Detection of platelet activation with monoclonal antibodies and flow cytometry Changes during platelet storage R. FIJNHEER, P.W. MODDERMAN, H. VELDMAN,W.H. OUWEHAND, H.K. NIEUWENHUIS, D. Roos, AND D. DE KORTE To investigate whether changes in platelet condition during platelet storage correlate with an altered expression of platelet membrane proteins, the binding of monoclonal antibodies (MoAbs) to fresh platelets was compared with MoAbs' binding to thrombin-activated platelets and to platelets stored as platelet concentrates. The MoAbs included antibodies against the platelet glycoprotein (GP) Ilb/llla complex and against two activation-dependent antigens, one of which was a component of the internal platelet alpha-granule membrane (GMP 140) and the other of which was a 53-kD protein derived from platelet lysosomes. The binding of MoAbs to platelets fixed with 1 percent paraformaldehyde was measured by flow cytometry. In thrombin-activated platelets, a threefold increase was found in the expressionof GP llbillla over that in fresh platelets. The binding of the activation-dependent MoAbs increased from 2 to 3 percent to 70 to 80 percent of the platelets. Storage of platelet concentrates for 5 days resulted in a 60 percent increase in GP Ilb/llla expression compared to Day 0 and increased binding of the MoAbs directed against GMP-140 from 3 to 16 percent and against the 53-kD protein from 2 to 8 percent of the platelets, respectively. These changes correlated with modifications in platelet morphology (decrease in swirling), leakage of lactate dehydrogenase, and release of P-thromboglobulin. These data indicate that platelets become activated and are damaged during the storage of platelet concentrates. Suboptimal storage conditions (1.4 x lo9 platelets/mL) can be distinguished from optimal storage conditions (1.O x 10' platelets/mL) with the aid of these assays. The immediate fixation of the platelets and an analysis of the antigen expression by flow cytornetry provide a very useful test system for platelet activation. TRANSFUSION 1990;30:20-25.
the plasma membrane. As a result of this process, a 140-kD a-granule protein (GMP 140) appears on the plasma membrane.''-'4 GMP 140 is a useful marker for platelet activation, because its surface expression correlates with the release of a-granule content, and it is not reinternalized following secretion. Moreover, Nieuwenhuis et al.I5 found that a 53-kD protein from lysosomelike granules is also exposed on the surface of activated platelets. GP IIb and IIIa form a calcium-dependent complex in the membrane of resting platelet^.'^^'^ On platelets stimulated with agonists such as ADP or thrombin, the GP IIb/IIIa complex functions as a receptor for fibrinogen and other adhesive proteins. The binding of fibrinogen to activated platelets mediates platelet aggregation. As the storage of platelets results in a decrease in the capacity of the platelets to aggregate upon stimulation with ADP or thrombin, changes in the expression of the GP IIb/IIIa complex may reflect the functional condition of stored platelets. From experiments with MoAbs against the GP IIb/IIIa complex and against activation-dependent antigens, we conclude that platelets become activated during storage in platelet concentrates (PCs).
NUMEROUSR E P O R T S ' ~ have demonstrated a progressive decline in platelet function during storage. These changes may be associated with changes in glycolysis, loss of membrane components, alteration in contractile proteins, decreased levels of metabolic ATP, and altered expression of platelet surface glyc~proteins.~-~ Variables such as P-thromboglobulin (P-TG) release, morphologic changes, and the progressive expression of an activationdependent marker" suggest that the platelets become activated during storage. With the availability of monoclonal antibodies (MoAbs) against platelet surface glycoproteins, the investigation of these glycoproteins becomes possible. To investigate whether the loss of platelet condition during storage correlates with changes in membrane glycoprotein (GP) IIb/IIIa complex and the activation-dependent antigens, we studied the expression of these glycoproteins on the platelet surface during storage. One of the changes occurring at the platelet surface during activation is the fusion of the a-granule membrane with
From the Military Blood Transfusion Service, Amsterdam; the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and the Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam; and the University Hospital Utrecht, Utrecht, The Netherlands. Supported by a grant from Netherlands Laboratory for the Production of Blood Transfusion Equipment and Infusion Fluids (NPBI, B.V.), EmmerCompascuum, The Netherlands. Received for publication January 10, 1989;revision received June 19, 1989, and accepted June 19, 1989.
Materials and Methods The following materials were obtained commercially: bovine serum albumin (BSA; Organon Teknika B.V., Oss, The Netherlands), human a-thrombin (Sigma Chemical Company, St. Louis, MO), and a P-TG radioimmunoassay (RIA) kit (The Radiochemical Centre, Amersham, UK).
20
TRANSFUSION 1990-Vol. 30. No. 1
PLATELET ACTIVATION DURING STORAGE
Monoclonal antibodies MoAb C17 (CDw41), a previously described'" MoAb, reacts with GP IIIa when GP IIb-IIIa is a complex and inhibits platelet aggregation and the binding of fibrinogen to ADP- or collagenactivated platelets. The antibody is complex specific, i.e., it does not bind to GP IIb or to GP IIIa after EDTA-induced dissociation of the GP IIb/IIIa complex.'g*20 MoAb Y2 was obtained commercially (Dakopatts, Copenhagen, Denmark). It binds to GP IIIa and is not complex specific.21 MoAb C8 is of the IgGl subclass and recognizes a 140 kD glycoprotein that becomes associated with the platelet surface during secretion. This glycoprotein has previously been designated platelet activation-dependent granule-external membrane (PADGEM) protein, l 4 or granule membrane protein, (GMP 140).13 MoAb 2.28 is of the IgG2b subclass and was prepared as described. l5 This MoAb reacts with a 53-kD protein located in a subclass of platelet granules, probably lysosomes. The antigen is expressed on the platelet membrane upon thrombin stimulation. We used concentrations of MoAbs that gave maximal binding to the platelets.
Preparation of the platelets Blood samples were obtained from healthy volunteers. Platelet-rich plasma (PRP) was obtained by centrifugation (280 X g for 15 min at room temperature) of blood anticoagulated with CPDA in plastic tubes or from blood fixed immediately by collection in excess paraformaldehyde (PFA, 1% vol/vol, final concentration) and incubated for 15 minutes at room temperature. Platelets were pelleted from PRP by centrifugation (2200 X g, 7 min) and washed three times with PBS/EDTA (PBS: 140 mM NaCI, 9.2 mMNa2HP04, 1.3 mM NaH,PO,, pH 7.4; 5 mM EDTA). Thrombin-activated platelets were prepared as follows: unfixed washed platelets in PBS/EDTA were incubated for 30 minutes at 37°C at 3 X 10" platelets per mL; thrombin (3 UlmL) was added and the incubation was continued for 2 minutes without stirring. The platelets were then fixed with PFA (l%, vol/vol) and washed three times with PBS/EDTA. Platelet concentrates were prepared from blood collected into CPDA in polyvinylchloride/diethylhexyl phthalate bags (PVC/ D E W , Nederlands Productie Laboratorium voor Bloedtransfusie-apparatuur en Infusi Vloeistoffen B .V. [NPBI], EmmerCompascuum, The Netherlands). The bag was centrifuged (2960 x g, 10 min, room temperature) and both the plasma and the buffy coat were collected.22 After addition of 40 mL of plasma, the buffy coat was centrifuged again (380 X g, 6 min, room temperature). The supernatant (concentrated platelets in plasma) was expressed into an empty bag, as described previously.22 The white cell content of these PCs was lower than 1.0 X 10 , and the pH on Day 10 was >6.7. The PCs were stored at room temperature on a horizontal rotator (1 cycle/sec). During storage, small samples were taken aseptically via a sample site coupler (NPBI), and these samples were fixed with PFA (I%, vol/vol, final concentration), incubated for 15 minutes, and washed twice with PBSAZDTA. Fixed and washed platelets were diluted to a concentration of 3 X 10' per mL, and 20 p L of platelet suspension was incubated with 20 p L of antibody for 30 minutes at room temperature. We then washed the platelets three times with PBS/EDTA and incubated them with 20 WLof goat anti-mouse IgG conjugated with fluorescein isothiocyanate (G/M-FITC, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service,
21
Amsterdam, The Netherlands). The mixture was incubated in the dark for 30 minutes. We subsequently washed the platelets with PBS/EDTA (three times), resuspended them in 125 p L of PBS/EDTA, and stored them (maximally 24 h, 4"C, in the dark) for flow cytometry analysis.
Flow cytometry analysis Blood samples were analyzed in a flow cytometer (FACSCAN, Becton Dickinson, Mountain View, CA). The instrument is equipped with an argon laser and operated at 15 mW power at a wavelength of 488 nm. FITC fluorescence pass filter. The instrument was calibrated for fluorescence and light scatter daily with beads (Calibrite, Becton Dickinson). Cell samples were passed through the laser beam via a curette. Light scatter and fluorescence data were obtained with gain settings in the logarithmic mode, and the data were analyzed on a computer with the Consort 30 program (Becton Dickinson). The platelets were distinguished from the other cells on the basis of their forward and 90" light scatter profile. Debris or "machine noise" was excluded from the analysis by setting the appropriate forwardscatter threshold. A gate was set around the platelets, and 5000 cells were analyzed for FITC fluorescence to quantitate the amount of platelet-bound antibody. For MoAbs C8 and 2.28, the binding was expressed as the percentage of platelets that bound more of these MoAbs than of the control MoAb of the same IgG subclass. Resting platelets did not bind MoAb C8 or 2.28. The control MoAbs were directed against tissue plasminogen activator and were of the IgGl and IgG2b subclass. MoAbs C17 and Y2 had already bound to resting platelets (see Results), and therefore, antibody binding of C17 and Y2 could be expressed only as the mean fluorescence intensity (MFI).
Statistical evaluation Statistical evaluation was performed with a t test for paired (for the same concentration) and unpaired (between the different concentrations) observations.
Release of p-TG and leakage of LDH P-TG was measured with an RIA. We measured released P-TG in the supernatant of platelets after centrifugation for 5 minutes at 12,000 X g and stored the samples at -80°C until the measurements were performed. The activity of lactate dehydrogenase (LDH) was measured spectrophotometrically, as described previously.23 The total amount of LDH in the platelets and the LDH activity in the supernatant were used to calculate the percentage of leakage of LDH. The total amount of LDH was measured after lysis of the platelets with detergent Triton X-100 (l%, vol/vol).
Platelet morphology Swirling patterns were determined in PCs during storage.24 We visually observed the patterns after a brief squeeze along the low border of the plastic bag containing the PCs. The degree of inhomogeneity observed was scored according to the following scale: 3: swirling inhomogeneity visible throughout the whole bag, with contrast observable as fine detail; 2: swirling inhomogeneity visible throughout the bag with good contrast; 1: some visible inhomogeneity, but only in a few places and with poor contrast; and
FIJNHEER ET AL.
33
0: homogeneous turbidity, remaining the same before and after the bag has been squeezed. Swirling patterns were judged by one person throughout this study.
Results Binding of MoAbs to resting and activated platelets To study the binding of the MoAbs to nonactivated platelets, we fixed platelets immediately by blood collection in excess PFA (1 vol of blood in 9 vol of 1% PFA, vollvol). Platelets prepared from this mixture showed an MFI of 102 f 9 with MoAb C17 and of 116 f 10 with MoAb Y2. Of these platelets, 2.7 f 0.8 percent bound MoAb C8 and 1.7 f 0.43 percent bound MoAb 2.28 above the negative control. All of these values are the mean 2 SD of five experiments. Nonspecific MoAbs of the same subclass bound to less than 1 percent of the platelets. In thrombin-stimulated platelets, a marked increase in the binding of the MoAbs occurred: the MFI with MoAbs C17 and Y2 had increased to 302 f 4.4 and 300 f 34, respectively. The percentage of cells positive for MoAb C8 had increased to 82 f 5.8 percent and for MoAb 2.28 to 70 f 4.0 percent. These values also represent the mean h SD of five observations. Representative flow cytometric fluorescence patterns for the various MoAbs are shown in Fig. 1.
TRANSFUSION Vol. 30, No. I - I Y Y O
Effect of platelet storage on GP IIblIIIa complex and activation-dependent antigens We stored PCs for 10 days at a platelet concentration of 1.O X lo9 per mL. At regular intervals, we took samples and measured the leakage of LDH and the release of P-TG in the cell-free supernatant. The leakage of LDH was 7 f 1.5 percent on Day 0, with a P-TG concentration of 6 p,g per mL 2 1.0 in the supernatant. These values gradually increased to 17 f 4.1 percent for LDH and 30 f 2.3 p,g per mL for P-TG on Day 10 (Table 1). Swirling patterns were scored at 3 on Day 0, and they gradually decreased to a value of 2 on Day 7. The binding of MoAb C17 against the GP IIb/IIIa complex slightly increased during the first 5 days of storage (Fig. 2). On Day 5, we found an MFI of 168 f 21, which is significantly higher (p < 0.005) than the Day 0 value. Binding of MoAb Y2 against GP IIIa showed a similar increase during storage time. On Day 0, 3.4 f 0.5 percent of the platelets bound MoAb C8 against GMP 140 and 2.4 f 0.6 percent bound MoAb 2.28 against a lysosomal protein. This binding increased to 16 +- 1.3 percent and 7.5 f 1.7 percent, respectively, at Day 5, with a further increase till Day 10 (Fig. 2). All values after 3 days of storage were significantly different (p < 0.0025) from the Day 0 value. Control MoAbs bound to stored platelets as poorly as to freshly collected platelets (less than 1% positive platelets). To test whether the changes in the binding of the Mo4bs correlated with other in vitro parameters, we also studied PCs stored under suboptimal conditions. These PCs were prepared
L 1
0
ntigen : GMP140
a W
m
5z
LOG FLUORESCENCE
antigen . lysosome-like granule protein
-
FIG.I . The expression of MoAbs C17, Y2, C8, and 2.28 as measured with a flow cytometer. The marker is set on a nonspecific MoAb of the same subtyr. Platelets were fixed immediately by blood collection in 1 percent PFA (-) or following incubation for 30 minutes at 37°C with thrombin (1
UllO platelets (+).
IRANSFUSION 19YO-Vul. 30, No. 1
23
PLATELET ACTIVATION DURING STORAGE
Table 1. Leakage of LDH and release of P-TG from stored PCs' Days 3
5
7
10
f 1.1 9 f 2.5
7 f 2.0 18 f 3.0
11 f 3.9 25 f 2.3
17 f 4.1 30 f 2.1
14 f 2.0 28 f 3.6
20 f 1.9 35 f 4.5
22 f 2.1 39 f 4.1
0
Platelets stored at normal concentration (1.0 X 109/mL) LDH (YO leakage) 7 f 1.5 P-thromboglobulin (pg/mL) 6 f 1.0 Platelets stored at high concentration (1.4 X 109/mL) LDH (YOleakage) 8 f 2.5 0-thromboglobulin (pg/mL) 8 f 2.4
5
*
9 2.4 11 f 1.9
Changes in mean values f SD of five experiments.
with less plasma, which resulted in a concentration of 1.4 x lo9 platelets per mL. After 5 days of storage, LDH leakage and P-TG release were similar to the values found for optimally stored platelets after 10 days (Table I). The leakage of LDH increased to 22 2 2.1 percent on Day 10, and the concentration of P-TG in the supernatant was 39 2 4.1 pg per mL (Table 1). With the high concentrations of platelets, the swirling pattern was scored at 1 on Day 7 . The binding of MoAb C 17 increased during storage. The Day 1 MFI of 104 2 6 increased to 376 2 37 on Day 7. A similar increase in binding was found for MoAb Y2 (Fig. 2). The binding of MoAb C8 increased progressively after 3 days of storage. MoAb 2.28 followed the same pattern,
although the increase was somewhat less pronounced (Fig. 2). The binding of MoAbs to platelets stored at a high concentration was significantly different on Days 5 and 7 from the binding of Mohbs to platelets stored at normal concentrations (p < 0.005).
Discussion Our data demonstrate changes in the platelet surface during the storage of PCs. For the GP IIb/IIIa complex, i t is known that platelets prepared from freshly collected blood expose the fibrinogen receptor upon stimulation
500
100
400
"1
901
Ag: G M P 1 4 0
70
300
200
40
30
! I
100
; I
V
2
W
1
0
3
1
V
.NORMAL
v) W
a
+
0
7
5
10
0
1
CONCENTRATION
3
7
5
10
0 - NORMAL CONCENTRATION
H I G H CONCENTRATION
+-
u.
3
3 2
H I G H CONCENTRATION
Y
z
g-
a
7
W
400
loo[ 90
$Ag:
LYSOSOME-LIKE GRANULE PROTEIN
60-
T
5040
-
30 20.L I
1
0
1
3
5
7
10
0
1
3
7
5
10
STORAGE TIME (DAYS)
FIG.2. Platelet activation during storage. Platelet concentrates were stored for 10 days at aconcentration of I .O X lo9 per mL ( 0 - 0 ) or 1.4 x lo9 per mL (*-*). Aseptic samples were taken at Days 0, I , 3,5,7, and 10. The platelets were fixed immediately in I percent PFA. After incubation for 15 minutes at room temperature, the platelets were washed and stained by the addition of MoAbs and G/M FITC. For MoAb C 17 (GP IIb/lIIa) and MoAb Y2 (GP Ma), the mean fluorescence was used. For MoAb C8 (GMP140 from the &-granules) and MoAb 2.28 (lysosome-like antigen), the percentage of positive cells above the negative control (nonspecific MoAb of the same subclass) was used. Values are the mean SD of five experiments.
*
24
TRANSFUSION
FIJNHEER ET AL.
with a variety of aggregating agents.25 We analyzed the changes occurring during activation with thrombin and during storage of platelet concentrates. The binding of both MoAb C17, which binds to GP IIIa, when GP IIb-IIIa is a complex, and MoAb Y2, which binds to GP IIIa alone, increased after thrombin stimulation. For these MoAbs, the MFI increased about threefold compared to that with platelets fixed immediately after blood collection in excess PFA. These results suggest that GP IIb/IIIa is present on the surface of resting platelets as a complex and increases after thrombin stimulation. Indeed, Woods et a1.26 reported an intracellular pool of GP IIb/IIIa in resting platelets, which can be exposed after thrombin stimulation. Gogstad et al.27have suggested the presence of a significant pool of a-granule-associated GP IIb/IIIa complex. In PCs stored under normal conditions, we found a significant increase in MoAb binding after 5 days of storage, which indicated GP IIb/IIIa exposure after this period. P-TG release, LDH leakage, and swirling patterns showed that the condition of the platelets deteriorated from that time. MoAbs C17 and Y2 both increased at the same rate, so no evidence was found for dissociation of the GP IIb/IIIa complex during storage. MoAbs C8 and 2.28 both bind to thrombin-activated platelets. C8 reacts with a 140-kD protein from the a-granules, which is probably the same antigen as the target of antibody S12. l 1 This a-granule membrane glycoprotein becomes exposed on the platelet membrane concurrent with granule secretion. MoAb 2.28 is directed against a 53-kD protein probably derived from the l y ~ o ~ o m which e ~ , ~is~ also expressed on the platelet plasma membrane after stimulation. During thrombin activation, a clear increase in the binding of these MoAbs occurs, although a subpopulation of platelets did not show this phenomenon (of the platelets, about 20% for MoAb C8 and 10%for MoAb 2.28). These results may represent heterogeneity of platelet granule content28or may be due to platelet aging in the circulation with a gradual loss of membrane responsivene~s.~~ During storage of PCs under optimal conditions (at a concentration of about 1.0 X 109/mL),binding of both MoAbs C8 and 2.28 increased during storage time, and on Day 5, this increase was significant compared to Day 0. Recently, George et al.30 also reported an increase of GMP-140 on the platelet surface during storage. To investigate whether there is a correlation between the condition of the platelets and MoAb binding, we compared PCs stored at 1.O X lo9and 1.4 X 199 per mL. The PCs stored at high concentrations showed clear deterioration of the platelets between Days 3 and 5, as judged by the increase in the release of P-TG, the percentage of leakage of LDH, and a decrease in swirling patterns, whereas the PCs stored at normal concentrations showed this deterioration between Days 5 and 7. Both the
Vol. 30, No. 1--1990
MoAb directed against GP IIb/IIIa complex and the activation-dependent MoAb showed a significant increase in binding compared to that for platelets stored under optimal conditions. Thus, this study shows clear platelet activation during storage of platelets in PCs, as measured by the increased accessibility of the GP IIb/IIIa complex and a-granule and lysosome release. The immediate fixation of the platelets and analysis of the antigen expression by flow cytometry provide a test system for measuring platelet activation during storage. Other applications will be the monitoring of activation during the preparation of platelets or during platelet filtration and the determination of platelet activation as a reflection of disease activity. l5
References 1. Kunicki TJ, Tuccelli M, Becker GA, Aster RH. A study of
variables affecting the quality of platelets stored at “room temperature.”Transfusion 1975;15:414-21. 2. Holme S, Vaidja K, Murphy S. Platelet storage at 22°C: effect of type of agitation on morphology, viability, and function in vitro. Blood 1978;52:425-35. 3. DiMinno G, Silver MJ, Murphy S. Stored human platelets retain full aggregation potential in response to pairs of aggregating agents. Blood 1982;59:563-8. 4. Fijnheer R, Pietersz RNI, de Korte D, Roos D. Monitoring of platelet morphology during storage of platelet concentrates. Transfusion 1989;29:36-40. 5 . Filip DJ, Eckstein JD, Sibley CA. The effect of platelet concentrate storage temprature on adenine nucleotide metabolism. Blood 1975;45:749-56. 6. Kim BK, Baldini MG. Glycolytic intermediates and adenine nucleotides of human platelets. 11. Effect of short-term storage at 4°C. Transfusion 1972;12:1-8. 7. George JN. Platelet membrane glycoproteins: alteration during storage of human platelet concentrates. Thromb Res 1976; 8:719-24. 8. Lucas RC, Lawrence J, Stracher A. On the preservation of contractile proteins during storage of human platelets. Blood 1981;57:1005-10. 9. Dhar A, Ganguly P. Altered expression of platelet surface glycoproteins during storage. Br J Haematol 1988;70:71-5. 10. Nugent DJ, Kunicki TJ, Berglund C, Bernstein D. A human monoclonal autoantibody recognizes a neoantigen on glycoprotein IIIa expressed on stored and activated platelets. Blood 1987;70:16-22. 1 1 . McEver RP, Martin MN. A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 1984;259:9799-804. 12. Hus-Lin SC, Berman CL, Furie BC, August D, Furie B. A platelet membrane protein expressed during platelet activation and secretion. J Biol Chem 1984;259:9121-6. 13. Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha-granule membrane protein (GMP- 140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 8 8 0 6 . 14. Berman CL, Yeo EL, Wencel-Drake JD, Furie BC, Ginsburg MH, Furie B. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein. J Clin Invest 1986;78:130-7. 15. Nieuwenhuis HK, van Oosterhout JJ, Rozemuller E, van Iwaarden F, Sixma JJ. Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. Blood 1987;70:838-45.
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PLATELET ACTIVATION DURING STORAGE
16. Kunicki TJ, Pidard D, Rosa JP, Nurden A. The formation of Ca++-dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution determined by crossed immunoelectrophoresis. Blood 1981;58:268-78. 17. Fitzgerald L, Phillips DR. Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex. J Biol Chem 1985;260:11366-74. 18. Tetteroo PAT, Lansdorp PM, Leeksma OC, von dem Borne AEG. Monoclonal antibodies against human platelet glycoprotein IIIa. Br J Haematol 1983;55:509-22. 19. Modderman PW, Huisman JG, van Mourik JA, von dem Borne AEGK. A monoclonal antibody to the human platelet glycoprotein IIb/IIIa complex induces platelet activation. Thromb Haemost 1988;60:68-74. 20. Pidard D, Didry D, Kunicki TJ, Nurden AT. Temperaturedependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability. Blood 1986;67:604-11. 21. Modderman PW, van Mourik JA, van Berkel W, et al. Decreased stability of the residual platelet glycoprotein IIb/IIIa complex in Glanzmann’s thrombasthenia. Br J Haematol, in press. 22. Pietersz RNI, Loos JA, Reesink HW. Platelet concentrates stored in plasma for 72 hours at 22°C prepared from buffycoats of citratephosphate-dextrose blood collected in a quadruple-bag salineadenine-glucose-mannitolsystem. Vox Sang 1985;49:81-5. 23. Noll F. Determinations with LDH, GPT and NAD. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis, vol3. Weinheim: Chemie Verlag, 19741475-9. 24. Fratantoni JC, Poindexter BJ, Bonner RF. Quantitative assessment of platelet morphology by light scattering: apotential method for the evaluation of platelets for transfusion. J Lab Clin Med 1984;103:620-3 1. 25. Di Minno G, Thiagarajan P, Perussia B, et al. Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex. Blood 1983;61:140-8. 26. Woods VL, Wolff LE, Keller DM. Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins. J Biol Chem 1986;261:15242-51.
25
27. Gogstad GO, Hagen I, Korsmo R, Solum NO. Characterization of the proteins of isolated human platelet a-granules: evidence for a separate a-granule pool of the glycoproteins 11, and 111,. Biochim Biophys Acta 1981;670:150-62. 28. Johnston GI, Picket EB, McEver RP, George JN. Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometq. Blood 1987;69:1401-3. 29. Corash L, Tan H, Gralnick HR. Heterogeneity of human whole blood platelet subpopulations. I. Relationship between buoyant density, cell volume, and ultrastructure. Blood 1977;49:71-87. 30. George JN, Pickett EB, Heinz R. Platelet membrane glycoprotein changes during the preparation and storage of platelet concentrates. Transfusion 1988;28:123-6.
Rob Fijnheer, MD, Research Associate, Department of Blood Cell Chemistry, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, University of Amsterdam, Amsterdam, The Netherlands. Piet W. Modderman, PhD, Research Fellow, Department of Immunohematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. Henk Veldman, technician, Department of Blood Cell Chemistry, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. Willem H. Ouwehand, MD, PhD, Specialist in Immunohematology, Department of Immunohematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. H. Karel Nieuwenhuis, MD, PhD, Specialist in Internal Medicine, University Hospital Utrecht, Utrecht, The Netherlands. Dirk Roos, PhD, Head of Department of Blood Cell Chemistry, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. Dirk de Korte, PhD, Research Fellow, Department of Blood Cell Chemistry, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. [no reprints available]
the plasma membrane. As a result of this process, a 140-kD a-granule protein (GMP 140) appears on the plasma membrane.''-'4 GMP 140 is a useful marker for platelet activation, because its surface expression correlates with the release of a-granule content, and it is not reinternalized following secretion. Moreover, Nieuwenhuis et al.I5 found that a 53-kD protein from lysosomelike granules is also exposed on the surface of activated platelets. GP IIb and IIIa form a calcium-dependent complex in the membrane of resting platelet^.'^^'^ On platelets stimulated with agonists such as ADP or thrombin, the GP IIb/IIIa complex functions as a receptor for fibrinogen and other adhesive proteins. The binding of fibrinogen to activated platelets mediates platelet aggregation. As the storage of platelets results in a decrease in the capacity of the platelets to aggregate upon stimulation with ADP or thrombin, changes in the expression of the GP IIb/IIIa complex may reflect the functional condition of stored platelets. From experiments with MoAbs against the GP IIb/IIIa complex and against activation-dependent antigens, we conclude that platelets become activated during storage in platelet concentrates (PCs).
NUMEROUSR E P O R T S ' ~ have demonstrated a progressive decline in platelet function during storage. These changes may be associated with changes in glycolysis, loss of membrane components, alteration in contractile proteins, decreased levels of metabolic ATP, and altered expression of platelet surface glyc~proteins.~-~ Variables such as P-thromboglobulin (P-TG) release, morphologic changes, and the progressive expression of an activationdependent marker" suggest that the platelets become activated during storage. With the availability of monoclonal antibodies (MoAbs) against platelet surface glycoproteins, the investigation of these glycoproteins becomes possible. To investigate whether the loss of platelet condition during storage correlates with changes in membrane glycoprotein (GP) IIb/IIIa complex and the activation-dependent antigens, we studied the expression of these glycoproteins on the platelet surface during storage. One of the changes occurring at the platelet surface during activation is the fusion of the a-granule membrane with
From the Military Blood Transfusion Service, Amsterdam; the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and the Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam; and the University Hospital Utrecht, Utrecht, The Netherlands. Supported by a grant from Netherlands Laboratory for the Production of Blood Transfusion Equipment and Infusion Fluids (NPBI, B.V.), EmmerCompascuum, The Netherlands. Received for publication January 10, 1989;revision received June 19, 1989, and accepted June 19, 1989.
Materials and Methods The following materials were obtained commercially: bovine serum albumin (BSA; Organon Teknika B.V., Oss, The Netherlands), human a-thrombin (Sigma Chemical Company, St. Louis, MO), and a P-TG radioimmunoassay (RIA) kit (The Radiochemical Centre, Amersham, UK).
20
TRANSFUSION 1990-Vol. 30. No. 1
PLATELET ACTIVATION DURING STORAGE
Monoclonal antibodies MoAb C17 (CDw41), a previously described'" MoAb, reacts with GP IIIa when GP IIb-IIIa is a complex and inhibits platelet aggregation and the binding of fibrinogen to ADP- or collagenactivated platelets. The antibody is complex specific, i.e., it does not bind to GP IIb or to GP IIIa after EDTA-induced dissociation of the GP IIb/IIIa complex.'g*20 MoAb Y2 was obtained commercially (Dakopatts, Copenhagen, Denmark). It binds to GP IIIa and is not complex specific.21 MoAb C8 is of the IgGl subclass and recognizes a 140 kD glycoprotein that becomes associated with the platelet surface during secretion. This glycoprotein has previously been designated platelet activation-dependent granule-external membrane (PADGEM) protein, l 4 or granule membrane protein, (GMP 140).13 MoAb 2.28 is of the IgG2b subclass and was prepared as described. l5 This MoAb reacts with a 53-kD protein located in a subclass of platelet granules, probably lysosomes. The antigen is expressed on the platelet membrane upon thrombin stimulation. We used concentrations of MoAbs that gave maximal binding to the platelets.
Preparation of the platelets Blood samples were obtained from healthy volunteers. Platelet-rich plasma (PRP) was obtained by centrifugation (280 X g for 15 min at room temperature) of blood anticoagulated with CPDA in plastic tubes or from blood fixed immediately by collection in excess paraformaldehyde (PFA, 1% vol/vol, final concentration) and incubated for 15 minutes at room temperature. Platelets were pelleted from PRP by centrifugation (2200 X g, 7 min) and washed three times with PBS/EDTA (PBS: 140 mM NaCI, 9.2 mMNa2HP04, 1.3 mM NaH,PO,, pH 7.4; 5 mM EDTA). Thrombin-activated platelets were prepared as follows: unfixed washed platelets in PBS/EDTA were incubated for 30 minutes at 37°C at 3 X 10" platelets per mL; thrombin (3 UlmL) was added and the incubation was continued for 2 minutes without stirring. The platelets were then fixed with PFA (l%, vol/vol) and washed three times with PBS/EDTA. Platelet concentrates were prepared from blood collected into CPDA in polyvinylchloride/diethylhexyl phthalate bags (PVC/ D E W , Nederlands Productie Laboratorium voor Bloedtransfusie-apparatuur en Infusi Vloeistoffen B .V. [NPBI], EmmerCompascuum, The Netherlands). The bag was centrifuged (2960 x g, 10 min, room temperature) and both the plasma and the buffy coat were collected.22 After addition of 40 mL of plasma, the buffy coat was centrifuged again (380 X g, 6 min, room temperature). The supernatant (concentrated platelets in plasma) was expressed into an empty bag, as described previously.22 The white cell content of these PCs was lower than 1.0 X 10 , and the pH on Day 10 was >6.7. The PCs were stored at room temperature on a horizontal rotator (1 cycle/sec). During storage, small samples were taken aseptically via a sample site coupler (NPBI), and these samples were fixed with PFA (I%, vol/vol, final concentration), incubated for 15 minutes, and washed twice with PBSAZDTA. Fixed and washed platelets were diluted to a concentration of 3 X 10' per mL, and 20 p L of platelet suspension was incubated with 20 p L of antibody for 30 minutes at room temperature. We then washed the platelets three times with PBS/EDTA and incubated them with 20 WLof goat anti-mouse IgG conjugated with fluorescein isothiocyanate (G/M-FITC, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service,
21
Amsterdam, The Netherlands). The mixture was incubated in the dark for 30 minutes. We subsequently washed the platelets with PBS/EDTA (three times), resuspended them in 125 p L of PBS/EDTA, and stored them (maximally 24 h, 4"C, in the dark) for flow cytometry analysis.
Flow cytometry analysis Blood samples were analyzed in a flow cytometer (FACSCAN, Becton Dickinson, Mountain View, CA). The instrument is equipped with an argon laser and operated at 15 mW power at a wavelength of 488 nm. FITC fluorescence pass filter. The instrument was calibrated for fluorescence and light scatter daily with beads (Calibrite, Becton Dickinson). Cell samples were passed through the laser beam via a curette. Light scatter and fluorescence data were obtained with gain settings in the logarithmic mode, and the data were analyzed on a computer with the Consort 30 program (Becton Dickinson). The platelets were distinguished from the other cells on the basis of their forward and 90" light scatter profile. Debris or "machine noise" was excluded from the analysis by setting the appropriate forwardscatter threshold. A gate was set around the platelets, and 5000 cells were analyzed for FITC fluorescence to quantitate the amount of platelet-bound antibody. For MoAbs C8 and 2.28, the binding was expressed as the percentage of platelets that bound more of these MoAbs than of the control MoAb of the same IgG subclass. Resting platelets did not bind MoAb C8 or 2.28. The control MoAbs were directed against tissue plasminogen activator and were of the IgGl and IgG2b subclass. MoAbs C17 and Y2 had already bound to resting platelets (see Results), and therefore, antibody binding of C17 and Y2 could be expressed only as the mean fluorescence intensity (MFI).
Statistical evaluation Statistical evaluation was performed with a t test for paired (for the same concentration) and unpaired (between the different concentrations) observations.
Release of p-TG and leakage of LDH P-TG was measured with an RIA. We measured released P-TG in the supernatant of platelets after centrifugation for 5 minutes at 12,000 X g and stored the samples at -80°C until the measurements were performed. The activity of lactate dehydrogenase (LDH) was measured spectrophotometrically, as described previously.23 The total amount of LDH in the platelets and the LDH activity in the supernatant were used to calculate the percentage of leakage of LDH. The total amount of LDH was measured after lysis of the platelets with detergent Triton X-100 (l%, vol/vol).
Platelet morphology Swirling patterns were determined in PCs during storage.24 We visually observed the patterns after a brief squeeze along the low border of the plastic bag containing the PCs. The degree of inhomogeneity observed was scored according to the following scale: 3: swirling inhomogeneity visible throughout the whole bag, with contrast observable as fine detail; 2: swirling inhomogeneity visible throughout the bag with good contrast; 1: some visible inhomogeneity, but only in a few places and with poor contrast; and
FIJNHEER ET AL.
33
0: homogeneous turbidity, remaining the same before and after the bag has been squeezed. Swirling patterns were judged by one person throughout this study.
Results Binding of MoAbs to resting and activated platelets To study the binding of the MoAbs to nonactivated platelets, we fixed platelets immediately by blood collection in excess PFA (1 vol of blood in 9 vol of 1% PFA, vollvol). Platelets prepared from this mixture showed an MFI of 102 f 9 with MoAb C17 and of 116 f 10 with MoAb Y2. Of these platelets, 2.7 f 0.8 percent bound MoAb C8 and 1.7 f 0.43 percent bound MoAb 2.28 above the negative control. All of these values are the mean 2 SD of five experiments. Nonspecific MoAbs of the same subclass bound to less than 1 percent of the platelets. In thrombin-stimulated platelets, a marked increase in the binding of the MoAbs occurred: the MFI with MoAbs C17 and Y2 had increased to 302 f 4.4 and 300 f 34, respectively. The percentage of cells positive for MoAb C8 had increased to 82 f 5.8 percent and for MoAb 2.28 to 70 f 4.0 percent. These values also represent the mean h SD of five observations. Representative flow cytometric fluorescence patterns for the various MoAbs are shown in Fig. 1.
TRANSFUSION Vol. 30, No. I - I Y Y O
Effect of platelet storage on GP IIblIIIa complex and activation-dependent antigens We stored PCs for 10 days at a platelet concentration of 1.O X lo9 per mL. At regular intervals, we took samples and measured the leakage of LDH and the release of P-TG in the cell-free supernatant. The leakage of LDH was 7 f 1.5 percent on Day 0, with a P-TG concentration of 6 p,g per mL 2 1.0 in the supernatant. These values gradually increased to 17 f 4.1 percent for LDH and 30 f 2.3 p,g per mL for P-TG on Day 10 (Table 1). Swirling patterns were scored at 3 on Day 0, and they gradually decreased to a value of 2 on Day 7. The binding of MoAb C17 against the GP IIb/IIIa complex slightly increased during the first 5 days of storage (Fig. 2). On Day 5, we found an MFI of 168 f 21, which is significantly higher (p < 0.005) than the Day 0 value. Binding of MoAb Y2 against GP IIIa showed a similar increase during storage time. On Day 0, 3.4 f 0.5 percent of the platelets bound MoAb C8 against GMP 140 and 2.4 f 0.6 percent bound MoAb 2.28 against a lysosomal protein. This binding increased to 16 +- 1.3 percent and 7.5 f 1.7 percent, respectively, at Day 5, with a further increase till Day 10 (Fig. 2). All values after 3 days of storage were significantly different (p < 0.0025) from the Day 0 value. Control MoAbs bound to stored platelets as poorly as to freshly collected platelets (less than 1% positive platelets). To test whether the changes in the binding of the Mo4bs correlated with other in vitro parameters, we also studied PCs stored under suboptimal conditions. These PCs were prepared
L 1
0
ntigen : GMP140
a W
m
5z
LOG FLUORESCENCE
antigen . lysosome-like granule protein
-
FIG.I . The expression of MoAbs C17, Y2, C8, and 2.28 as measured with a flow cytometer. The marker is set on a nonspecific MoAb of the same subtyr. Platelets were fixed immediately by blood collection in 1 percent PFA (-) or following incubation for 30 minutes at 37°C with thrombin (1
UllO platelets (+).
IRANSFUSION 19YO-Vul. 30, No. 1
23
PLATELET ACTIVATION DURING STORAGE
Table 1. Leakage of LDH and release of P-TG from stored PCs' Days 3
5
7
10
f 1.1 9 f 2.5
7 f 2.0 18 f 3.0
11 f 3.9 25 f 2.3
17 f 4.1 30 f 2.1
14 f 2.0 28 f 3.6
20 f 1.9 35 f 4.5
22 f 2.1 39 f 4.1
0
Platelets stored at normal concentration (1.0 X 109/mL) LDH (YO leakage) 7 f 1.5 P-thromboglobulin (pg/mL) 6 f 1.0 Platelets stored at high concentration (1.4 X 109/mL) LDH (YOleakage) 8 f 2.5 0-thromboglobulin (pg/mL) 8 f 2.4
5
*
9 2.4 11 f 1.9
Changes in mean values f SD of five experiments.
with less plasma, which resulted in a concentration of 1.4 x lo9 platelets per mL. After 5 days of storage, LDH leakage and P-TG release were similar to the values found for optimally stored platelets after 10 days (Table I). The leakage of LDH increased to 22 2 2.1 percent on Day 10, and the concentration of P-TG in the supernatant was 39 2 4.1 pg per mL (Table 1). With the high concentrations of platelets, the swirling pattern was scored at 1 on Day 7 . The binding of MoAb C 17 increased during storage. The Day 1 MFI of 104 2 6 increased to 376 2 37 on Day 7. A similar increase in binding was found for MoAb Y2 (Fig. 2). The binding of MoAb C8 increased progressively after 3 days of storage. MoAb 2.28 followed the same pattern,
although the increase was somewhat less pronounced (Fig. 2). The binding of MoAbs to platelets stored at a high concentration was significantly different on Days 5 and 7 from the binding of Mohbs to platelets stored at normal concentrations (p < 0.005).
Discussion Our data demonstrate changes in the platelet surface during the storage of PCs. For the GP IIb/IIIa complex, i t is known that platelets prepared from freshly collected blood expose the fibrinogen receptor upon stimulation
500
100
400
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901
Ag: G M P 1 4 0
70
300
200
40
30
! I
100
; I
V
2
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1
0
3
1
V
.NORMAL
v) W
a
+
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7
5
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1
CONCENTRATION
3
7
5
10
0 - NORMAL CONCENTRATION
H I G H CONCENTRATION
+-
u.
3
3 2
H I G H CONCENTRATION
Y
z
g-
a
7
W
400
loo[ 90
$Ag:
LYSOSOME-LIKE GRANULE PROTEIN
60-
T
5040
-
30 20.L I
1
0
1
3
5
7
10
0
1
3
7
5
10
STORAGE TIME (DAYS)
FIG.2. Platelet activation during storage. Platelet concentrates were stored for 10 days at aconcentration of I .O X lo9 per mL ( 0 - 0 ) or 1.4 x lo9 per mL (*-*). Aseptic samples were taken at Days 0, I , 3,5,7, and 10. The platelets were fixed immediately in I percent PFA. After incubation for 15 minutes at room temperature, the platelets were washed and stained by the addition of MoAbs and G/M FITC. For MoAb C 17 (GP IIb/lIIa) and MoAb Y2 (GP Ma), the mean fluorescence was used. For MoAb C8 (GMP140 from the &-granules) and MoAb 2.28 (lysosome-like antigen), the percentage of positive cells above the negative control (nonspecific MoAb of the same subclass) was used. Values are the mean SD of five experiments.
*
24
TRANSFUSION
FIJNHEER ET AL.
with a variety of aggregating agents.25 We analyzed the changes occurring during activation with thrombin and during storage of platelet concentrates. The binding of both MoAb C17, which binds to GP IIIa, when GP IIb-IIIa is a complex, and MoAb Y2, which binds to GP IIIa alone, increased after thrombin stimulation. For these MoAbs, the MFI increased about threefold compared to that with platelets fixed immediately after blood collection in excess PFA. These results suggest that GP IIb/IIIa is present on the surface of resting platelets as a complex and increases after thrombin stimulation. Indeed, Woods et a1.26 reported an intracellular pool of GP IIb/IIIa in resting platelets, which can be exposed after thrombin stimulation. Gogstad et al.27have suggested the presence of a significant pool of a-granule-associated GP IIb/IIIa complex. In PCs stored under normal conditions, we found a significant increase in MoAb binding after 5 days of storage, which indicated GP IIb/IIIa exposure after this period. P-TG release, LDH leakage, and swirling patterns showed that the condition of the platelets deteriorated from that time. MoAbs C17 and Y2 both increased at the same rate, so no evidence was found for dissociation of the GP IIb/IIIa complex during storage. MoAbs C8 and 2.28 both bind to thrombin-activated platelets. C8 reacts with a 140-kD protein from the a-granules, which is probably the same antigen as the target of antibody S12. l 1 This a-granule membrane glycoprotein becomes exposed on the platelet membrane concurrent with granule secretion. MoAb 2.28 is directed against a 53-kD protein probably derived from the l y ~ o ~ o m which e ~ , ~is~ also expressed on the platelet plasma membrane after stimulation. During thrombin activation, a clear increase in the binding of these MoAbs occurs, although a subpopulation of platelets did not show this phenomenon (of the platelets, about 20% for MoAb C8 and 10%for MoAb 2.28). These results may represent heterogeneity of platelet granule content28or may be due to platelet aging in the circulation with a gradual loss of membrane responsivene~s.~~ During storage of PCs under optimal conditions (at a concentration of about 1.0 X 109/mL),binding of both MoAbs C8 and 2.28 increased during storage time, and on Day 5, this increase was significant compared to Day 0. Recently, George et al.30 also reported an increase of GMP-140 on the platelet surface during storage. To investigate whether there is a correlation between the condition of the platelets and MoAb binding, we compared PCs stored at 1.O X lo9and 1.4 X 199 per mL. The PCs stored at high concentrations showed clear deterioration of the platelets between Days 3 and 5, as judged by the increase in the release of P-TG, the percentage of leakage of LDH, and a decrease in swirling patterns, whereas the PCs stored at normal concentrations showed this deterioration between Days 5 and 7. Both the
Vol. 30, No. 1--1990
MoAb directed against GP IIb/IIIa complex and the activation-dependent MoAb showed a significant increase in binding compared to that for platelets stored under optimal conditions. Thus, this study shows clear platelet activation during storage of platelets in PCs, as measured by the increased accessibility of the GP IIb/IIIa complex and a-granule and lysosome release. The immediate fixation of the platelets and analysis of the antigen expression by flow cytometry provide a test system for measuring platelet activation during storage. Other applications will be the monitoring of activation during the preparation of platelets or during platelet filtration and the determination of platelet activation as a reflection of disease activity. l5
References 1. Kunicki TJ, Tuccelli M, Becker GA, Aster RH. A study of
variables affecting the quality of platelets stored at “room temperature.”Transfusion 1975;15:414-21. 2. Holme S, Vaidja K, Murphy S. Platelet storage at 22°C: effect of type of agitation on morphology, viability, and function in vitro. Blood 1978;52:425-35. 3. DiMinno G, Silver MJ, Murphy S. Stored human platelets retain full aggregation potential in response to pairs of aggregating agents. Blood 1982;59:563-8. 4. Fijnheer R, Pietersz RNI, de Korte D, Roos D. Monitoring of platelet morphology during storage of platelet concentrates. Transfusion 1989;29:36-40. 5 . Filip DJ, Eckstein JD, Sibley CA. The effect of platelet concentrate storage temprature on adenine nucleotide metabolism. Blood 1975;45:749-56. 6. Kim BK, Baldini MG. Glycolytic intermediates and adenine nucleotides of human platelets. 11. Effect of short-term storage at 4°C. Transfusion 1972;12:1-8. 7. George JN. Platelet membrane glycoproteins: alteration during storage of human platelet concentrates. Thromb Res 1976; 8:719-24. 8. Lucas RC, Lawrence J, Stracher A. On the preservation of contractile proteins during storage of human platelets. Blood 1981;57:1005-10. 9. Dhar A, Ganguly P. Altered expression of platelet surface glycoproteins during storage. Br J Haematol 1988;70:71-5. 10. Nugent DJ, Kunicki TJ, Berglund C, Bernstein D. A human monoclonal autoantibody recognizes a neoantigen on glycoprotein IIIa expressed on stored and activated platelets. Blood 1987;70:16-22. 1 1 . McEver RP, Martin MN. A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 1984;259:9799-804. 12. Hus-Lin SC, Berman CL, Furie BC, August D, Furie B. A platelet membrane protein expressed during platelet activation and secretion. J Biol Chem 1984;259:9121-6. 13. Stenberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha-granule membrane protein (GMP- 140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 8 8 0 6 . 14. Berman CL, Yeo EL, Wencel-Drake JD, Furie BC, Ginsburg MH, Furie B. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein. J Clin Invest 1986;78:130-7. 15. Nieuwenhuis HK, van Oosterhout JJ, Rozemuller E, van Iwaarden F, Sixma JJ. Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. Blood 1987;70:838-45.
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PLATELET ACTIVATION DURING STORAGE
16. Kunicki TJ, Pidard D, Rosa JP, Nurden A. The formation of Ca++-dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution determined by crossed immunoelectrophoresis. Blood 1981;58:268-78. 17. Fitzgerald L, Phillips DR. Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex. J Biol Chem 1985;260:11366-74. 18. Tetteroo PAT, Lansdorp PM, Leeksma OC, von dem Borne AEG. Monoclonal antibodies against human platelet glycoprotein IIIa. Br J Haematol 1983;55:509-22. 19. Modderman PW, Huisman JG, van Mourik JA, von dem Borne AEGK. A monoclonal antibody to the human platelet glycoprotein IIb/IIIa complex induces platelet activation. Thromb Haemost 1988;60:68-74. 20. Pidard D, Didry D, Kunicki TJ, Nurden AT. Temperaturedependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability. Blood 1986;67:604-11. 21. Modderman PW, van Mourik JA, van Berkel W, et al. Decreased stability of the residual platelet glycoprotein IIb/IIIa complex in Glanzmann’s thrombasthenia. Br J Haematol, in press. 22. Pietersz RNI, Loos JA, Reesink HW. Platelet concentrates stored in plasma for 72 hours at 22°C prepared from buffycoats of citratephosphate-dextrose blood collected in a quadruple-bag salineadenine-glucose-mannitolsystem. Vox Sang 1985;49:81-5. 23. Noll F. Determinations with LDH, GPT and NAD. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis, vol3. Weinheim: Chemie Verlag, 19741475-9. 24. Fratantoni JC, Poindexter BJ, Bonner RF. Quantitative assessment of platelet morphology by light scattering: apotential method for the evaluation of platelets for transfusion. J Lab Clin Med 1984;103:620-3 1. 25. Di Minno G, Thiagarajan P, Perussia B, et al. Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex. Blood 1983;61:140-8. 26. Woods VL, Wolff LE, Keller DM. Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins. J Biol Chem 1986;261:15242-51.
25
27. Gogstad GO, Hagen I, Korsmo R, Solum NO. Characterization of the proteins of isolated human platelet a-granules: evidence for a separate a-granule pool of the glycoproteins 11, and 111,. Biochim Biophys Acta 1981;670:150-62. 28. Johnston GI, Picket EB, McEver RP, George JN. Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometq. Blood 1987;69:1401-3. 29. Corash L, Tan H, Gralnick HR. Heterogeneity of human whole blood platelet subpopulations. I. Relationship between buoyant density, cell volume, and ultrastructure. Blood 1977;49:71-87. 30. George JN, Pickett EB, Heinz R. Platelet membrane glycoprotein changes during the preparation and storage of platelet concentrates. Transfusion 1988;28:123-6.
Rob Fijnheer, MD, Research Associate, Department of Blood Cell Chemistry, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, University of Amsterdam, Amsterdam, The Netherlands. Piet W. Modderman, PhD, Research Fellow, Department of Immunohematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. Henk Veldman, technician, Department of Blood Cell Chemistry, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. Willem H. Ouwehand, MD, PhD, Specialist in Immunohematology, Department of Immunohematology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. H. Karel Nieuwenhuis, MD, PhD, Specialist in Internal Medicine, University Hospital Utrecht, Utrecht, The Netherlands. Dirk Roos, PhD, Head of Department of Blood Cell Chemistry, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. Dirk de Korte, PhD, Research Fellow, Department of Blood Cell Chemistry, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service. [no reprints available]
