James G. White, Departments of Laboratory Medicine and Pathology,Pediatrics, University of Minnesota Medical School, UMHC Box 490,420 Delaware Street S.E., Minneapolis, MN 55455, USA; GinesEscolar, Servicio de Hemoterapiay Hemostasia, Hospital Clinico yProvincial, Villarroel. 170, 08036 Barcelona, Spain.

Correspondence to: James G. White, M.D., Departments of Labo-ratory Medicine and Pathology, Pediatrics, University of MinnesotaMedical School, UMHC Box 490, 420 Delaware Street S.E.,Minneapolis, MN 55455, USA.

EDTA-induced changes in platelet structure andfunction: adhesion and spreading

James G. White, Gines Escolar

Ethylenediamine tetraacetic acid (EDTA) is an effective anticoagulant, but unfortunately causes structural,biochemical and functional damage to human platelets. Some of the functional injuries, such as adhesion to andspreading on surfaces, are considered irreversible. The present investigation has evaluated that hypothesis. Ourfindings indicate that platelets from EDTA platelet-rich plasma (PRP) or CCD PRP to which EDTA has beenadded do not adhere to glass or plastic surfaces. However, when platelets from EDTA PRP or CCD PRPcontaining added EDTA are washed and resuspended under conditions reported to cause irreversible dissociationof the fibrinogen receptor, GPIIb/IIIa, then washed and resuspended in buffer containing Ca2+ and Mg2+ ions willadhere and spread in the same manner as platelets not exposed to EDTA. The ability to recover adhesive functionmay explain why EDTA platelets are able to sustain clot retraction as well as CCD platelets.

Introduction

Ethylenediamine tetraacetic acid (EDTA), a divalentcation chelating agent, has been used for many years asan anticoagulant to maintain the fluidity of blood.1– 3

However, EDTA causes structural, biochemical andfunctional injuries to platelets.4 –7 Platelets from bloodcollected in EDTA or blood obtained in citrate anticoag-ulant to which EDTA has been added develop narrow, aswell as dilated channels of the open canalicular system(OCS).4,5,8 The narrow, elongated channels resemble‘zippers’ and have a pentilaminar appearance due tocondensation of the glycocalyx lining channel mem-branes into small spheres. Physical changes in EDTAplatelets are associated with loss of their aggregationresponse to adenosine diphosphate (ADP), epinephrine,A23187, vasopressin and serotonin, and their ability toadhere to glass.6 EDTA platelets also aggregate poorly

with thrombin and collagen, and have a reduced ability tobind fibrinogen. These effects are associated with amarked decrease in binding of monoclonal antibodies toglycoprotein (GP) a IIb b 3 (GPIIb/IIIa) and irreversibledissociation of GPIIb/IIIa into its GPIIb and GPIIIacomponents. 6,8 – 10

Despite these undesirable effects, EDTA platelets areable to retract clots normally.6 This finding suggestedthat effects of EDTA on platelet function may not be asirreversible as previously considered. Our first investiga-tion showed that EDTA-treated platelets in suspensioncould take up particulates, including latex spherules andfibrinogen-coated gold particles that require GPIIb/IIIafor binding and uptake.11 The present study has exam-ined the possibility that irreversible loss of EDTAplatelet ability to adhere to and spread on glass and othersurfaces can be reversed. Results indicate that EDTAplatelets can recover their adhesive function.

Materials and methods

Preparation of platelets

Blood from the present study was obtained afterinformed consent from well-characterized, normaldonors who had not taken aspirin for at least 10 days.12

ISSN 0953-7104 print/ISSN 1369-1635 online/00/010056-06 © 2000 Taylor & Francis Ltd

Platelets (2000) 11, 56– 61

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Following venipuncture, the samples were mixed imme-diately with citrate–citric acid–dextrose (CCD), pH 6.5,in a ratio of nine parts blood to one part anticoagulant.Other samples were mixed with 0.1 M/l EDTA in thesame ratio of blood to anticoagulant.13 Platelet-richplasma (PRP) was separated by centrifugation at roomtemperature for 20 min at 100´ g. PRP samples werecombined with equal volumes of CCD anticoagulant,then washed twice and resuspended in Hank’s balancedsalt solution (HBSS) containing 10 mmol/l EDTA.11 TheEDTA platelets and CCD platelets not exposed to EDTAwere incubated at 37°C for 30 min at pH 7.8. EDTA andCCD samples were then sedimented, rinsed, and resus-pended in HBSS with or without Ca2+ and Mg2+ ions, butno added EDTA for 30 min.

Interaction of EDTA and CCD platelets with glass

Drops of CCD PRP, EDTA PRP, CCD PRP with EDTA,EDTA or CCD PRP washed and resuspended in HBSSwith or without EDTA for 30 min at 37°C, and EDTA orCCD PRP washed and resuspended with EDTA for30 min, then washed and resuspended in HBSS with orwithout Ca2+ and Mg2+ ions for 30 or 60 min at 37°Cwere placed on glass slides, cover slips added andexamined under oil in an interference phase contrastmicroscope with a 100 ´ oil immersion objective at 10,20 and 30 min.

Interaction of CCD and EDTA platelets with plastic

Drops of the platelet preparations mentioned in theprevious section were placed on formvar-coated, electronmicroscope grids and allowed to interact with the surfaceat 37°C for 20 min. After this period unattached plateletswere washed away by passing the grid through succes-sive drops of HBSS and then fixed in 0.01%glutaraldehyde. 14

Identification of receptors on spread EDTA and CCDplatelets

Monoclonal antibody, API, against GPIb/IX, and 7E3against GPIIb/IIIa were kindly provided by Tom Kunicki(Scripps Institute, La Jolla, CA) and Barry Coller (MountSinai, New York, NY), respectively. Fibrinogen-coatedgold particles (Fgn/Au) to detect GPIIb/IIIa wereprepared in the manner described by Loftus andAlbrecht,15 by Leistikow et al.16 and by Escolar et al.17

Fixed grids were washed in phosphate-buffered saline(PBS) and exposed to one of the primary antibodies (APIor 7E3) at a 1:100 dilution (10 m g/ml) in PBS for 60 min.After repeated washing the grids were exposed for60 min to goat antimouse IgG (Amersham International)coupled to 10- and 20-nm gold particles. After the lastincubation grids were washed successively with PBS anddistilled water and air dried. Grids incubated with Fgn/Au for 20 min were washed, excess fluid removed withfilter paper and air dried.

Cross-sections of spread EDTA and CCD platelets

Lab-Tek 4 chamber slides (Nunc, Inc.) were used toprepare cross-sections of surface-activated platelets.18

The surface of each chamber was coated initially with0.1% polylysine for 5 min and washed several timesbefore being air dried. Volumes (0.5 ml) of the severalpreparations of EDTA and CCD platelets describedabove were added to chambers and incubated at 37°C for30 min. The chambers were rinsed well and carriedthrough fixation, or exposed to Fgn/Au for 30 min at37°C and then fixed. The chambers were fixed again in3% glutaraldehyde in White’s saline (a 10% solution ofa 1:1 mixture of: (1) 2.4 mmol/l NaCl, 0.1 mmol/l KCl,46 mmol/l MgSO4 , 64 mmol/l Ca(No3 )2·4H2O; and (2)0.13 mol/l NaHCO3 , 8.4 mmol/l NaH2PO4 and 0.1 g/l ofphenol red, pH 7.4 (5) for 15 min and then with 1%osmic acid combined with 1.5% potassium ferrocyanidein distilled water for 30 min at room temperature. Afterfixation the samples were washed and dehydrated in aseries of alcohols and exposed to propylene oxide briefly.The spread cells were quickly transferred from chambersto a tube containing additional propylene oxide, and thenincreasing concentrations of epon in alcohol. After thefinal rinse in 100% epon, the chambers were drained, and0.3 ml of 100% epon was added to each chamber. Someof the epon was saved in a syringe for final embedding.The chambers were placed in a 45°C oven overnight andin a 60°C oven for 1–2 days. Slabs of embedded plateletswere cut into rectangular wedges, mounted in holders,and sectioned on an ultramicrotome. Thin sections werestained with lead citrate and uranyl acetate to enhancecontact.

Interaction of EDTA and CCD platelets with latexspherules

Latex particles with an average diameter of 6.4 m mand 1.7 m m in diameter were washed and suspended inHBSS as described previously.11 ,1 4 Latex (0.1 ml) wasadded to 0.9 ml of the CCD or EDTA platelet suspen-sions indicated above. The experiment of importancewas the latex combined with EDTA platelets washedand resuspended in HBSS with EDTA for 30 min, thenwashed and resuspended in HBSS containing Ca2+ andMg2+ ions together with latex for 30–60 min.

Results

Effects of EDTA on platelet structure

Platelets from blood collected in EDTA anticoagulantand platelets from blood collected in CCD to whichEDTA was subsequently added develop significantphysical changes.4,5 EDTA platelets become irregularin form, the ‘spiny spheres’ described by Zucker andBorrelli.19 Examination of thin sections of EDTAplatelets in the electron microscope revealed conver-

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sion of many OCS channels into long narrow seg-ments (Figure 1). Condensation of the glycocalyxcovering the channel membranes into microspheresgave the narrow segments a pentalaminar appearance(Figure 2A,B). Other OCS channels, frequently con-nected to narrow segments, become dilated. CCDplatelets to which EDTA was added retained theirdiscord shape, but developed the same OCS changesas EDTA cells.

Interactions of EDTA and CCD platelets with glass

Platelets in PRP separated from blood collected in EDTAdo not adhere to glass, nor do platelets from bloodanticoagulated first with CCD to which EDTA (10 mmol/l) was then added (Figure 3A). EDTA and CCD plateletswashed and resuspended in HBSS without Ca2+ or Mg2+ ,but containing 10 mmol/l EDTA, then washed andresuspended in HBSS without Ca2+ and Mg2+ ions

58 EDTA-INDUCED CHANGES IN PLATELET STRUCTURE: ADHESION AND SPREADING

Figure 1. Platelet from blood collected in citrate (CCD) antico-agulant and then combined with EDTA, washed and resus-pended in HBSS with EDTA for 30 mins. The discoid cell hasbeen sectioned in the equatorial plane. A circumferential coil ofmicrotubules (MT) lies just under the surface membrane. Manyalpha granules (G) and a few dense bodies (DB) fill thecytoplasma. Channels of the open canalicular system (OCS)have been altered by EDTA. Toruous elements have beenconverted into long, narrow channels (NC) and dilated channels(DC) which are often connected. Original magnification,´ 30 000.Figure 2. (A,B) The platelet in (A) reveals several of the narrowOCS channels (NC) caused by exposure to EDTA. Some of thechannels have been tangentially sectioned yielding a zipper-likeappearance (ZC). Four EDTA platelets in close cell contact areshown in (B). The zones of association between four cells havethe zipper-like appearance, and between two others the tube-likeform (*) of narrow channels of OCS. Original magnification(A) ´ 40 000; (B) ´ 24 000.

Figure 3. (A–C) Interference phase contrast micrographs ofplatelets at stages of EDTA treatment and recovery. The cell in(A) was in EDTA PRP. Platelets in (B) are from EDTA PRP,washed and resuspended in HBSS with EDTA for 30 mins, thenwashed and resuspended in HBSS without Ca2+ or Mg2 + ions.The cells have adhered to the glass and extended pseudopods,but have not spread. Platelets in (C) were prepared in the samemanner as those in (B), but resuspended in HBSS with Ca2 + andMg2 + ions. The cells have adhered to glass and spread as well ascontrol CCD platelets. Original, magnification: (A) ́ 800; (B) ́ 800;(C) ´ 800.Figure 4. Platelet from EDTA PRP, washed and resuspended inbuffer with EDTA, then washed and resuspended in HBSS withCa2 + and Mg2 + ions. The cell was spread on formvar and stainedfor GPIb with immunogold methods. Ten- and 20-nm goldparticles indicating sites of GPIb/IX receptor complexes areevenly dispersed over the cell surface from edge to edge.Original magnification ´ 27 000Figure 5. Platelet prepared in the same manner as the cell inFigure 4, but stained for GPIIb/IIIa. Immunogold particlesindicating sites of the GPIIb/IIIa complex are randomly dispersedover the platelet surface from edge to edge. Original magnifica-tion ´ 20 000.

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present adhered to glass and converted to dendritic forms(Figure 3B). If the final resuspension was in HBSS withCa2+ and Mg2+ ions, the platelets would adhere andspread (Figure 3C).

Interaction of EDTA and CCD platelets with plastic

Platelets from CCD PRP spread fully during 20 mincontact with formvar. EDTA PRP platelets did not attachto grids. EDTA and CCD platelets washed and resus-pended in HBSS containing EDTA for 30 min thenwashed and resuspended in HBSS for 30 min couldadhere to formvar and change into dendritic forms, but

did not spread. If Ca2+ and Mg2+ ions were present in thefinal resuspension and incubation continued for 30 minthe platelets were able to adhere and spread fully.

Identification of receptors on spread EDTA and CCDplatelets

Examination of washed and resuspended preparations ofEDTA and CCD platelets spread on formvar and stainedwith APl and goat anti-mouse IgG coupled to 10- and20-nm gold particles revealed random distribution ofGPIb receptors over both types of cells form edge toedge (Figure 4). The density of gold particles was the

PLATELETS 59

Figure 6. Spread platelet prepared in the way as cells inFigures 4 and 5, but stained for GPIIb/IIIa by exposure tofibrinogen coated gold particles (Fgn/Au). GPIIb/IIIa receptorsare evenly dispersed over the spread cell surface. Original mag´ 25 000.Figure 7. Thin section of platelet from EDTA PRP, washed andincubated with EDTA in suspension then washed and resus-pended in HBSS with Ca2 + and Mg2 + ions. The cell has attachedto the plastic surface (PS) and is in the process of spreading.Narrow channels (NC) are still present in the cytoplasma, but willdisappear as channels of the open canalicular system areevaginoted during spreading. Original magnification ´ 20 000.

Figure 8. Platelets from sample prepared in the same manneras the cell in Figure 7, but combined with 0.1 U/ml of thrombinbefore placing on the plastic surface (PS). After fixation thesample was incubated with Fgn/Au to identify GPIIb/IIIa receptorcomplexes. Fgn/Au particles are evenly dispersed over theexposed surfaces of the activated and spread EDTA platelets.Original magnification ´ 20 000.Figure 9. Platelet from sample of EDTA PRP incubated insuspension with EDTA then washed and resuspended in HBSSwith Ca2+ and Mg2 + ions and incubated with large latex (L)spherules for 30 min. One platelet has spread completely over alarge latex spherule. An adjacent platelet contains narrowchannels (NC) of OCS in its cytoplasma. Original magnification´ 20 000.Pl

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same on spread CCD and EDTA platelets. Evaluation ofspread EDTA and CCD platelets stained with 7E3against GPIIb/IIIa followed by anti-mouse IgG coupledto 10 nm gold also revealed a uniform distribution ofgold particles indicating sites of the receptor complex onboth cell types (Figure 5). Incubation of spread EDTAand CCD cells with Fgn/Au revealed uniform distribu-tion of this marker for GPIIb/IIIa on both cells (Figure6). The density of the gold marker was essentially thesame on EDTA and CCD spread platelets.

Cross-sections of EDTA and CCD platelets spread onplastic

The chamber method permits examination of CCD andEDTA platelets spread on glass, fixed and embedded forthin sectioning.18 Cross-sections of EDTA and CCDplatelets from washed, resuspended preparationsrevealed extension of peripheral margins out onto theplastic surface. Central regions revealed persistence ofthe narrow OCS channels in spread EDTA platelets andtheir absence in CCD cells (Figure 7). Examination ofthe EDTA and CCD spread cells fixed and incubatedwith Fgn/Au revealed persistence of the GPIIb/IIIareceptors on EDTA spread platelets (Figure 8).

Interaction of EDTA and CCD platelets with latexspherules

Previously we have shown that EDTA platelets washedand resuspended in HBSS containing Ca2+ and Mg2+

ions can take up small latex particles into narrow anddilated OCS channels.11 In the present study EDTAplatelets treated in a similar manner were reacted withlarge latex spherules 6.4 m m in diameter. Despite pres-ence of narrow, elongated OCS channels in EDTAplatelets, the cells were able to spread normally over thelatex particle surfaces (Figure 9).

Discussion

The ability to selectively bind divalent cations in blood,thereby preventing clot formation, is the only knowneffect of EDTA and similar chelating agents likeEGTA.1– 3 Yet, in contrast to other anticoagulants thatbind calcium, such as trisodium citrate, EDTA causesinjury to platelets considered to be irreversible.4– 8

Structural changes include conversion of many tortuousOCS channels into narrow elongated segments, anddililation of others connected or not to the narrowcanaliculi. 4,5,8 The glycocalyx covering membranes lin-ing the narrow channels of OCS in EDTA platelets iscondensed into microspheres.8 Their periodicity suggeststhe presence of a separate layer between juxtaposedmembranes yielding a pentalaminar appearance.

A similar organization is not seen on membraneslining dilated segments of OCS, or on the exposedsurface membrane of the cell. However, when EDTAplatelets come in close cell contact, the same pentalami-

nar appearance observed in narrow channels of the OCSis visible at the zone of association.20 The physicalchanges are not reversible under the conditions describedin this study.

EDTA also causes platelets to lose their discord formand convert into irregular shapes with filoform pseudo-pods, the ‘spiny spheres’ described by Zucker and co-workers.19 ,21 However, addition of EDTA to plateletsfrom blood collected previously in CCD anticoagulantretain their discord form, even though they develop thesame alterations in the OCS as platelets from bloodanticoagulated with EDTA.4 ,5 The basis for the shapechange caused by EDTA and how CCD prevents itremain uncertain.

The physical changes in EDTA platelets are associatedwith permanent loss of their aggregation response toadenosine diphosphate (ADP), epinephrine, A23187,vasopressin and serotonin, and their ability to adhere toglass.6 EDTA-affected platelets aggregate poorly whenstirred with thrombin or collagen, and exhibit a dimin-ished capacity to bind fibrinogen. These effects havebeen related to a marked decrease in binding ofmonoclonal antibodies to GPIIb/IIIa, and to irreversibledissociation of GPIIb/IIIa into its components, GPIIband GPIIIa.6,9 ,1 0

The structure and biochemical damage induced inplatelets by EDTA compromises their functional capac-ity, and many of the limitations are considered irreversi-ble.6 ,2 1 However, some functions of platelets are notimpaired by EDTA,6,7 and others may not be asirreversible as suggested previously.11 EDTA-treatedplatelets, for example, retract clots as rapidly andcompletely as CCD platelets.6 EDTA was used as theanticoagulant of choice when platelets for transfusionwere stored in the cold.7 The cells did not survive as wellas do CCD platelets maintained at room temperature, butthey did appear to improve hemostasis in thrombocy-topenic patients. Recently we have shown that EDTA-treated platelets can interact with particulates and clearthem from exposed surfaces to channels of the OCS inthe same manner as CCD cells.11 Based on these findingsit seemed worthwhile to determine just how irreversibleother lesions induced by EDTA really are.

The present study has examined the ability of EDTA-treated platelets to interact with surfaces. A majorobservation made by Zucker and Grant6 was that EDTA-treated platelets irreversibly lose their ability to adhere toglass. Platelets in EDTA anticoagulated PRP or in theCCD PRP to which EDTA has been added are unable tobind to glass as these authors suggested. However, ifplatelets in EDTA PRP or CCD-PRP to which EDTA wasadded are washed and resuspended in HBSS with orwithout EDTA for 30 min, the cells will adhere to glass. Ifthe platelets washed and resuspended in HBSS with orwithout EDTA are washed again and resuspended inHBSS with Ca2+ and Mg2+ ions they will not only adhereto glass, but spread as well as platelets which have neverseen EDTA. If they are sedimented and resuspended inplatelet poor plasma (PPP) prepared from EDTA PRP, the

60 EDTA-INDUCED CHANGES IN PLATELET STRUCTURE: ADHESION AND SPREADING

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cells will no longer adhere or spread. Thus the presence ofplasma, as well as EDTA, appears to be an importantfactor in loss of adhering and spreading activity.

The ability of EDTA-treated platelets to interact withplastic surfaces was similar to that described for glass.When washed and resuspended in HBSS with or withoutEDTA, the EDTA PRP cells or platelets from CCD PRPto which EDTA had been added could adhere to formvaror chamber plastic. If they were washed and resuspendedagain in HBSS with Ca2+ and Mg2+ ions they adheredand spread as well as platelets that had not been exposedto EDTA.

Glycoprotein receptors on EDTA platelets appeared tobe present. Spread EDTA platelets exposed to either 7E3to detect GPIIb/IIIa or API to identify GPIb followed bygoat anti-mouse IgG coupled to gold were as wellcovered by the receptors as spread CCD platelets.Fibrinogen-coated gold particles also bound to spreadEDTA platelets in the same manner as CCD platelets,and, as shown previously,11 can be translocated tonarrow and dilated channels of the OCS. The earlierwork proved that EDTA platelets washed and resus-pended in HBSS with Ca2+ and Mg2+ could bind andinternalize small latex particles. Our present study hasshown that washed and resuspended EDTA platelets bindand spread on large latex spherules despite narrowedchannels of OCS.

The findings obtained in this investigation are puzzl-ing in light of published observations by others. Zuckerand Grant6 attempted to reverse the effects of EDTA onaggregability by prolonged incubation with CaCl2 ,adding normal plasma or washing the platelets withoutsuccess. It is possible that their washing procedure wasnot as effective as ours, because washing was critical forrestoring platelet functions inhibited by exposure of PRPto EDTA.

Irreversible dissociation of GPIIb/IIIa into its sub-units, GPIIb and GPIIIa was suggested as a possiblebasis for the functional losses caused by EDTA6.Fitzgerald and Phillips10 and Pidard et al.9 have shownthat exposure of platelets to excess EDTA for 30 min at37°C results in dissociation of GPIIb and GPIIIa, andtheir conversion into polymers that remain irreversible.We have no basis for disagreement with their findings.However, washed and resuspended EDTA platelets inHBSS with Ca2+ and Mg2+ incubated at 37°C for 1 h andspread on formvar grids or on plastic chambers readilybound Fgn/Au and an antibody (7E3) to GPIIb/GPIIIa.Our findings may help to explain why EDTA plateletsare able to bind to fibrinogen and retract fibrin clots asrapidly and completely as CCD platelets.

In conclusion, the results of the present study haveshown that the ability of platelets to adhere to and spreadon glass, as well as other surfaces, is not irreversiblyblocked after exposure to EDTA6. Washing and resus-pending EDTA PRP or CCD PRP in HBSS with orwithout EDTA, incubating at 37°C, pH 7.8, for 30 minfollowed by washing and resuspension in HBSS contain-ing Ca2+ and Mg2+ ions restores the adhesive and

spreading functions inhibited by EDTA. Restoration offunction to EDTA platelets is associated with recovery oftheir ability to bind Fgn/Au and monoclonal antibodiesagainst GPIIb/III. These studies may help to explain theeffects of EDTA on platelets and restore confidence inthe agent for use in long-term platelet preservation.

AcknowledgementsThis work has been supported by a grant from the Graduate School ofthe University of Minnesota.

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nismus organischer substanzen. Ztschr Ges Exp Med 1942; 110:412–7.

2. Proescher F. Anti-coagulant properties of ethylene-bis-imino-diacetic acid. Proc Soc Exp Biol Med 1951; 76: 619–20.

3. Trianlaphyllopoulos D C, Quick A J, Greenwalt T J. Action ofdisodium ethlenediamine tetracetate on blood coagulation: evi-dence of the development of heparinoid activity during incubationor aeration of plasma. Blood 1955; 10: 534–44.

4. White J G. Effects of ethlenediamine tetracetic acid (EDTA) onplatelet structure. Scand J Haematol 1968; 5: 241– 50.

5. White J G. physiochemical dissection of platelet structuralphysiology. In: Baldini M G, Ebbe S, Eds. Platelets: ProductionFunction Transfusion and Storage. New York: Grune and Stratton,1974; 235– 52.

6. Zucker M B, Grant R A. Nonreversible loss of platelet aggreg-ability induced by calcium depletion. Blood 1978; 52: 505–14.

7. Slichter S J. Preservation of platelet viability and function duringstorage of concentrates. In: Greenwalt T J, Jamieson G A, Eds. TheBlood Platelet in Transfusion Therapy. New York: Alan R. Liss,1978; 83–100.

8. Gachet C, Hanau D, Spehner D, Brison C, Garaud J C, Schmitt DA, Ohlmann P, Cazenave J P. a IIb b 3 integrin dissociation inducedby EDTA results in morphological changes of the platelet surfaceconnected canalicular system with differentiated location of thetwo sub units. J Cell Biol 1993; 120: 1021– 30.

9. Pidard D, Didry D, Kenicki T J, Nurden A T. Temperature-dependent effects on EDTA on the membrane glycoprotein Iib–IIIa complex and platelet aggregability. Blood 1986; 67: 604–11.

10. Fitzgerald L A, Phillips D R. Calcium regulation of the plateletmembrane glycoprotein GPIIb–IIIa complex. J Biol Chem 1985;260: 11366–74.

11. White J G, Krumwiede M D, Escolar G. EDTA induced changes inplatelet structure and function: influence on particle uptake.Platelets 1999; 10: 327–37.

12. White J G. Interaction of membrane systems in blood platelets. AmJ Pathol 1972; 66: 295– 312.

13. Gerrard J M, Phillips D R, Rao G H R, Plow E F, Walz D A, RossR, Harker L A, White J G. Biochemical studies of two patients withthe gray platelet syndrome: sellective deficiency of plateleta -granules. J Clin Invest 1980; 66: 102– 9.

14. White J G. Functional significance of mobile receptors on humanplatelets. Atheroscler Thromb 1993; 13: 1236– 43.

15. Loftus J C, Albrecht R M. Redistribution of the fibrinogen receptorof human platelets after surface activation. J Cell Biol 1984; 99:822–9.

16. Leistikow E A, Barnhart M I, Escolar G, White J G. Receptorligand complexes are cleared to the open canalicular system ofsurface activated platelets. Br J Haematol 1990; 74: 93–100.

17. Escolar G, Leistikow E, White J G. The fate of the platelet opencanalicular system (OCS) during surface activation. Blood 1989;74: 1983–8.

18. White J G, Krumwiede M. The downside of platelet surface. EurJ Cell Biol 1994; 65: 178– 88.

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