Ann Hematol (1992) 64:292-298
Hematology 9 Springer-Verlag 1992
Original article Stabilization of standard platelet concentrates and minimization of the platelet storage lesion by a prostacyclin analogue M. Elias ~, A. Heethuis ~, M. Weggemans 1, V. Born 2, N. Blom 2, R.L. McShine 1, M . R . Halie 2, and C.T. Smit Sibinga 1 i Red Cross Blood Bank Groningen-Drenthe, Groningen, The Netherlands; 2 Dept. of Hematology, University of Groningen, The Netherlands Received October 4, 1991/Accepted March 26, 1992
Summary. Platelet concentrates were pretreated with a
stable synthetic prostacyclin analogue (Iloprost) at two different concentrations before the second centrifugation step (pelleting step) of preparation. This resulted in loss of platelet sensitivity to aggregating agents. To mimic the situation after transfusion and to assess the reversibility o f platelet inhibition, platelets were washed during and after storage and resuspended in fresh-frozen autologous plasma. The Iloprost-treated and washed platelets exhibited an increased sensitivity to the aggregating agents, compared with the control platelets (p < 0.01). Post-storage recovery of the synergistic aggregation was more than 80% of prestorage aggregation. 13-thromboglobulin (13TG) release and thromboxane B2 (TXB2) formation were significantly inhibited in Iloprost-treated platelets (p < 0.01). After the second centrifugation step, 15TG release was 0.7% + 0.3%, compared with 2.7% + 0.9% for the controls. TXB2 was 99 + 91 pg/ml, compared with 495 + 356 pg/ml for the controls. Platelet morphology and ultrastructure were well preserved during 5-day storage. In addition, Iloprost exerted a cytoprotective effect, as evidenced by the significant reduction in lactate dehydrogenase leakage. Post-storage L D H was 378 +_ 159 and 415 _+ 239 U/1 respectively by the two Iloprost concentrations, compared with 1180 + 937 U/1 for the control platelets. The inhibitory and cytoprotective effects of Iloprost were sustained throughout storage, in contrast to the effect of PGE1 (Prostin) which was limited to the early phase of storage.
The concept of preventing ex vivo platelet activation dates back to the 1960s and 1970s. Methods attempted [7, 19, 25, 30], such as pH lowering, C a + + and M g + + exclusion, or the addition of chelating agents or metabolic inhibitors, were all confounded by the resulting unsatisfactory platelet quality. It has been shown that addition of prostacyclin (PG12) - the most potent antiplatelet agent so far - to platelet-rich plasma preserved platelet function appreciably during the preparation of washed platelet suspensions . Later, prostacyclin was tried in the preparation of standard  and cryopreserved  platelet concentrates. However, the short half-life of prostacyclin and its poor storage characteristics, together with its potent vasodilatory effect, limited its use for this purpose. Since Iloprost, the stable carbacyclin derivative of prostacyclin, is the only prostaglandin analogue reported in which the platelet inhibition potential is dissociated from its vasodilatory action, we investigated whether Iloprost would be of practical value in the stabilization of standard platelet concentrates during preparation and storage. A preliminary study was performed to select the Iloprost concentration to be used in the preparation of PC. A previously described study using another inhibitor prostaglandin E~ (Prostin) has been reproduced and is reported here as well.
Key words: Platelet storage lesion - Platelet activation
Materials and methods
inhibition - Prostacyclin and platelets
Address for correspondence: M. Elias, P.O. Box 1191,NL-9701 BD Groningen, The Netherlands
Prostin (PGEp study
Prostin was kindly provided by the Upjohn company (Netherlands) as 500 pg in 1 ml dehydrated alcohol. PRP was prepared from three ABO-identical blood donations, pooled, and equally redivided into three transfer packs "C", "E", and "E+R". The study (n = 6) was carried out as described by Menitove et al. . Briefly, PGE1 (6x 10-8) M final concentration) was added to packs "E" and "E+R", mixed, and incubated for 30 min. Pack "C" served as control. The three platelet-rich plasmas (PRPs) (first spin 1000x g for 9 min) were concentrated by the second centrifuga-
293 tion step (1300• for 30 nfin). All but 70 ml platelet-poor plasma (PPP) was removed. Platelet concentrate (PC) " E + R " was immediately placed on the rotator. PC "E" and control PC "C" were allowed to rest for 90 min at room temperature before being rotated. Samples were drawn from control and test PC after 15 min aggitation, and after 48 and 96 h of storage.
Iloprost (PGIz) study A preliminary study (n = 5) was performed to define the dose of the prostacyclin analogue ZK 36374 (Iloprost) to be used for the preparation of PCs: Iloprost (a generous gift of Schering, Germany) was provided in 0.1 mg/ml vials (277 pM) with Tris buffer in 0.9% saline used as diluent. Iloprost concentrations ranging from 0.0001 to 0.1 pM in l-log increments were incubated for 1 min with PRP. These concentrations were based on a study by Fisher et al.  comparing the effect of three eicosanoids on human platelet function. Aggregation in response to single (5- and 10-pM ADP) and dual agonists (5 pM ADP + 5/~M epinephrine (Ep) proceeded for 5 rain for the control and the test PRPs. The aim of this preliminary study was to determine the lowest dose of Iloprost that would inhibit platelet activation during storage while maintaining the capacity of the transfused platelets to take part in hemostasis.
4. Lactate dehydrogenase leakage  was used as a marker of cell lysis. 5. The occurrence of the release reaction from a-granules was expressed as the percentage of 13TGreleased in relation to the total content of lysed platelets (13-TGRadioimmunoassay Kit, Amersham International plc, Amersharn, UK). 6. The activity of the cyclo-oxygenase pathway (with and without Iloprost) was assessed by the spontaneous thromboxane B2 (TBX2) production (Cayman chemical EIA kit 419031, CAMPRO Scientific). 7. Inhibition and recovery of platelet function after Iloprost wash were assessed by single and synergistic aggi'egation responses (laBOR Apact aggregometer, V1.2 Hamburg). 8. Preservation of platelet morphology, followed up by the morphology score  and electron microscopy study, was performed on three series.
Statistics Data are expressed as mean _+ SD and differences were determined using the Friedman test.
Use of Iloprost for preparation of PC Experiments (n = 13) were set up using three ABO-identical platelet concentrates, prepared as follows: Three compatible whole-blood donations (450 ml blood + 63 ml CPDA-1, USP) were collected in a triple-bag system (FGR 1367 B, Baxter, Le Ch~trtre). PRP was prepared by room-temperature centrifugation at 1000xg for 9 rain (Hereaus Christ cryofuge 8000, Ostenrode, Germany). Three units of PRP were pooled in a 1-1transfer bag, mixed, and redivided into equal-volume polyolefin containers (PL 732, Fenwal). Unit "A" served as control; units "B" and " C " were injected respectively with 1 p M and 0.1 pM final concentrations of Iloprost. After 15 min incubation, PRP were subjected to the second spin at 1300• for 30 rain. All but 70 ml of supernatant PPP was expressed and snap-frozen in plastic tubes. Autologous PPP was used for platelet wash and resuspension during storage. Iloprost-treated platelets "B" and " C " were immediately agitated on an end-overend rotator (Helmer Laboratories Inc, USA) without a rest period. This allowed for better mixing with Iloprost. The control bag "A" was, according to routine, left undisturbed for 60-90 min at 22 ~C. The platelet button was then resuspended for 15 rain on the rotator. PC units were subsequently agitated and stored in PL-732 containers for 5 days in a controlled-temperature incubator (Farina Scientific, USA).
Results o f Prostin study
In vitro assessments
Morphology. T h e m o r p h o l o g y score was higher in b o t h
In order to mimic the situation after transfusion and to demonstrate the reversible nature of Iloprost-inhibition of platelet function, samples were taken anaerobically using a plasma-exchange coupler about halfway into the storage period (day 2) and at the end of storage (day 5), washed off the inhibitor at 2500 rpm• rain (Sigma 3E-l), and resuspended in Iloprost-free autologous freshfrozen plasma (FFP). The restoration of platelet aggregability was assessed by the response to single (ADP 10 pM) and dual (ADP 5 pM + epinephrine 5/~M) agonists. The following assays were performed on PRP as baseline values and concurrently on control and test PC, on days 0, 2,, and 5: 1. Platelet and leukocyte counts were determined by Sysmex PL 110; mean platelet volume (MPV) and platelet distribution curves were determined electronically (Coulter S +, Coulter Electror~cs). 2. The metabolic status was assessed by pH, HCO3 (178 pH/ bloodgas analyzer, Corning, Baxter), and glucose (hexokinase method, DuPont aca) and lactate (Marbach and Weil enzymatic method, DuPont, aca) levels. 3. Since both ionized and total calcium increase in stored PC , we examined the effect of Iloprost on the total calcium level during storage by the Complexone method .
P G E l - t r e a t e d PCs (Table 1) t h r o u g h o u t storage.
Inspection. The Prostin-treated P C " E + R", which was i m m e d i a t e l y agitated, showed n o aggregates at all at the time o f sampling. Prostin-treated P C " E " , which was left u n d i s t u r b e d , showed tiny aggregates even after 15 m i n agitation. C o n t r o l P C " C " was n o t fully disaggregated even after 4 h a g i t a t i o n a n d showed gross macroscopic aggregates. Light microscopy showed microaggregates in P C " C " a n d " E " , whereas " E + R " was fully disaggregated.
Cell counts. I n contrast to the reference study , we f o u n d a significant difference in platelet c o u n t between the control a n d P G E l - t r e a t e d platelets (Table 1). This difference, however, disappeared w h e n platelets were c o u n t e d after 48 h. Leukocyte counts were initially higher in the c o n t r o l P C . T h e 48- a n d 96-h c o u n t s were n o t different.
Aggregation responses. Response to a single agonist o n day 0 was inhibited by 68% a n d 65% in P C " E "
Table 1. Pre- and post-storage values of platelet count, morphology and c~-granulerelease in PGEl-treated PC (Prostin 0.06 pM), stored for 4 days, with and without rotation for resuspension
Control: PGEI: PGEI: + rotat:
pre post pre post pre post
I3TG % release
640 _+115 583+126 688 _+ 162 717 _+55 784 _+30 732 _+61
268 _+27 222_+24 329 + 46 302 _+21 365 _+7 319 _+10
61 _+9 51_+6 67 + 20 60 _+6 84 _+3 65 _+5
3.4 _+0.9 5.8_+3.4 1.4 + 0.6 3.7 + 1.2 1.5 + 0.7 4.5 _+0.9
2 IJM I i
;IL imlf 8TG
2 Storage days
Fig. l. The aggregation response of PGEl-treated platelets to dual agonists was partially inhibited during the first 2 days, then equally lost in control and PGE~-treated platelets. Control platelets (black bars), PGE-treated platelets with (white bars) and without (hatched bars) rotation
" E + R " , respectively. The response to dual agonists (Fig. 1) was inhibited by 11% and 15%, respectively. After 48 and 96 h of storage, both A D P and synergistic stimulation were equally lost in all PC.
Fig. 2. The fffG % release was significantly inhibited in PGE1treated platelets only during the first 2 days of storage. Control platelets (black bars), PGE-treated platelets with (white bars) and without (hatched bars) rotation
flTG release. The percentage of TG release was significantly higher in the control PC (p < 0.05) up to day-2 measurements ( p < 0 . 0 1 ) . Post-storage difference in fffG% release was not significant (p < 0.1) between the control and the Prostin-treated PC (Fig. 2). No significant p H difference was found between control and test PC.
Results of Iloprost study The aim of the preliminary study was to find the dose of Iloprost which totally inhibits the response to single agonists (in vitro situation) while only partially inhibiting the synergistic response, which most probably reflects the in vivo situation . For the controls 5 p M A D P produced 68.6%0 +_ 15.3% aggregation, 10/~M A D P produced 76.0% + 8.5% aggregation, and the dual agonists produced 83.60/0 +_ 7.7% aggregation (Fig. 3). In each of the five experiments it was found that P R P aggregation in response to 5 p M A D P was partially inhibited with 0.0001 and 0.001/tM Iloprost and totally inhibited by 0.01 and 0.1 pM. The last two concentrations resulted in 48.6% and 79.9% inhibition of the synergistic aggregation, respectively, and only the concentration of 0.1 p M pro-
0.;01 Iloprost IJM
Fig. 3. Platelet aggregation percentages of control and Iloprosttreated PC, induced by single and dual agonists. Aggregation by 5 pM ADP (white bars) and by 10 ~ ADP (hatched bars) was totally inhibited at concentrations of 0.01 and 0.1//3//Iloprost, Only at 0.1/.tM was the synergistic aggregation by 5/~M ADP + 5/tM epinephrine (black bars) inhibited by more than 50070 in all series
duced more than 50% inhibition in all series. Therefore, 0.1/JM and 1/~M were chosen as final concentrations for preparation of PCs in the actual study. A higher concentration was used because the concentration of platelets in PCs is about 4 times that in PRP, and because PCs undergo an additional centrifugation step. The pre- and post-storage parameters of Iloprost-treated platelets are shown in Table 2.
2. Pre- and post-storage values of platelet count, morphology, release, activation and lysis of Iloprost-treated PCs (0.01/zM) stored for 5 days
Control: PGI 2:
pre post pre post
1013 + 239 997 +__222 1307 + 324 1234 +_324
311 +_18 278 + 34 353 + 20 331 + 22
62 + 7 52 + 9 78 + 6 70 + 6
2.7 +__0.9 6.5 + 1.0 0.7 + 0.3 3.7 _+0.4
495 + 356 4344 + 4043 99 +__91 487 + 316
219 +__68 1180 + 937 146 + 21 415 + 239
Inspection. Complete resuspension of the Iloprost-treated PC " B " and " C " was observed within 10 rain of rotation. Control PC "A" continued to show gross aggregates after 4 h on the rotator.
" C " Iloprost-treated platelets after the pelleting step. Post-storage LDH level was 358 and 414 U/I, respectively, in test PC versus 1180 U/1 in control PC.
Metabolic parameters. No significant difference in pH Platelet yield from PRP. The yield was significantly higher (p < 0.01) in Iloprost-treated platelets. In contrast to PGEl-treated platelets, the increased platelet count was sustained till the end of storage (19< 0.01). Unexpectedly, no difference was observed in the mean platelet volume (MPV) or the platelet distribution width (PDW).
Leukocytes. The leukocyte count was significantly higher (0.01) in control PC on day 0. This difference disappeared when leukocytes were counted on day 2 and on day 5.
Morphology. The morphology score was significantly higher in the Iloprost-treated PC (17 < 0.01). The difference between the test and control PC remained significant (p < 0.01) during the whole storage period. After 5-day storage, 70~ _+ 6.8~ and 70.4% _+ 5.8~ of Iloprosttreated platelets were discoid compared with 51.8% _+ 9.7% in the control PC.
Platelet ultrastructure. Figure 4 shows the electron microscopic findings with test and control platelets after preparation (above) and during storage (below): Control platelets exhibit signs of activation and increased platelet fragments. Many degranulated cells and others with disrupted membranes were seen on day 5. Iloprost-treated platelets show preservation of the discoid configuration till day 5, with normal number and distribution of organelles. However, a more widely open canalicular system was observed.
Platelet function. The inhibition of platelet aggregation by Iloprost persisted for the whole storage period. The difference in aggregation inhibition observed with the two Iloprost doses was not significant. The aggregability on day 5 was equally inhibited in control and test platelets. After Iloprost had been washed out and platelets resuspended in autologous FFP, platelets from " B " and " C " groups restored their sensitivity to aggregation better (17 < 0.01) than the control platelets "A". The effect of rejuvenation with fresh plasma on the control platelets was not significant. The improved recovery of the aggregation potential of Iloprost-washed PC was more marked after synergistic stimulation. Synergistic aggregation of " B " and " C " on day 5 was 82.3~ and 83.9% of the initial maximal aggregation, respectively (Fig. 5), compared with 32.7~ and 31.5~ in response to ADP.
giG release and TXB2 formation. The data obtained from measurement of the grG 070release and TXB 2 levels indicated a significant inhibition (p < 0.01) of activation and occurrence of the release reaction of Iloprost-treated platelets during the pelleting step. The difference between test and control PC persisted throughout storage.
Cell lysis. LDH leakage was significantly reduced (p < 0.01 and p < 0.05, respectively) in both " B " and
level, lactate production, or bicarbonate use was observed between control and test PC. Glucose consumption, however, was significantly higher in both concentrations of Iloprost-treated PC after 2 days (p < 0.01) and in the higher Iloprost concentration after 5 days (p < 0.05). A significant correlation (p < 0.01) was found between glucose consumption and the synergistic aggregation after washing. The total calcium rise was significantly reduced (p < 0.05) in Iloprost-treated PC.
The progressive deterioration in platelet function during storage, characterizing the platelet storage lesion, is well documented [9, 14, 17]. This lesion is manifested in vivo by impaired hemostatic capacity  and shortened circulation time . There is accumulating evidence attributing rapid clearance of stored platelets from the circulation to ex vivo platelet activation. Recent studies [10, 26] demonstrated that platelets exposing activation-dependent antigens on their plasma membranes are rapidly cleared from the circulation, whereas those exposing G P I I b / I I I a complex are not. Accordingly, agents that prevent ex vivo platelet activation should be able to improve in vivo platelet function and survival. It appeared from the Prostin study that addition of PGE~ to PRP during PC preparation increased the platelet recovery from PRP by facilitating suspension of the aggregates and improved in vitro platelet parameters. Immediate agitation of PC allows better mixing of the inhibitor with the pellet and saves the time otherwise meeded for spontaneous resuspension. The limitation of the Prostin effect to the early phase of storage, however, makes it unattractive as an additive medium to prevent platelet activation during preparation and storage of PC. Moreover, this transient elevation in cyclic AMP was shown to have no effect on platelet behavior after transfusion . To sustain the transient increase in cyclic AMP caused by PGE2 or PGI2 for a longer time, Bode and Miller  added inhibitors of the phosphodiesterase enzymes that break down cAMP, together with a specific inhibitor of thrombin. Storage of platelets under conditions that reduce the potential for activation  has been shown to reduce the metabolic rate. The reduced metabolic rate was related to improved maintenance of respiratory activity, ATP levels, GPIb levels, and other in vitro properties related to platelet viability . In our study, inhibition of platelet activation and release by Iloprost was not paralleled by inhibition of platelet metabolism as expected. Unchanged anaerobic glycolytic metabolism (unchanged lactate release) in the presence of Iloprost was also observed in studies on myocardial ischemia . The contribution of oxygen-derived free radicals to tissue injury is becoming increasingly recognized. These toxic oxygen metabolites have been ascribed to mitochon-
296 drial sources, xanthine oxidase, prostaglandin biosynthesis, and polymorphs . In addition, platelets produce potentially harmful products such as thromboxane A 2 and serotonin. Iloprost, by virtue of its potential to inhibit all activation pathways, its cytoprotective effect, and its metabolic stability, minimized the platelet storage lesion beyond the effect achieved by other inhibitory agents [11, 22]. Like prostacyclin, Iloprost seems to protect
h u m a n platelets during manipulations connected with preparation and storage. This action is mediated by an increase in cyclic A M P levels. In turn, cAMP is known to prevent the formation and release of oxygen-free radicals. However, the cytoprotective effect of Iloprost appears not to be dependent on its effect on platelets . It is most likely a direct scavenger-like effect on oxygen-free radicals .
Fig. 4 a - d . Electron microscopy of test and control platelets after preparation and after 5-day storage, a Control platelets exhibit signs
of activation and increased platelet fragements, b After storage many cells are degranulated, and others have a disrupted platelet membrane. e Iloprost-treated platelets show preserved membrane integrity; d the majority of cells show preservation of the discoid configuration up to the end of the storage period, with normal number and distribution of organelles. However, a more widely open canalicular system was observed
297 Iloprost-treated P C is paralleled by an improved clinical efficacy. Intolerance or secondary side effects should be excluded as well before Iloprost is considered as an adj u n c t to reduce platelet activation and subsequent loss o f function during the various necessary ex vivo manipulative procedures and during storage.
ADP 51JM * EP 6pM 100"
"""-.. ...... :)
Acknowledgements. The authors thank Henry Carper, Eddy Ligeon, Jietze Medema, Robert Bloem, Willy Struiksma, W. Sluiter, and Maya ten Cate and are most grateful to the volunteer donors.
ADP 61JM * Ep 6gM 100
;'8 50 25 0 0
Fig. 5 a, b. The synergistic aggregation response exhibited by test and control platelets, before (a) and after (b) wash and resuspension in FFP. Control ( . . . . ); Iloprost 1 ztM (--); Iloprost 0.t/zM
The unique property o f cytoprotection was d e m o n strated by limitation o f cell damage, evidenced by the significantly lower L D H leakage, in Iloprost-treated PC. The higher leukocyte count in the control PC, together with the lower platelet counts, might be explained by the presence o f platelet microaggregates counted as leukocytes by the electronic cell counter. H y p o t e n s i o n coupled with P G E I a n d PGI2 is a limiting factor for their extended use clinically. Iloprost represents an expection to the rule that the more potent an agent is as a platelet inhibitor, the more potent it is as a vasodilator [23, 27]. A pool o f six freshly prepared P C s would contain 2.2 p g (0.01 p M ) o f Iloprost; 80% o f this dose is present in the P P P during preparation. A s s u m i n g that the platelet transfusion were to a 60-kg patient administered at a rate o f 10 m l / m i n , this a m o u n t o f Iloprost would result in a concentration o f 0.2 n g / k g / m i n , a dose having no cardiovascular effect [2, 18]. The present m e t h o d for the p r o d u c t i o n o f platelet concentrates by incubation with Iloprost before the pelleting step yields a stable, responsive, and substantially improved platelet preparation protected against spontaneous decay during storage. This protection is extended beyo n d the chemical and biological half-life o f Iloprost  and is n o t explained by the molecular presence o f Iloprost in the suspensions. However, the protective effect o f ecosanoids ex vivo does n o t necessarily m e a n better recovery and survival in vivo . Clinical studies are warranted to assess whether the improved ex vivo quality o f
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