ing platelet activation is associated with the shedding ofmicroparticles from platelet membranes, which can fur-ther support platelet procoagulant activity (6,11,12).

The development of the procoagulant surface may bemonitored using annexin V, a placental protein with ahigh affinity and a strict specificity for aminophosphoricgroups of membrane PL in the presence of physiologicalconcentrations of calcium ions. Several features make an-nexin V an attractive tool for studying the surface expo-sure of PS by platelets. Annexin V binds to the membranesof platelets in which lipid asymmetry is lost in response toactivation (13). The binding of annexin depends on thetype of platelet activator, ranging from mild (like throm-bin or TRAP alone) to strong (collagen) or very strong(collagen with thrombin; 13,14). Conversely, the use ofcommon protocols for annexin V binding has some limi-tations and disadvantages. These include its unspecificbinding to platelet components other than PS (e.g., sphin-gomyelin, protein kinase C) and possible excessive plate-let activation. Despite its simplicity, labeling protocols forannexin require a lengthy incubation of unfixed plateletsin a buffer containing a high concentration of Ca2�. Thisis understandable, considering that the interaction of an-nexin V with membrane PL occurs through ligation ofCa2� ions with carboxyl oxygens of annexin and phos-phoryl oxygens of PL (15). These staining procedures areintimately associated with artefactual platelet activationand increased formation of platelet aggregates. Ca2� sen-sitivity for annexin V binding is determined by the molarfraction of PS in the platelet membrane lipid bilayer.However, with varying Ca2� concentrations, PS may notbe the only membrane bilayer PL to which annexin Vspecifically binds (14).

In this study, we propose that merocyanine 540(MC540), an anionic nonpermeant fluorescence probewith a high affinity to the lipid component of cell mem-branes, may be an alternate probe for monitoring theplatelet membrane procoagulant surface. Because of thepresence of the negative charge on the MC540 molecule,an increased negative cell surface charge density may leadto reduced interaction of the dye with platelet mem-branes. The abundance of negatively charged PS in theouter leaflet increases upon platelet activation and Ca2�

mobilization. Therefore, the uptake of MC540 by the ac-tivated platelets should be decreased, resulting in a re-duced intensity of MC540 fluorescence.

Our objectives were to determine (a) whether MC540interaction with the membrane lipid bilayer can be mon-itored with the use of flow cytometry in whole bloodplatelets, i.e., with minimal artefactual platelet activation;(b) whether it is sensitive to Ca2� mobilization in plate-lets; and (c) whether alterations in the staining of plateletswith MC540 correspond to the changes in the binding ofannexin V, which is a recognized marker of the increasedplatelet membrane PL symmetry.

MATERIALS AND METHODSChemicals

All chemicals were from Sigma (St. Louis, MO), unlessstated otherwise. Tubes for blood collection containing0.105 mol/L sodium citrate (Vacutainer tubes) were pro-vided by Becton-Dickinson (Mountain View, CA). Bovinethrombin for flow cytometry was obtained from Dade-Behring (Marburg, Germany). Adenosine-5-diphosphate(ADP), collagen fibrils (type I) from equine tendons forwhole blood aggregometry was obtained from Chrono-Log (Havertown, PA). 3-sn-phosphatidylserine from freshegg yolks (containing 48% unsaturated fatty acids) and3-sn-phosphatidylserine (52% unsaturated fatty acids)from bovine brains were provided by Fluka (Buchs, Swit-zerland). Chloroform was from Polish Chemicals (POCh;Gliwice, Poland). 3,3’-dipentyloxacarbocyanine iodide[DiOC5(3)] and pyrene were provided by MolecularProbes (Eugene, OR). Clexane was obtained from AventisPharma (Bridgewater, NJ).

Mouse monoclonal antibodies (mAbs) anti-human plateletGPIIIa (anti-CD61, fluorolabeled with fluorescein isothiocya-nate [CD61/FITC]), mouse mAbs anti-human platelet P-selec-tin (anti-CD62, fluorolabeled with phycoerythrin [CD62/PE]), mouse isotype IgG1 (fluorolabeled with PE), annexin V(fluorolabeled with PE), FACSFlow (optimized phosphate-buffered saline [PBS] for cell preparation and washing) andCellFix (phosphate-buffered fixative containing 10% v/vformaldehyde and 1% w/v sodium azide) were obtained fromBecton-Dickinson. Water used for solution preparation andglassware washing was passed through an Easy Pure UFwater purification unit (Thermolyne Barnstead, Sparks, NV).

Subjects

Eighteen healthy volunteers aged 22–34 years (27 � 6years) were enrolled in this study. None of the selected

120 WATALA ET AL.

individuals had a history suggesting any underlying hemo-static disorder and none had taken aspirin or any otherdrug that is known to affect platelet function within 10days before the study. Blood was collected under theguidelines of the Helsinki Declaration for human researchand the studies were approved by the committee on theEthics of Research in Human Experimentation at the Med-ical University of Łodz.

Blood Collection

Blood from each examined subject was collected bydripping blood from an 18-gauge needle placed in a pe-ripheral vein into a tube containing either 0.1 mg/mlClexane or 3.2% sodium citrate.

Blood was drawn with special caution to avoid undesir-able artefactual platelet activation. Donors rested for20–30 min before blood was drawn. The first 0.5 ml wasdiscarded before the collection of 5 ml of blood that wasused for further platelet function assays. All platelet reac-tivity measurements were performed within 1 h afterblood was drawn.

Preparation of PL Vesicles

Two PL, 3-sn-phosphatidylcholine (estimated averagemolecualr weight [MW] 801.7) and 3-sn-phosphatidylser-ine (estimated MW 802.7), were used without furtherpurification. The degree of lipid oxidation was estimatedfrom ultraviolet (UV) absorbance (16), based on the ratioof the peak for dienes at 230 nm to the monodiene peakat 200 nm (�0.1).

For the preparation of liposomes by the injectionmethod, we used a protocol described by Disalvo et al.(17). The PL mixture containing increasing proportions ofPS was dissolved in chloroform and injected into 10 ml ofbuffer (10 mmol/L Tris-HCl, pH 7.0, or annexin buffer; seebelow for the composition) kept at 70°C to a final con-centration of 2 mg/ml. Nitrogen was bubbled continu-ously through the solution during injection. Finally, thesolution was annealed for 1 h at 70°C and the vesiclesuspension cooled to room temperature (RT). Preparedsamples contained vesicles of an average diameter ofabout 300–500 nm.

Blood Incubation With the Modulators of theGeneration of the Procoagulant Surface

All experiments were performed on whole blood plate-lets, which reduces the risk of undesirable artefactualplatelet activation (18,19). Immediately after venipunc-ture,10 or 25-�l aliquots of whole blood were added to 0.1or 0.25 ml 10-fold diluted CellFix, mixed by gentle movingforth and back, left at RT for 1 h, and further used toevaluate the extent of platelet activation in circulatingblood.

Aliquots of fresh whole blood anticoagulated with ei-ther Clexane or citrate were supplemented with one ofthe following agonists or modulators: 25–50 �g/ml equinetendon collagen type I (1 min) in the absence or presenceof 5 mmol/L EGTA (5 min), 100 �mol/L VO4

(�3) (5 min),0.15 U/ml bovine thrombin (1–4 min) and 2.5 mM Gly-

Pro-Arg-Pro (GPRP), or 1 �mol/L thrombin receptor-acti-vating peptide (TRAP; SFLLRNPNDKYEPF) (4 min) with 1�mol/L thapsigargin (5 min). Incubations were carried outat RT.

Following incubation, the samples of resting or acti-vated platelets were stained with either annexin V orMC540, labeled with a gating antibody, and fixed as de-scribed below. Unless otherwise stated, all determinationsin whole blood samples were made using blood anticoag-ulated with Clexane.

Detection of Annexin V and MC540 InteractionWith Blood Platelets by Flow Cytometry

Aliquots of circulating blood or whole blood incubatedwith agonists/modulators were diluted eight times in theannexin buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 2.5mmol/L CaCl2) and gently mixed. Ten microliters of thediluted suspension was added immediately to 3 �l ofanti-CD61/FITC and 3 �l of either annexin V/PE orIgG1/PE and incubated for 30 min in the dark. Cells werefixed with 150 �l of 1% CellFix for 1 h in the dark anddiluted with FACSFlow before measurements were per-formed.

For labeling with MC540, aliquots of whole blood werediluted three times in PBS. One volume of the diluted cellsuspension was transferred immediately into seven vol-umes of annexin buffer containing MC540 and incubatedin the dark for 15 min. The final MC540 concentration inthe stained sample was 50 �mol/L. Next, the cell suspen-sion was diluted 10-fold with 1% CellFix, fixed for 1 h atRT in the dark, and used for staining with anti-CD61antibodies. The aliquots of 20 �l of fixed cells were addedvery gently to 2 �l anti-CD61/FITC. The staining wasallowed to proceed for 20 min at RT in the dark. Finally,cells were resuspended in 0.3 ml FACSFlow solution. Toset up the above protocol, we used MC540 in the concen-tration range of 1–100 �mol/L and incubation times of5–30 min.

Flow Cytometry Measurements of PlateletProcoagulant Activity by Annexin V or MC540

All flow cytometry measurements were performed atthe Flow Cytometry Unit, 2nd Clinic of Children Diseases,Institute of Paediatrics, Medical University of Łodz. Theywere fluorescence compensated on a daily basis for eachset of measured samples using calibration beads (Becton-Dickinson) to ensure that there was no considerablegreen, orange, and red fluorescence overlap.

Flow cytometric assays of annexin V and MC540 wereperformed within 3 h after staining on fixed blood cellswith light scatter and FL1 fluorescence gates set on theplatelet fraction (threshold 315 and 251, respectively).Green, orange, or red fluorescence of stained plateletswas recorded for FITC (channel FL1 filter transmitting at530 nm with a bandwidth of 30 nm), PE (channel FL2 filtertransmitting at 585 nm with a bandwidth of 42 nm), orMC540 (channel FL3 filter transmitting above 650 nm),respectively, on a FACSCalibur instrument (Becton-Dick-inson) with the excitation set at 488 nm. For the analysis

121PLATELET MEMBRANE ASYMMETRY MONITORED WITH MC540

of annexin V or MC540, the photomultiplier (PMT) of thedetector in FL1 was set at 560 V, FL2 at 524 V, and FL3 at616 V. FL1-FL2 compensation was 1.0%, FL2-FL1 compen-sation was 21.4%, and FL3-FL2 compensation was 20.7%.A minimum of 5,000 cells were analyzed per sample. Alldata were processed using Lysis II (Becton-Dickinson)software. The percentage of the specific fluorescence-positive platelets was obtained after the subtraction ofnonspecific mouse IgG1 binding or MC540-free cells forannexin V and MC540, respectively. Intensities of thespecific bound fluorescence were expressed in fluores-cence arbitrary units. The extent of forward light scatter(FSC) was employed to evaluate the fractions of plateletmicroparticles and aggregates following functional plate-let assays in whole blood (18–20). The formation of abun-dant subpopulations of microparticles is determined fol-lowing accelerated platelet consumption upon thecontact of platelets with artificial surfaces in a dynamicmodel of platelet activation (agitation with a rotary stirrer:1 h, RT, eight 360° rotations per minute equivalent to

7–8-s cycles) (20). The extended formation of plateletaggregates is read upon platelet stimulation with 20�mol/L ADP in whole blood anticoagulated with a non-calcium chelating anticoagulant (LMWH Clexane, 0.1 mg/ml). Following this experimental approach, we observedthe true subpopulations of microplatelets and aggregatesassigned to separate modes (21) in an FSC histogram plotof an overall platelet population. Assigned ranges of chan-nels were 0–30 for microparticles and 180–255 for aggre-gates on the log 4 decade 256-channel scale (Fig. 1). Thefractions of platelet microparticles and aggregates sub-tracted from the overall analyzed platelet population gavethe central FSC cohort of normal “free” platelets or nor-moplatelets.

Flow Cytometry Measurementsof Transmembrane Potential

To monitor how the transmembrane potential (TMP) inwhole blood platelets is affected in the course of plateletactivation, DiOC5(3) was used, which is more reliable for

FIG. 1. Flow cytometric analysis of platelet microparticles and aggregates in resting (A), ADP-activated (B), resting annexin V-stained (C), andcollagen-activated annexin V-stained platelets (D) in whole blood. Blood was anticoagulated with 0.1 mg/ml Clexane. The final concentrations of ADP andcollagen were 20 �mol/L and 25 �g/ml, respectively. Platelet microparticles (red/blue dots) and aggregates (green dots) were assigned to the regionsframed by red and green rectangular dashed outlines on an FSC (channels 0–30 and 180–255, respectively) of the log 4 decade 256-channel scale. Bluedots within the frame of microparticles represent annexin V-stained platelets. For further experimental details, see Materials and Methods.

122 WATALA ET AL.

the analysis of plasma membrane potential rather than forstudies on transmembrane electrical potential in mito-chondria (22). As the cationic molecule, DiOC5(3) travelsto negative sites and is released when the charge changes.When the cell inside becomes more negatively polarized(hyperpolarized), higher amounts of DiOC5(3) accumu-late. Conversely, when the membrane potential is lost, the

dye escapes the cell (22,23). When excited at 488 nm, theprobe emission may be recorded in FL1. Whole bloodanti-CD61/peridin chlorophyll protein (PerCP)-labeledplatelets (20 min, RT in the dark) were incubated in theannexin buffer with 10–20 nmol/L DiOC5(3) (1 mmol/Lstock solution dissolved in ethanol) for 5 min at 37°C.Next, 10- �l aliquots of the labeled cell suspensions wereadded to 1 ml FACSFlow and the fluorescence of pair-matched samples (resting and activated platelets) wasmonitored every 3 min for up to 15 min.

For the analysis of a minimum of 5,000 of the DiOC5(3)-positive platelets, the PMT of the detector in FL1 was setat 570 V, FL2 at 524 V, and FL3 at 616 V, with thecompensation set as stated above.

Flow Cytometry Analysis of VesiclesLabeled With MC540

Suspensions of PL vesicles containing increasing pro-portions of PS (2 mg/ml total PL in the annexin buffer)were incubated with MC540. Briefly, one volume (10 �l)of vesicle suspension was mixed with one volume of theannexin binding buffer containing 6 �mol/L MC540, in-cubated in the dark (20 min, RT), and diluted in 1 mlFACSFlow before use.

Flow cytometric analysis of MC540 bound to the vesiclesurface was performed with the FSC gate set on theliposome fraction (threshold 200). Laser excitation wasset at 488 nm and the orange fluorescence of at least10,000 vesicles stained with MC540 was recorded withthe PMT of the detector in FL1 set at 560 V, FL2 at 650 V,and FL3 at 616 V. The compensation was set as statedabove. The percentage of the fluorescing vesicles withvarying ratios of PS/phosphatidylcholine (PC) was ob-tained after the subtraction of nonspecific autofluores-cence of MC540-free vesicles.

Electronic Spin Resonance (ESR) and FluorescenceMeasurements of Lipid Fluidity in PS/PC Vesicles

Ethanol 10�3 mol/l solutions of [2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy] free radical (5-

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™FIG. 2. a: Effect of MC540 concentration on the staining with MC540 of

resting (triangles) and activated blood platelets (circles) in the absence(solid symbols) or presence (open symbols) of EGTA (semi-log plot). Datapresented as mean � SEM, n � 5 experiments. Aliquots of whole bloodwere incubated without or with 5 mmol/L EGTA (5 min) and activatedwith 50 �g/ml collagen for 1 min at RT. To stop the activation, thesamples were diluted 25-fold with PBS and then incubated with MC540 inthe annexin buffer in the dark for 15 min. After fixation (1% CellFix, 1 h,RT in the dark), cells were stained with anti-CD61/FITC (20 min, RT, inthe dark) and resuspended in 0.3 ml FACSFlow before analysis. Option-ally, resting (squares) or activated platelets (diamonds) were labeled withMC540 in the presence of 5 mmol/L EGTA (dashed lines). Insert: semilogplots of the formation of platelet microparticles (upper) and aggregates(lower) in resting (solid line with triangles) and collagen-activated plate-lets (dashed line with circles) labeled with MC540 in the presence (solidcircles) or absence (open circles) of calcium. b: Effect of MC540 concen-tration on the formation of microparticles and aggregates in resting (leftcolumn) and collagen-activated platelets (right column) in whole blood.The M, M1, and M2 gates demarcate the fractions of MC540-positiveplatelets, platelet microparticles, and platelet aggregates (evaluated basedon FSC), respectively. For further experimental details, see Materials andMethods. Data, presented as mean � SEM (n � 5), for resting andactivated platelets are shown in Table 1.

123PLATELET MEMBRANE ASYMMETRY MONITORED WITH MC540

doxylstearic acid, 5-DOXYL-Ste) or [2-(14-carboxytetrade-cyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy] free radical(16-doxylstearic acid, 16-DOXYL-Ste) were added to chlo-roform solutions of either PC or PS at a final concentrationof 50 �mol/l and vortexed vigorously. Spin-labeled PL mix-tures containing increasing proportions of PS were used forthe preparation of liposome suspensions, as describedabove. ESR measurements were performed in a Bruker SX-300E spectrometer as described by Watala et al. (24).

To monitor pyrene mobility in the vesicle membranelipid bilayer, 3 �l of the ethanol solution of 2 mmol/Lpyrene was introduced per 2 ml of PS/PC vesicle suspen-sion (1 mg/ml PL) containing increasing amounts of PSand incubated for 20 min at RT. Fluorescence of pyrene-labeled PS/PC vesicles was excited at 339 nm and emis-sion recorded in the range of 350–550 nm (2.5-nm slit).Pyrene mobility was reflected as the yield of the excimerformation (excimer fluorescence-to-monomer fluores-cence ratio read at 395 nm and 470 nm, respectively),which directly depends on the collisional rate and dis-tances of label molecules in the vesicle membrane lipidbilayer (25). Measurements were performed with a PerkinElmer (Oak Brook, IL) LS50B spectrofluorometer.

Statistical Analysis

Means � SEM are given for the normally distributedparameters. Data that showed the right-skewed distribu-tion and met the remaining criteria of normal distributionwere transformed logarithmically and analyzed with therelevant parametric tests. As all the raw and transformeddata showed no departures from normality (according tothe Shapiro-Wilk’s test), we used either the Student’s t-testfor independent or pair-matched samples or analysis ofvariance (ANOVA) randomized complete block designand Scheffe’s test depending on the number of groupscompared. Pearson’s linear correlation and partial corre-lation analysis was used to determine the associationsbetween variables. To verify how far the variability ofvarious interrelated parameters explained the variability inthe fraction of annexin V or MC540-positive whole bloodplatelets, we employed multiple regression analysis. Pear-

son’s chi-square test was employed to evaluate the mea-sures of diagnostic precision and reliability (26,27).

RESULTSFluorescence of MC540 Associated With Platelets

Figure 2, Table 1, and Figures 3 and 4 show the effectsof MC540 concentration, incubation time, and blood dilu-

FIG. 3. Effect of incubation time on the binding of MC540 to resting(solid line with triangles) and activated blood platelets (dashed line withcircles). Data presented as mean � SEM, n � 5 experiments. Aliquots ofwhole blood were activated with 50 �g/ml collagen, diluted with PBS, andincubated with 50 �mol/L MC540 in the annexin buffer in the dark for 5, 10,20, or 30 min. For further experimental details, see Figure 2. Insert: Theformation of platelet microparticles (solid line, Pslope � 0.0001) and aggre-gates (dashed line) in collagen-activated platelets versus incubation time.

FIG. 4. Effect of blood dilution on the binding of the increasing con-centrations of MC540 to resting (solid line) and activated blood platelets(dashed line). Data presented as mean � SEM, n � 5 experiments.Aliquots of whole blood diluted 3 (solid triangle), 20 (open diamond), or50 times (open circle) were activated with 50 �g/ml collagen for 1 min atRT. To stop the activation, one volume of cell suspension was transferredimmediately into 7 volumes of annexin buffer containing 50 �mol/LMC540 and incubated in the dark for 15 min. The resultant dilution ofwhole blood was, respectively, 24 (solid triangle), 160 (open diamond),or 400 times (open circle). For further experimental details, see Figure 2.

Table 1Effect of MC540 Concentration on the Formation of

Microparticle and Aggregates in Restingand Activated Platelets*

MC540(�M)

Resting Activated

M1 (%) M2 (%) M1 (%) M2 (%)

1 2.9 � 0.7 2.0 � 0.7 12.3 � 1.7 7.8 � 1.55 3.8 � 1.2 2.0 � 0.6 12.6 � 2.0 7.7 � 1.010 2.8 � 0.8 1.8 � 0.6 13.1 � 1.9 6.3 � 1.250 3.2 � 0.9 2.5 � 0.9 17.2 � 0.9 4.7 � 1.2100 4.0 � 0.9 2.6 � 0.9 17.6 � 1.5 5.0 � 1.2

*Significance of differences for pair-matched groups of restingversus activated cells, estimated by means of Student’s t-test ontransformed data, was P � 0.04 or less. The values of significanceof regression slopes for formation of microparticles and aggre-gates in activated platelets versus MC540 concentration wereP � 0.002 and P � 0.02, respectively.

124 WATALA ET AL.

tion on MC540-positive resting and collagen-activatedwhole blood platelets. The fraction of MC540-positivecells in both resting and activated platelets did not in-crease significantly within the range of lower dye concen-trations (1–10 �mol/L, F value for linearity NS) reachedsubmaximal values at 50 �mol/L and leveled above 50�mol/L (Fig. 2a). In the presence of collagen, whichtriggers Ca2� mobilization and underlies membrane PLredistribution, the fraction of MC540-positive platelets re-mained depressed but was restored when Ca2� was che-lated by EGTA before platelet activation (Fig. 2a). How-ever, the staining of resting or activated platelets withMC540 was not sensitive to EGTA-induced Ca2� depletionin the Ca2�-free annexin buffer.

In activated, but not in resting platelets, there was aproportional trend for the increase in platelet micropar-ticles (Pslope � 0.002) and a reciprocal trend for plateletaggregates (Pslope � 0.02) with increasing MC540 concen-trations (Fig. 2a inserts,b). The distinct discrimination be-tween the fractions of MC540-positive cells in resting andactivated platelets was observed when monitored in theFL2 channel (Fig. 5). In the course of the incubation ofresting cells with 50 �mol/L MC540, the maximal fractionof the dye-positive cells was achieved already with 5 minof incubation and did not increase significantly followingfurther incubation. Otherwise, collagen-activated plateletsshowed submaximal staining after 5 min of incubation.Subsequently, the fraction of MC540-positive cells gradu-ally decreased with up to 20 min of incubation (Fig. 3). Nostatistically significant effect of prolonged incubationtimes on the formation of microparticles or aggregates inresting platelets was revealed. However, for incubationtimes longer than 10 min, a significantly increased forma-tion of microparticles in collagen-activated platelets oc-curred (F for linearity P � 0.003; see insert in Fig. 3).There was no influence of the initial dilution of whole

blood sample subjected to the incubation with the in-creasing concentrations of MC540 on the efficiency ofstaining with MC540 in either resting platelets or cellsactivated with collagen (Fig. 4).

For further analysis, we chose submaximal staining withMC540 in resting platelets and moderate labeling withMC540 of platelets activated with strong agonists inducinga vast PL redistribution (collagen alone or in a combina-tion with other agonists): MC540 concentration of 50 �M,an incubation time of 15 min, and a sample dilution of25-fold.

Fractions of Annexin V and MC540-PositiveActivated Platelets in Overall Platelet Population

Opposite tendencies were observed for annexin V andMC540 in the course of platelet activation. Whereas thebinding of annexin V was augmented, the staining withMC540 was reduced in activated platelets (Fig. 6). Colla-gen was the most potent agonist facilitating symmetriza-tion of the platelet PL bilayer, as demonstrated by theaugmented annexin V binding and vastly reduced fluores-cence of MC540 bound to platelets (Fig. 7). Collagen-induced symmetrization was maintained in the presenceof thrombin or TRAP, although neither thrombin norTRAP was efficient when acting alone. Thapsigargin, theinhibitor of platelet tubular system Ca2� ATP-ase, whichelevates intraplatelet Ca2� concentration, led in the pres-ence of TRAP to the increased fraction of annexin-positiveand the reduced fraction of MC540-positive platelets. Theaddition of VO4

(�3), the known inhibitor of membraneaminophospholipid translocase, to platelets primarily ac-tivated with collagen significantly inhibited the restorationof membrane PL asymmetry. It resulted in even moresignificantly enhanced annexin binding and was associ-ated with further reduction in MC540 fluorescence (Fig.7). The chelating of outside Ca2� with EGTA before plate-let activation with collagen almost completely depressedthe effects of the collagen-induced platelet membrane PLsymmetrization. The translocation of inner leaflet PS wasreduced markedly, which resulted in a low fraction ofannexin V-positive and a high fraction of MC540-positiveplatelets. Conversely, when evaluating the effects of EGTAon the yield of the labeling of whole blood platelets witheither annexin or MC540, we demonstrated that EGTAmarkedly reduced the binding of annexin V (11.0 � 1.8%versus 27.0 � 4.4%), but did not affect the fraction ofMC540-positive cells in collagen-activated platelets(46.1 � 3.1% versus 47.1 � 5.1%, ns; Fig. 2). In citratedblood samples, not only collagen alone (52.6 � 3.7% forClexane versus 50.6 � 4.2% for citrate) or in a combina-tion with thrombin (55.4 � 4.3% for Clexane versus47.6 � 4.5% for citrate) or TRAP (56.9 � 5.8% for Clexaneversus 44.3 � 3.0% for citrate), but also thrombin (79.5 �2.8% for Clexane versus 63.3 � 3.2% for citrate) or TRAP(81.4 � 2.7% for Clexane versus 73.9 � 5.3% for citrate)alone, induced a marked reduction (82.6 � 1.8% for Cl-exane versus 85.2 � 2.8% for citrate in resting platelets) inMC540-positive platelets.

FIG. 5. Fluorescence of MC540 bound to resting (triangles) and acti-vated blood platelets (circles) recorded in the flow cytometer channel FL2(solid line) or FL3 (dashed line). Data presented as mean � SEM, n � 5experiments. For experimental details, see Figure 2.

125PLATELET MEMBRANE ASYMMETRY MONITORED WITH MC540

In general, in the presence of platelet agonists thatinduced a strong PL symmetrization (referred to as “strongprocoagulants”), the fraction of annexin V-positive plate-lets increased, whereas that of MC540-positive cells de-creased. In resting platelets or in the presence of agonistsinducing inconsiderable PL symmetrization (“weak proco-agulants”), the binding of annexin V was low and MC540fluorescence high (Fig. 7).

Fractions of Annexin V and MC540-PositiveActivated Platelets in Subpopulations of PlateletMicroparticles, Normoplatelets, and Aggregates

Significantly increased annexin V binding and reducedstaining with MC540 were observed in normoplatelets(overall platelet population minus subpopulations of mi-croparticles and aggregates) when whole blood plateletswere activated with the strong procoagulants and the

pattern of changes matched very well that recorded forthe overall platelet population (Table 2).

In general, an increased fraction of annexin-positivemicroparticles and a reduced fraction of MC540-positivemicroparticles were observed in the samples subjected toincubation with strong procoagulant agents. However, nosignificant discrimination between various agonists wasrevealed. A much better discrimination between weak andstrong procoagulants was found for MC540-labeled micro-particles. Platelet aggregates in the samples incubatedwith strong procoagulants bound significantly more an-nexin V, whereas no differences occurred in the sampleslabeled with MC540 for all activators but thapsigargin �TRAP (Table 2).

Formation of Platelet Microparticles andAggregates in Whole Blood Stained With Annexin

V or MC540

Strong procoagulants induced the enhanced formationof platelet microparticles to a much higher extent in thesamples incubated with MC540 than annexin V. Other-wise, more abundant fractions of platelet aggregates oc-curred in the samples incubated with annexin V com-pared with MC540 (Table 3).

Figure 8 shows the formation of microparticles andaggregates in resting, collagen-activated, and ADP-acti-vated platelets in whole blood stained with either anti-Pselectin mAbs, annexin V, or MC540. In the referencesystem used in this study, which was the sample labeledwith anti-CD62-PE mAbs, platelet activation in wholeblood with either collagen or ADP led to a significantincrease in the formation of microparticles and aggregates(Fig. 8, plots 1A–C). Compared with reference samples,the nonactivated samples incubated with annexin V butnot MC540 showed markedly enhanced aggregates (P �0.0001; Fig. 8, plot 2A), which were nearly the same asthose observed in the samples incubated without annexin

FIG. 6. Flow cytometric analysis of blood platelet staining with annexinV or MC540. The horizontal marker on the histogram plot denotes thespecific staining with either annexin V/PE (A) or MC540 (B) to resting(outline) and collagen-activated (solid fill) platelets in whole blood. TheM1 and M2 gates demarcate the fractions of platelet microparticles andplatelet aggregates (evaluated based on FSC), respectively. For furtherexperimental details, see Materials and Methods.

FIG. 7. Staining with annexin V and MC540 of resting and activatedplatelets in whole blood. Mean � SEM, numbers given in Tables 2 and 3.Significance of differences, estimated by ANOVA randomized completeblock design and Scheffe’s test or one-tailed Student’s t-test for pair-matched samples, was as follows: annexin V: �weak � �strong, P � 0.0001;�con � �TRAP, P � 0.001; �

thr� �TRAP, P � 0.001; �coll � �VO4

, P � 0.001;MC540: �weak � �strong, P � 0.0001; �

con� �coll�thr, P � 0.03; �coll �

�VO4, P � 0.03.

126 WATALA ET AL.

V in the annexin buffer (30.1 � 1.3%). In platelets acti-vated with collagen, microparticles were particularly highin the samples stained with MC540 (P � 0.003; Fig. 8, plot3B). In collagen-activated samples, the aggregates weresignificantly higher for samples stained with annexin V(P � 0.0001) and reduced in the presence of MC540 (P �0.02; Fig. 8, plots 2B, 3B). Similarly,, in whole bloodplatelets activated with ADP, the fraction of aggregateswas significantly elevated for annexin V (P � 0.0001) andlowered for MC540 (P � 0.002; Fig. 8, plots 2C, 3C).

Correlations Between Annexin Binding andStaining With MC540 in Resting

and Activated Platelets

In the overall data matrix, including both strong andweak procoagulants, the fluctuations in the reduced

MC540 fluorescence correlated negatively with the en-hanced annexin V binding (r � �0.682, R2

cor � 0.459,P � 0.0001). The same was true, although to a lowerextent, if we included for the analysis only the strongprocoagulants (r � �0.560, R2

cor � 0.300, P � 0.0001),but there was no such a correlation for the agonists ofweak procoagulant activity (r � 0.077, R2

cor � 0.005, NS).Based on determination coefficients, we deduced that theoverall variability in the fluorescence attributed to plate-lets stained with annexin V or MC540 was mainly ex-plained by the variations in the formation of platelet mi-croparticles (rpartial � 0.918 and �0.748 for annexin V andMC540, respectively; P � 0.0001 for both) and was almostcompletely independent of the formation of platelet ag-gregates (Table 4). In the annexin V-positive platelets, theportions of annexin V bound to normoplatelets and aggre-

Table 2Staining With Annexin V and MC540 B in Whole Blood Platelets*

Annexin V binding MC540 binding

Microparticles Normoplatelets Aggregates Microparticles Normoplatelets Aggregates

Weakprocoagulants 40.3 � 3.1 2.6 � 0.4 4.8 � 0.9 14.1 � 2.4 88.9 � 2.1 95.8 � 3.5

Resting platelets 44.8 � 7.1 2.3 � 0.5 4.3 � 1.3 17.5 � 3.1 85.5 � 3.4 92.6 � 6.8Thrombin 34.4 � 4.9 1.6 � 0.3 3.4 � 0.7 13.8 � 3.4 89.3 � 3.6 92.8 � 6.4TRAP 43.2 � 4.4 4.8 � 0.4 8.1 � 1.4 12.8 � 4.0 87.4 � 3.5 91.7 � 7.9Strong

procoagulants 52.8 � 2.9 29.3 � 2.1 38.9 � 2.3 6.8 � 0.9 59.1 � 2.9 93.5 � 2.7Collagen 48.8 � 5.6 22.4 � 4.5 34.1 � 5.8 6.9 � 1.8 65.0 � 5.8 99.6 � 2.0Collagen �

thrombin 50.5 � 6.6 24.7 � 4.0 35.9 � 5.3 6.3 � 1.3 62.5 � 5.1 89.2 � 7.2Thapsigargin �

TRAP 59.3 � 3.5 23.6 � 3.9 30.4 � 4.4 7.4 � 1.6 70.0 � 4.5 83.8 � 8.5VO�3

4 53.4 � 6.3 37.0 � 3.9 43.9 � 3.5 6.6 � 2.0 52.2 � 6.6 97.4 � 2.3

*Mean � SEM, n � 11 for all cases except resting platelets, collagen-activated platelets (n � 18), and weak (n � 38) and strongprocoagulants (n � 60). Significance of differences, estimated by ANOVA randomized complete block design and Scheffe’s test or byone-tailed Student’s t-test for pair-matched samples, was as follows: (1) annexin V binding to normoplatelets: �weak � �strong, P ��0.0001; �con � �TRAP, P � 0.03; �thr � �TRAP, P � 0.002; �coll � �VO4

, P � 0.04; annexin V binding to aggregates: �weak � �strong, P�� 0.0001; �con � �TRAP, P � 0.03; �thr � �TRAP, P � 0.02; (2) MC540 binding to microparticles: �weak � �strong, P � 0.003; MC540binding to normoplatelets: �weak � �strong, P �� 0.0001.

Table 3Formation of Platelet Microparticles and Aggregates in Whole Blood Platelets*

Incubation with annexin V Incubation with MC540

Microparticles Aggregates Microparticles Aggregates

Weak procoagulants 0.3 � 0.1 29.6 � 1.6 3.4 � 0.3 6.8 � 1.0Resting platelets 0.5 � 0.2 24.6 � 3.0 2.5 � 0.4 6.0 � 2.6Thrombin 0.3 � 0.1 28.5 � 2.5 3.3 � 0.4 5.5 � 1.7TRAP 0.5 � 0.1 32.1 � 2.5 4.3 � 0.5 7.2 � 2.6Strong procoagulants 3.3 � 0.3 33.2 � 1.2 14.6 � 0.9 9.2 � 1.1Collagen 2.6 � 0.5 29.0 � 3.3 13.8 � 1.6 5.9 � 0.6Collagen � thrombin 3.3 � 0.7 32.0 � 2.1 14.1 � 1.8 11.0 � 2.5Thapsigargin �

TRAP 2.4 � 0.5 40.8 � 2.1 9.6 � 1.5 16.1 � 3.1VO�3

4 3.8 � 0.4 34.3 � 1.6 17.5 � 1.7 6.2 � 0.9

*Mean � SEM, n � 11 for all cases except resting platelets, collagen-activated platelets (n � 18), and weak (n � 38) and strongprocoagulants (n � 60). Significance of differences, estimated by ANOVA randomized complete block design and Scheffe’s test or byone-tailed Student’s t-test for pair-matched samples, was as follows: (1) microparticles in annexin V-positive platelets: �weak � �strong,P �� 0.0001; �thaps/TRAP � �VO4

, P � 0.05; microparticles in MC540-positive platelets: �weak � �strong, P �� 0.0001; �con � �TRAP,P � 0.02; �thaps/TRAP � �coll, P � 0.03; �thaps/TRAP � �VO4

, P �� 0.0001; (2) aggregates in annexin V-positive platelets: �weak � �strong,P � 0.04; aggregates in MC540-positive platelets: �thaps/TRAP � �coll, P � 0.003; �thaps/TRAP � �VO4

, P � 0.001.

127PLATELET MEMBRANE ASYMMETRY MONITORED WITH MC540

gates were the major components, each explaining over90% of the total annexin V binding. The largest portions ofthe variability of the overall MC540 fluorescence wereexplained by the fractions of microparticles (rpartial ��0.643, P � 0.0001) and normoplatelets.*

Measures of Diagnostic Precision and Reliabilityfor the Methods With Annexin V and MC540

The ranges within which the populations of annexinV-negative and MC540-positive platelets (relevant to cellswith unaltered PL asymmetry) are likely to lie were esti-mated from the observed sample means and their standarderrors (SE) and equaled 2.93%–3.79% for annexin V-posi-tive and 78.21%–84.23% for MC540-positive nonactivatedplatelets. The calculated upper (annexin V) or lower(MC540) 95% confidence limits were 3.79% and 78.21%,

respectively. Based on these critical values, we calculatedthe measures of diagnostic reliability using two methodsfor monitoring platelet membrane PL asymmetry. Theyreflect the associations between the quality of the methodto detect membrane PL distribution and true plateletmembrane PL asymmetry. Table 5 shows that the MC540method is more reliable. Both methods are equally sensi-tive (100%), but specificity was only moderate for MC540and poor for annexin V: respectively, 22 and 45 cases perevery 100 samples of nonactivated platelets with unal-tered PL asymmetry are likely to be falsely classified asplatelets with enhanced procoagulant activity. Thechance of detecting a negative result in samples withunaltered membrane PL asymmetry is significantly higherfor MC540. Accordingly, the diagnostic precision is verygood for MC540 and relatively good for annexin V. The

FIG. 8. Flow cytometric analysis of the formation of microparticles and aggregates in resting (A), collagen-activated (B), and ADP-activated platelets (C)in whole blood stained with anti-P selectin mAbs (1A–C), annexin V (2A–C), or MC540 (3A–C). The M1 and M2 gates demarcate the fractions of plateletmicroparticles and platelet aggregates evaluated based on FSC (channels 0–30 and 180–255 on the log 4 decade of the 256-channel scale, respectively).For further experimental details, see Materials and Methods. Data, presented as mean � SEM (n � 5), were as follows: CD62-PE resting: M1 � 2.7 � 0.2%;M2 � 1.9 � 0.5%; P � 0.05 versus M1coll, M1ADP; P � 0.0001 versus M2coll, M2ADP; collagen-activated: M1 � 4.9 � 0.8%; M2 � 11.4 � 1.3%; ADP-activated:M1 � 5.4 � 0.3%; M2 � 17.8 � 0.7%; P � 0.01 versus M2coll; annexin-PE: resting: M1 � 1.1 � 0.2%; M2 � 34.6 � 1.5%; P � 0.05 versus M2coll; P � 0.0001versus M2ADP; collagen-activated: M1 � 4.2 � 0.3%; M2 � 39.9 � 1.4%; P � 0.0001 versus M1rest, M1ADP; P � 0.005 versus M2ADP; ADP-activated: M1 �2.0 � 0.2%; M2 � 47.8 � 1.8%; MC540: resting: M1 � 3.1 � 0.9%; M2 � 1.9 � 0.9%; collagen-activated: M1 � 22.9 � 6.2%; M2 � 4.3 � 1.5%; P � 0.015versus M1rest, M1ADP; ADP-activated: M1 � 4.2 � 1.1%; M2 � 4.0 � 1.9%.

128 WATALA ET AL.

likelihood to confirm the altered membrane PL asymmetryin platelet samples with a positive test result (i.e., thechance that the PL asymmetry in a sample classified asmore asymmetrical is really enhanced) is significantlyhigher for the MC540 method compared with the annexinV method.

Effect of Platelet Activation on the Fraction ofCarbocyanine-Positive Cells

To characterize the changes in TMP in the course ofplatelet activation, we monitored the accumulation andfluorescence of carbocyanine dye inside resting or acti-vated whole blood platelets. Upon the addition of valino-mycin (100 nmol/L) to nondiluted whole blood anticoag-ulated with Clexane and stained with DiOC5(3), we

observed the decreased fraction of DiOC5(3)-positiveplatelets. The activation of DiOC5(3)-stained whole bloodplatelets with either collagen or collagen and TRAP alsoresulted in a significant reduction in the fraction ofDiOC5(3)-positive platelets (Fig. 9).

Effect of PS Partition on Staining PLVesicles With MC540

We observed a linear dependence between the fractionof MC540-positive PS/PC vesicles and the molar partitionof PS, i.e., vesicles with a higher amount of PS bound lessMC540 (Fig. 10).

Figure 11 shows the effect of MC540 concentration onthe staining of vesicles containing different amounts of PS.Vesicles containing lower PS partition of 5% mol werecharacterized by higher MC540 fluorescence comparedwith 20% mol PS vesicles. A significant discriminationbetween vesicles with various PS/PC ratios occurred forthe range of MC540 concentrations of 1–20 �mol/L (P �0.01 or less).

Effect of PS Partition on Bilayer Lipid Fluidity inPS/PC Liposomes

To verify how far the PS partition affects the dynamicparameters of the lipid bilayer of PS/PC liposomes in thepresence and absence of Ca2�, we used ESR and fluores-cence techniques. Using two spin label probes, 5-Doxyl-Ste and 16-Doxyl-Ste, which reflect the mobility of mem-brane PL fatty acid chains in the near-the-surface or deeperregions of the liposome lipid bilayer, we found a signifi-

Table 4Multiple Regression Analysis of the Parameters Explainingthe Overall Variability in Annexin V or MC540 Binding to

Whole Blood Platelets*

B Rcor2

Annexin V-positive plateletsAnnexin in microparticles 0.507 0.249Annexin in normoplatelets 0.987 0.973Annexin in aggregates 0.973 0.946Fraction of microparticles 0.904 0.815Fraction of normoplatelets �0.277 0.067Fraction of aggregates 0.074 0.005

MC540-positive plateletsMC540 in microparticles 0.677 0.452MC540 in normoplatelets 0.933 0.869MC540 in aggregates 0.213 0.035Fraction of microparticles �0.807 0.647Fraction of normoplatelets 0.679 0.455Fraction of aggregates �0.105 0.000

*The coefficient B is a normalized regression coefficient (nor-malized regression slope); Rcor

2 denotes the corrected determina-tion coefficient (the ratio of the variation explained by the re-gression to the overall variation)

Table 5Measures of Diagnostic Reliability of Two Methods for the

Monitoring of Platelet Membrane PL Asymmetry*

Annexin V MC540 Significance

Test sensitivity 100.0% 100.0% n.s.Test specificity 55.2% 78.3% 0.0004Diagnostic precision 79.6% 90.5% 0.001True frequency of altered

asymmetry 54.5% 56.1% n.s.Expected frequency with

positive prediction 72.7% 85.5% 0.004Expected frequency with

negative prediction 0.0% 0.0% n.s.Likelihood of false-negative

result 0.0% 0.0% n.s.Likelihood of false-positive

result 44.8% 21.7% 0.002

*All measures were calculated based on the critical values of�95% CI � x� � z’ � SE for annexin V (3.79%) and MC540(�78.21%). Significance of differences estimated with the one-tailed test for two proportions with a normal approximation.

FIG. 9. Fractions of DiOC5(3)-positive cells in resting and activatedwhole blood platelets. Mean � SEM, n � 6. Aliquots of whole bloodlabeled with 20 nmol/L DiOC5(3) (5 min, 37°C) and anti-CD61/PerCP (20min, RT in the dark) were 100-fold diluted in FACSFlow. The fluorescencevalues of pair-matched samples (resting and activated platelets) for theoverall platelet population (left lane) and normoplatelets (right lane) wereread after an additional 15 min. For further experimental details, seeMaterials and Methods. Significance of differences, estimated by ANOVArandomized complete block design, was as follows: �control � �valinomycin

� �collagen � �coll � TRAP, P(1) � 0.002 for overall platelet population, and�control � �collagen, �coll � TRAP, �valinomycin, P(1) � 0.001 for normoplate-lets.

129PLATELET MEMBRANE ASYMMETRY MONITORED WITH MC540

cant influence of Ca2� (P � 0.002), but no significanteffect of PS partition on the h�1/h0 ratios of 5-Doxyl-Steand 16-Doxyl-Ste. Similarly, the excimer/monomer fluo-rescence ratio in pyrene-labeled PS/PC vesicles was notinfluenced by the PS partition.

DISCUSSIONIn this study, we report the alternate use of MC540 to

monitor the generation of the procoagulant surface oncellular membranes of activated blood platelets. In addi-tion, we compared this new technique with the standardtechnique of staining procoagulant platelets with annexinV. First, we revealed a very significant negative correlation

between staining with annexin V and MC540, whichsupports our reasoning that MC540 may be used success-fully to monitor platelet surface membrane procoagulantproperties. We found that the reductions in MC540 fluo-rescence associated with activated cells were mostly ex-plained by the variability in the formation of micropar-ticles and discriminated well along with various types ofplatelet activators and procoagulant factors. They rangedfrom weak (thrombin or TRAP alone) or moderate (thap-sigargin � TRAP) to strong (collagen � thrombin) or verystrong (collagen � VO4

�3). Second, this reciprocal corre-lation between the fractions of MC540 and annexin V-positive platelets holds particularly for the cases whenplatelet membrane PL asymmetry was disturbed drasti-cally, i.e., for moderate and higher binding of annexin Vand moderate or weaker staining with MC540. Third, therecognized blockers of Ca2�–ATP-ases and inhibitors ofaminophospholipid translocase considerably affectMC540 uptake and fluorescence. This means that MC540is sensitive enough to detect intracellular events underly-ing membrane PL symmetrization, including mobilizationof the intraplatelet Ca2�.

When explaining the obtained fluorescence data forMC540, we have to be aware that the interaction ofMC540 molecules with cell membranes involves severalfactors (28,29). The n-butyl side chains of the dye drawthat end of the molecules into the hydrophobic interior oflipid membranes. This, however, is countered by the po-larity of the amide linkages and the negative charge on thesulphonate, which acts as a pivot (29). Therefore, MC540binding is limited to the outer leaflet of the plasma mem-brane lipid bilayer and is predominantly sensitive to thesurface charge of a membrane lipid bilayer, which origi-nates from the abundance of negative PS. MC540 bound tolipid membranes exists in two differently colored species

FIG. 11. Effect of MC540 concentration on its binding to the PS/PCvesicles. Data presented as mean � SEM, n � 12 experiments. Dyebinding was expressed as MC540-positive vesicles containing 5 (triangles)or 20%/mol PS (circles). Differences for all the pairs marked as solidsymbols were at least P � 0.01 by the pair-matched Student’s t-test.

FIG. 10. Effect of PS partition on MC540 binding to PS/PC vesicles. a:Linear regression plot of the dye binding to vesicles containing increasingfractions of PS. Data presented as mean � SEM, n � 6 experiments.MC540 concentration was 3 �mol/L. Dye binding was expressed asMC540-positive vesicles. PS partition was expressed in mol/PS per 100mol/PL. The estimated coefficients of the linear regression y � ax � bwere a � �0.776 � 0.117 (P � 0.0001), b � 30.3 � 1.4 (P � 0.0001).b: Flow cytometric analysis of MC540 binding to PS/PC vesicles. Histo-gram plots of MC540-positive vesicles containing 5 (grey solid fill), 10, or20 mol/l PS (outlines from right to left). Insert: Dot plots of FSC versusMC540 binding (FL2) for PS/PC vesicles containing 5 (A), 10 (B), or20%/mol PS (C). The dashed lines demarcate the markers of the specificbinding of the dye to vesicles. For further experimental details, seeMaterials and Methods.

130 WATALA ET AL.

— monomers and dimers (30,31). Because only MC540monomers are fluorescent and the dimer fluorescence isnegligible, the observed MC540 fluorescence should beattributed to MC540 monomers that interact with theouter leaflet of the platelet membrane lipid bilayer. Thechanges in the fraction of MC540-positive platelets evokedby activation may be explained by the changes in theoverall amount of MC540 molecules that interact in someway with platelets and/or by the changes in MC540 mono-mer-dimer distribution. Although the absorption and flu-orescence spectral characteristics of MC540 might beshifted by an altered chemicophysical state of the mem-branes of activated platelets (32), the presented changesin the platelet-associated MC540 fluorescence monitoredby flow cytometry can be regarded as the changes in theinteraction of MC540 with plasma membranes occurringupon platelet activation.

Our study was based on the assumptions that the an-ionic MC540 has a high affinity to the membrane lipidcomponent and that it is sensitive to the alterations inmembrane physicochemical properties. Although not em-ployed previously in blood platelets, the dye has beenused successfully to study membrane PL asymmetry inother cell types, including RBC (33–35). MC540 bindspreferentially to fluid, loosely packed, cholesterol-free PLdomains in membranes (9) and a change in the naturalmembrane PL asymmetry results in altered MC540 bind-ing, i.e., more tight packing of the outer membrane leafletlead to the decreased dye penetration into lipid mem-branes and vice versa (36). With respect to any distortionsin membrane PL organization and changes in membranelipid fluidity, one has to be aware that a procoagulantactivity of blood platelets is not exclusively dependent onthe amount of surface-exposed PS. Other lipids such as PEsphingomyelin (SM) may also have modulating effects.Both of these are known bilayer rigidizers, so it might beexpected that a translocation of PE and/or SM to the outerleaflet in the course of platelet activation would inducealtered MC540 binding. Discrimination between the ef-fects of platelet membrane PL translocation and mem-brane lipid bilayer rigidization in blood platelets is impos-sible because both events are physiologically inseparable.However, we show that MC540 interaction(s) vanishesalong with the increased PS partition in PS/PC liposomes,although these changes were not accompanied by thealtered lipid fluidity in the vesicles.

Finally, the binding of MC540 is strongly dependent onTMP and on the interrelated surface membrane potential.In some tested cell types (i.e., RBC), the hyperpolarizationresulted in decreased MC540 binding (28). The alterationsin platelet membrane PL asymmetry are always accompa-nied by the mobilization of calcium ions in the course ofplatelet activation and release reaction. However, theproblem of whether such a calcium mobilization mightaffect platelet TMP and platelet membrane surface poten-tial depends on what order of magnitude are the changesin the ratio [Ca2�]in/[Ca2�]out compared with the otherpermeable ions occurring upon platelet activation (37).Considering that the changes in local Ca2� concentrations

in activated platelets are of a nanomolar order of magni-tude, one may not expect that they are likely to affectconsiderably MC540 binding. The reports on changes inTMP in activated platelets are inconsistent in claimingeither unchanged or decreased TMP upon platelet activa-tion with ADP, thrombin, or collagen (38–40). Our find-ings corroborate the latter, i.e., the observed reduction inthe DiOC5(3)-positive platelets suggests the decreasednegative polarization of platelets inside upon the additionof either valinomycin or collagen (41). Whereas the di-minished membrane–MC540 interaction(s) as a result ofthe hyperpolarization of platelets inside might be moreeasily speculated, no direct relevance between reduceddye binding and depolarization — both phenomena oc-curring upon platelet activation — was deduced. Further,in a recent study (unpublished data), we showed that thepartition of negatively charged PL was the major factorinfluencing the binding of MC540 to PS/PC vesicle mem-branes, whereas the contribution of TMP to MC540–membrane interaction(s) was minor. We believe that theobserved changes in MC540-positive platelets are lesslikely to be driven by changes in TMP and are attributed tothe ongoing symmetrization of platelet membrane PL. Themarked chelating of outside Ca2� by EGTA did not atten-uate the MC540 interaction with platelets. EGTA wasexpected to impair ionic balance and influence the TMPdue to the removal of the outside Ca2�. Because no con-siderable effect of EGTA on platelet–MC540 interaction(s)was seen, we conclude that in our system, the alterationsin TMP were probably the minor contributor to thechanges in the platelet-associated MC540 fluorescence.

The use of MC540 for monitoring the platelet proco-agulant surface has both advantages and disadvantagesover the use of annexin V. Compared with the moderatefluctuations in annexin binding between resting andstrongly procoagulant platelets (20%–30%), the range ofthe relevant extreme values for MC540 is roughly twofoldwider. Therefore, reasonable discrimination between var-ious procoagulant agents is more likely. In addition, thebinding of annexin V to negatively charged PS is unique inthat PL molecules recognized by annexin cannot beloosely dispersed in the lipid bilayer matrix, but must formlattices, with PS polar heads adjacent to each other (42).To gather such an orientation is possible only due to theelectrostatic interaction with Ca2� ions. Due to its verysmall size, MC540 may easily penetrate even to singlecharged PL molecules, which makes the dye binding moreproportional to the number of PS exposed. MC540 islikely to recognize (to be repulsed by) even single chargedPS molecules, which supports the much higher propor-tionality between MC540 binding and PS exposure. Theuse of annexin V is intimately connected with the tightcontrol of Ca2� concentrations. Both the specificity andproportionality of the dye binding to aminophosphoricgroups of PS is very Ca2�-sensitive and the enhancedbinding of annexin to a particle does not necessarily implyPS exposure. Electrostatic forces are also involved in theinteraction of MC540 molecules with cell membranes.Therefore, the dye interaction might be potentially af-

131PLATELET MEMBRANE ASYMMETRY MONITORED WITH MC540

fected also by factors other than the partition of negativelycharged PL, like TMP and surface membrane potential. Wesuggest that the latter are of minor significance in acti-vated blood platelets and that MC540 interaction is pre-dominantly driven by the changes in platelet membranePL asymmetry.

We have to be aware of all the potential benefits andobstacles of both dyes. There is a strong association be-tween staining with annexin V and maintaining high con-centrations of Ca2�, which leads to abundant fractions ofplatelet aggregates following the incubation procedure.We showed that the gradual increase in annexin V bindingto activated platelets, which is dependent on the type ofplatelet activator, is no longer maintained in the fractionof platelet aggregates. The portions of annexin V bound tonormoplatelets and aggregates were the largest compo-nents of the variability in annexin V binding, which wascompletely independent of the formation of aggregates.As there is marked artefactual platelet activation and ex-tensive formation of platelet aggregates in the course ofstaining the platelets with annexin V, large portions ofannexin V remain useless in defining the procoagulantproperties of activated platelets. We believe that only alimited subpopulation of bound annexin molecules maybe attributed to the alterations in platelet membrane PSexposure.

At higher concentrations, MC540 may favor the forma-tion of microparticles because of its potential cytotoxicity.Because platelet lysis causes exposure of PS present in theinner leaflet of plasma membranes, as well as in the mem-branes of intracellular structures, one should be aware ofcell integrity under various conditions of platelet activa-tion. The abundant fraction of platelet microparticles re-sults in the overestimation of platelet procoagulant activ-ity (defined as their ability to bind prothrombinasecomplex) because of the significant increase in the bind-ing of annexin V and the immensely reduced uptake ofMC540. We showed that the interaction of the dye withactivated platelets is due to a lesser extent by the forma-tion of platelet microparticles in the case of MC540 com-pared with annexin V.

As annexin V requires tight control of calcium, an im-portant consideration may be the choice of calcium-nonchelating blood anticoagulants. The alternate option isto use thrombin inhibitors. However, this requirementmakes the examination of the effects of thrombin (aloneor in a combination with collagen) unreliable. Conversely,MC540 does not require omission of calcium-chelatinganticoagulants because staining with MC540 is calcium-independent. Both dyes monitor membrane PL symmetri-zation, which is largely calcium-dependent. Therefore, itis likely that calcium chelators might affect platelet cal-cium mobilization and might potentially lead to the un-derestimation of MC540-positive platelets, even thoughthe staining itself would be unaffected. We believe thatthere is no gold standard to guide the use of this method-ology of determination of platelet procoagulant surfacewith some agonists, like thrombin. As we demonstrated inthis study, we may still overcome this problem by using

TRAP, which activates the same type of platelet receptor(PAR-1) and is insensitive to thrombin inhibitors.

The estimated diagnostic precision and reliability weresignificantly higher when MC540 was used to monitor theprocoagulant activity of activated platelets compared withannexin V staining. MC540 is more sensitive and specificand has more than a twofold lower risk of false-positiveresults.

Two problems remain unresolved, i.e., the molecularmechanism(s) of the MC540-supported depletion in plate-let aggregates following whole blood platelet activationwith recognized proaggregatory agents (collagen or ADP)and the apparent lack of the ameliorating effect of Ca2� onMC540 interaction with the negatively charged procoagu-lant surface of activated platelets. The first problem mayoriginate from the MC540-mediated effects on electro-static interactions because the antiaggregative effect ofMC540 was observed on lecithin liposome suspensions(43). The second problem might question whetherMC540 binding to the platelet membrane surface ismerely driven by electrostatic repulsion between anionicMC540 and negative PS. We speculate that the electro-static forces that define the surface charge density are ofprimary importance in determining the electrostatic repul-sion of anionic MC540 with the increased negative chargeof PS accumulating in the outer platelet membrane bilayerleaflet during platelet activation. We might presume thatdivalent cations (like Ca2�) would reduce the surfacemembrane potential by their screening effect (41). Be-cause MC540 is negatively charged, the presence of Ca2�

may result in a weaker repulsion of the dye from themembrane of activated platelets. However, we haveshown that the interaction of MC540 with the plateletsurface was equally efficient, regardless of the presence ofCa2�. One possible explanation might be that despiteselective recognition of negatively charged PS aminophos-phoric groups by MC540, the alterations in membranelipid dynamics and the reorganization in rigidly packedcellular membranes of activated platelets attenuate thedye interaction and considerably reduce its binding tomembrane surface.

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