Aggregation Response of Human Platelets Stored at 22 C as Platelet-Rich Plasma G. MOROFFA N D C. H. CHANG From the American Red Cross Blood Services, Cell Preservation Laboratory, Bethesda, Maryland

The eggregatlog response of human platckts stored at 22 C for 72 hours has been studkd. Platelets were stored M platekt-rich plasma in order to mdntain the p b m a pH essentially constant. The response to ADP and collagen decreased with time, but the decrease WM less with relatively high concentrntbns of the aggregating agents. The response to the bnophore A23187 was essentllly d t e r e d durlng the storage prbd. Synergistk aggregatbn with combinntbns of ADP, collagen and bnophore A23187 was observed with platckts stored for 72 hours under conditbns where singly each of the stlmuli caused llttk or no efkct. stored platckts underwent reversible aggmgatbn over a wide range of ADP concentrntbns but imversibk aggregatbn WM observed in the presence of maaggregating comvntratbns of collagen or bnophore AWJ87. T& lnhibitbn of aggngaUon by PGE, or cyclk AMP WM facilitated as a result of storage. It is suggested that the decreased response toward ADP and collagen reflects a reduced ability on the part of the platelets to mobilize calcium.

PLATELETS that are to be used for transfusion purposes are stored in concentrated suspensions as platelet concentrates. Storage of these concentrates at 22 C (room temperature storage) is associated with a decrease in plasma pH16*27*36 and a reduced response to aggregating agents. 1,7,33 The aggregation lesion appears to be related to changes in the platelets themselves since the addition of freshly collected platelet-poor plasma to stored platelets, or the addition of platelet-poor plasma derived from stored platelet-rich plasma to freshly collected Contribution No. 410 from the American Red Cross Blood Services, Cell Preservation Laboratory, Bethesda, Maryland 20014. Presented in part at the 30th Annual Meeting of the American Association of Blood Banks, Atlanta, Georgia, November, 1977. Received for publication August 7, 1978; accepted November 18, 1978. Supported in part by Research Grant No. HL/16627 and Biomedical Support Grant No. 51 SO71 RRO5737.

platelets, did not influence the aggregation response to ADP.25 It has not been clearly resolved whether the decreased aggregation response is due to storage as such or to the decreased pH to which the platelets are exposed.37Furthermore, the quantitative aspects of the decreased response have not been examined over an appropriate range of concentrations with a variety of aggregating agents and reasons for the storage-induced reduction in aggregation response have not been delineated. In this report, we have stored platelets as platelet-rich plasma in order to maintain the plasma pH essentially constant during a 72hour storage period. This ensures that the changes being measured reflect alterations due to storage as such while eliminating the additional effects caused by pH changes which occur when platelets are stored as platelet concentrates. In addition, we have measured the response to ADP, collagen and the ionophore A23 187 over a wide range of concentrations. The results indicate that platelets stored at 22 C have not lost the ability to aggregate but exhibit a decreased response toward aggregating agents due to alterations which prevent the triggering of aggregation via the mobilization of calcium. Materials and Methods 14C-serotonin(5-hydroxytryptaminecreatinine sulphate) was obtained from Amersham Searle. ADP, bovine tendon collagen and dibutyryl cyclic adenosine monophosphate were purchased from Sigma Chemical Company. Ionophore A23187 was a gift from Dr. Robert Hamill of the Lilly Research Laboratories. Prostaglandin El was a gift

0041-1132/79/1100/0704$01.25 0 J. B. Lippincott Co. Transhsion November-December 1979

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of Dr.John Pike of the Upjohn Pharmaceutical Company. Units of human whole blood collected in CPD anticoagulantwere obtained from the Washington Regional Blood Center- American Red Cross. Platelet-rich plasma (PRP) was separated at 22 C within 4 hours after blood collection by centrifugation at 2,645 x g for 3.5 minutes in a Sorvall RC-3 centrifuge (HG-4L rotor). The PRP was placed into PL- 146 polyvinyl chloride blood storage bags (Fenwal Laboratories) having a volume capacity of 300 ml. The volume of PRP was 200225 ml unless otherwise noted. Platelet counts obtained using phase microscopy and a Unopette diluting device (Becton-Dickinson)were between 200,000 and 400,000 platelets/pl. The PRP suspensions were stored in a Forma platelet incubator thermostatted at 22 2 0.5 C. Continuous mixing was achieved by means of a fenis wheel type of agitator that rotated at one rpm. Sampling of PRP was accomplished under sterile conditions with the aid of medication injection sites. PRP samples were stored in plastic syringes during experimentation. This is necessary since the pH of platelet suspensions stored in open test tubes increases,because of COz diff~sion.~ The pH of platelet suspensions was measured with a Corning Model 12 pH meter. In Vitro Viability Assays Viability was assessed with the hypotonic stress and "C-serotonin uptake assays that have been shown to correlate with in vivo viability.12* IsA1 The hypotonic stress assay was performed by adding 0.4 ml of distilled water to 0.6 ml of PRP with rapid mixing. This procedure causes a rapid decrease in optical density followed by an increase in optical density approaching the original absorbance value. The initial rate of the increase in optical density (referred to as the reverse reaction) is measured. A Cary 118 spectrophotometer set at 610 nm was used. The uptake of I4C-serotoninwas camed out essentially by the procedure of Lundberg et al. Is Platelet Aggregation

The aggregation response of platelets was measured at 37 C with a Chrono-log aggregometer. After a two minute preincubation, 10 pl of the aggregating agent was added to 0.5 ml PRP. When =El and cyclic AMP were utilized, they were incubated with the PRP samples following the initial preincubation period for specified times before the addition of the aggregatingagent. ADP was prepared in 0.01 M Tris-HC1 containing 0.9% NaCl, pH 7.0. Acid soluble bovine tendon

collagen was prepared basically by the method of Holmsen et ~ 1 . The ' ~ acetic acid content of the collagen preparations was 1 mM and the concentration of the collagen suspensionswere adjusted after protein content was assessed by a ninhydrin assay using bovine serum albumin as a standard. Stock solutions of calcium ionophore A23187 were prepared in ethanol. Dilutions of the stock solutions were made with 0.01 M Tris-HCI containing 0.9% NaCl. pH 7.0.

Results Platelets are stored in blood banks as platelet concentrates (PC) in which the platelet count per microliter is approximately five times that found in platelet-rich plasma (PRP). Storage of platelet concentrates results in a decrease in plasma pH16827,98 which has been shown to cause a reduction in post-transfusion ~ i a b i l i t y . ~In ~ *order * ~ to determine the aggregation response of stored platelets in the absence of significant changes in pH, platelets were stored as PRP; 200 ml or more of plasma were routinely added to a PL- 146 blood storage bag. This volume of PRP, as shown in Figure 1, prevents the pH from decreasing below 7.0 after 72 hours of storagefrom an original pH of 7.2. With smaller volumes, there is an increase in pH. The pH changes in PRP are caused by lactic acid production via glycolysisZeand COz diffusion from the p l ~ s r n aThe . ~ high pH values detected with low volumes of PRP probably reflect an increased diffusion of CO, due to the increased ratio of surface area to volume. The in vitro viability, as measured by assays for serotonin uptake and hypotonic stress, did not show any significant change during 72-hour storage when the PRP volume was 200 ml. In addition, the platelet count did not decrease more than 10 per cent with this volume during the experimental period. When the pH increased above 7.2, with lesser volumes of PRP, there was a significant decrease in in vitro viability. For example, when the volume of PRP was 50 ml, lysis was observed after 48 hours of storage. In this regard, it has been observed that when the pH increases to 7.3 and above during storage, platelets show morphologic changes including degranulation with a decrease in in vivo viability." Both the rate of aggregation and the extent of aggregation were maximal around pH 7.0 to 7.2 with ADP, collagen and ionophore A23187 (Fig. 2). This relationship was identical with either freshly prepared platelets or platelets stored for 24 hours at 22 C. The aggregation response of platelets toward ADP (1 - 100 pM) after 72 hours of storage was decreased compared to that ob-

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Incubation Time ( Hours ) FIG. 1. The influence of plasma volume on pH changesoccurring during72-hourstorage. Two units of platelet-rich plasma were pooled and indicated volumes were expressed into PL-146 blood storage bags.

served with freshly prepared platelets (Fig. 3). This decrease occurs in a progressive manner during 72-hour storage with the largest change occurring during the first 24 hours. The reduction occurs to a greater extent at lower ADP concentrations. For example, 5 p M causes the maximal response with freshly prepared platelets, while platelets stored for 72 hours show little or no aggregation. On the other hand, the response to 100 pM ADP was altered to a much less extent. With freshly collected platelets, reversible aggregation was seen with 1 to 2 p M ADP while after 72 hours of storage this type of aggregation pattern was seen with 20 p M ADP. The tendency to undergo reversible aggregation is variable. In some cases, reversible aggregation was detected after 72 hours of storage even with 100 pM ADP. The aggregation response of platelets toward collagen (0.1 to 22 pg) after 72 hours of storage was decreased compared to that observed with freshly prepared platelets (Fig. 4). As with ADP, the decrease was progressive during 72 hours of storage with the largest change occumng during the first 24 hours. The reduction occurs to a greater extent at lower collagen concentrations. For example, 0.2 pg causes the maximum re-

sponse with freshly prepared platelets while platelets stored for 72 hours exhibit no detectable aggregation with collagen concentrations of 0.9 pg or less. On the other hand, the response to 3.7 and 22 pg of collagen was altered only to a small degree. The aggregation response toward the calcium ionophore A23187 after 72 hours of storage was very similar to that observed with freshly prepared platelets (Fig. 5 ) . The lag period before the onset of aggregation has also been reported by White et aLU The aggregation with the ionophore A23187 was essentially an all or nothing phenomenon. The ionophore-induced aggregation of stored platelets was substantiated utilizing electron microscopic techniques.22 The decrease in response to ADP and collagen could not be reversed by the addition of freshly collected platelet-poor plasma to a platelet concentrate prepared from a unit of stored plateletrich plasma. Furthermore, this procedure had no effect on the aggregation induced by ionophore A23187. Removal of residual leukocytes and erythrocytes from freshly prepared platelet-rich plasma by low speed centrifugation was also without effect.

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-g

100

2

50

A

t

.-0

75

EOD

) .

0

FIG.2. The influence of pH on the aggregation response of platelets toward lOpM ADP(O), 0.45pg COIlagen (A) and 5 pM ionophore A23187 (0).Desired pH levels were obtained by the addition of small volumes of 0.1 M NaOH or 0.1 M HCI (prepared in 0.9% NaCI) to aliquots of PRP. The rate of aggregation (A) refers to the initial rate calculated as per cent light transmission per minute. Initial rates of aggregation were determined by drawing a tangent to the portion of the aggregation trace representative of the initial phase of aggregation.

a

a

25 0

6.0

6.5

7.0

7.5

0.0

7.5

8.0

PH

75

) .

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c

C c. X

a

W

25 0

6.0

A synergistic aggregation response was observed both with a combination of ADP and the ionophore A23187 and with a second combination of ADP and collagen with platelets stored for 72 hours (Fig. 6). The collagen-ionophore A23187 couple also induced a synergistic response. The response is dependent on the concentration of the agents. The ADP and collagen concentrations which are involved in the causation of the synergistic response singly induce

6.5

7.0

significant aggregation with freshly collected platelets. The synergistic response was greater when the two agents are added within seconds of each other. If 60 seconds elapsed before the addition of the second agent, little or no synergistic effect was seen. With ADP concentrations which exhibit reversible aggregation, an irreversible aggregation pattern was observed in the presence of concentrations of ionophore A23187 or collagen which themselves do not cause any sig-

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60 40

20 0 80 60

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Time (min) The ADP awegation reswnse of freshly prepared platelets (A) and of platelets stored for 72 hours (B). - ADP was idded at the arrowpoint.

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A

60 40 I

s

20

I

C

.-0

0

fa

8C

v)

C

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$

Em

fi-

6C

3.7ug

1 4c

1.8 rrg

2c 0

0.1Ug- 0.4 I

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I

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Time (min) FIG.4. The collagen aggregation responseof freshly preparedplatelets (A) and of platelets storedfor 72 hours (B). Collagen was added at the arrowpoint.

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A

60 40

20 0 80

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60

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2a 0

0

1

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Time (min) FIG.5 . The ionophore A23187 aggregation response of freshly prepared platelets (A) and of platelets stored for 72 hours (B). Ionophore A23187 concentrations: a. 2.5 pM; b. 5 pM; c. 10 p M . The ionophore A23187 was added at the arrowpoint. (Reprinted with permission from The Blood Platelet in Transfusion Therapy. Edited by T. J. Greenwalt and G.A. Jamieson. New York, Alan R. Liss, 1978.)

1

2 3 Time (min) 4

2.5 uM

B

A

O

-

10-

-

C

v)

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60

E 80-

.-v)

v)

.-0

C

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0

t

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-

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.

0.3 vg Collagen

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st the arrowpoint. D) Aggregation response with 0.3 pg collagen in combination with ADP (2.5 p M to 10 phi). Collagen was added at arr~ and ADP was added at arrowpoint b. (Reprinted with permission from The Blood Platelet in Transfusion Therapy. Edited by T. J. Greenwalt amieson. New York, Alan R. Liss, 1978.)

6. Synergistic aggregation exhibited by platelets stored for 72 hours. A) Aggregation response with 1 p M ionophore A23187 or 10 p M A1 ltingagent was ad& at the arrowpoint. B) Aggregation response with 1 pM ionopbre A23187 in Combination with ADP(2.5 pM to 10 c1M).The ionoph ded at arrowpoint a and ADP was added at arrowpoint b. C) Aggregation response with 10 pM ADP or 0.3 pg collagen. Aggregating agent 1

I

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1 uM lonophore

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cause ultrastructural changes identical to those observed with thrombin and collagen.6 Furthermore, the involvement of calcium mobilization in ADP-induced aggregation has been suggested by fluorescence studies with chlortetracycline.l7 Collagen aggregation appears to be triggered by calcium mobilization in that the aggregation per se is caused by processes which depend on calcium. It has been shown that collagen aggregation is caused by released ADP and the production of oxygenated products of arachidonic acid.15 Evidence cited above indicates that ADP causes the mobilization of calcium while the production of arachidonic acid metabolites appears to be initiated by the action of calcium as the ionophore A23 187 activates the phospholipase which releases arachidonic acid from phospholipids.15m The inhibition of aggregation by cyclic AMP Discussion appears to reflect a sequestration of calcium from the platelet cytoplasmll and, thus, This report shows that the decreased agfurther supports the role of calcium mobilizagregation response toward ADP and collagen tion in aggregation. occurs independently of the decreased pH Our work indicates that the storage lesion which occurs during the storage of platelets occurs at a stage earlier than the events which as platelet concentrates. Furthermore, it has depend on the mobilization of calcium since been shown that platelets stored for 72 hours have not lost their ability to aggregate. Sig- platelets stored for 72 hours respond to the ionophore A23187 in a similar fashion as do nificant aggregation was observed at relafreshly collected platelets. Furthermore, the tively high concentrations of ADP and collagen, with a wide range of ionophore A23 187 fact that nonaggregating concentrations of ionophore A23 187 influence the aggregation concentrations, and with combinations of response with low ADP and collagen conaggregating agents under conditions where centrations suggests that synergistic aggregasingly each of the agents cause little or no tion occurs as a result of adequate calcium aggregation. Therefore, it can be concluded mobilization. We propose, therefore, that that the stored platelets have undergone the decreased aggregation in response to changes which prevent aggregation from ocADP and collagen with stored platelets may curring under conditions which were effecbe due to a decreased ability to mobilize tive with freshly prepared platelets. calcium subsequent to the binding of the Results from other laboratories indicate stimulus to membrane receptors. This phethat the triggering of aggregation may be due nomenon could be due to either an alteration to the mobilization of c a l c i ~ m ~from * * ~ inin a step which stimulates the mobilization tracellular storage pools or the extracellular of calcium or to a lesion in the mechanism medium. This suggestion is based on the which is directly responsible for the moability of the ionophore A23187, which is bilization of calcium. Impaired calcium moknown to transport calcium across memhas been suggested recently as a branes, to induce a g g r e g a t i ~ n ~and .~.~ to~ . ~ bilization ~

nificant aggregation (Fig. 7). This phenomenon was seen with freshly prepared platelets and platelets stored for 72 hours. Lower concentrations of PGEl are needed to inhibit aggregation in platelets which have been stored at 22 C than in freshly collected platelets. The altered response when aggregation was induced by collagen or the ionophore A23187 is shown in Figure 8. Similar results were observed for ADP-induced aggregation. The effect appears to be maximal after only 24 hours of storage. High concentrations of ADP and collagen were used to induce aggregation so as to offset the decreased aggregation response which is enhanced at low concentrations of these agents. Lower concentrations of dibutyryl cyclic AMP are also needed to inhibit the aggregation of stored platelets. The altered response when aggregation is induced with the ionophore A23187 is shown in Figure 9. Similar results were obtained when the aggregationwas induced by ADP or collagen.

0

a,t bt

/'

1

1

I

1

1

0

1

1

3

2 3 Time (min)

1

Time (min 1

.

2

2.5 V M lonophore

J

4

4

A

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0 .

t

"F / 0

&I

I '

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.

Time (min)

. 2

0.75 ug Collagen

1

3

I

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1

I

Influence of nonaggregatingconcentrations of ionophore A23 187 and collagen on reversible ADP aggregation with platelets stored for 72 h gregation response with 2.5 pM ionophore A23187 or 10 pM ADP. Aggregating agent was added at the arrowpoint. B) Aggregation response hore A23187 and ADP in combination. The ionophore A23187 was added at arrowpoint a and ADP was added at arrowpoint b. C) Aggreg nse with 0.75 pg collagen or 10 p M ADP. Aggregating agent was added at the arrowpoint. D) Aggregation response with collagen and A1 ination. Collagen was added at arrowpoint a and ADP was added at arrowpoint b.

. 7.

3

0

0

0

0

0 0

0

0 0 0

~

6

600

PGEIhM)

60

6000

0 -

20

0.06

6

PGEl(nM)

0.6

A23187 1OpM

6

B. The inhibition of aggregation by PGE, with freshly collected platelets (0).after storage for 24 hours (A) and 72 hours (0).Aggregating age gen (3 pg); B) ionophore A23187 (10 pM);PGE,was added 30 seconds before the aggregating agent. The extent of the aggregation response I ed to assess the magnitude of the inhibition.

0.6

0

c

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100

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100 80 FIG. 9. The inhibition of ionophore A23 187 aggregation of dibutyryl cyclic AMP with freshly collected platelets (0).and after storage for 24 hours (A). The ionophore A23187 concentmtion was 10 pM. Dibutyryl cyclic AMP was added 5 minutes before the aggregating agent.

t 0 .-

.c

60

-t 8 40

20 0 0.4

0.0

1.2

1.6

2.0

CAMP (mM) reason for platelet dysfunction in three patients .z Platelets stored at 22 C have an increased tendency to undergo reversible aggregation with ADP compared to freshly collected platelets. Electron microscopic analysis has shown that platelets undergo a reversal of the ultrastructural changes caused by the addition of ADP following deaggregati~n.'~ The deaggregation phase could conceivably be caused by a sequestration of mobilized calcium before the initiation of the steps which lead to irreversible aggregation. Our observationZ' that the addition of PGE, to ADP-aggregated platelets causes deaggregation, if the compound is added when the maximum aggregation is just reached, supports this suggestion since cyclic AMP, produced as a result of the influence of PGE, on adenyl cyclase, stimulates calcium se-

questration.l l The conversion of reversible aggregation to the irreversible type by nonaggregating concentrations of ionophore A23187, suggests that the tendency to undergo irreversible aggregation is controlled by whether or not a threshold level of mobilized calcium is reached. Thus, the subthreshold concentration of calcium may reflect either a suboptimal stimulation of the mechanism responsible for calcium mobilization or an increased sequestration of calcium that has been mobilized. Either explanation could be responsible for the shift from irreversible to reversible aggregationexhibited by stored platelets. Following in vivo circulation of platelets stored at 22 C, it has been found that the aggregation response is enhanced and the aggregation pattern is of the irreversible type rather than the reversible type.25One

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Transfusion November-December 1979

an increased capacity to synthesize cyclic explanation consistent with the concepts AMP in response to PGE1l3 is consistent discussed in this report is that in vivo circuwith this suggestion. Since the aggregation lation allows the platelets to reacquire the response toward the ionophore A23 187 is ability to mobilize calcium in response to not influenced during the 72-hour storage moderate or weak stimuli. period, the facilitated ease by which its Synergistic aggregation has been observed with a variety of platelet-activating aggregation is inhibited suggests that independent storage lesions are responsible for agent^.^^*^^.^^.^^.^^ This is the first report that the decreased aggregationand the increased stored platelets exhibit this type of aggregaease by which aggregation is inhibited. tion. As mentioned previously, we are interIt is thought that platelets stored at 22 C preting the synergistic aggregation in terms of have a delayed hemostatic effectiveness alcalcium mobilization. Recently, it has been though documentation of this phenomenon shown that the arachidonate pathway is asis very limited. Storage lesions described sociated with various types of synergistic in this report are compatible with this aggregation.l 4 A synergistically-inducedinconcept. crease in calcium mobilization could be responsiblefor this phenomenon as it has been Acknowledgments shown that the ionophore A23187 activates the phospholipase activity which liberates The authors express appreciation to Dr. Graham Jamieson for valuable advice during the writing of this arachidonic acid from phospholipids and manuscript. allows for its oxygenation to endoperoxides and t h r o m b o x a n e ~ . ~ ~ * ~ ~ References Platelet aggregation by various activating agents is inhibited by cyclic AMP and 1. Becker, G. A., M. Tuccelli, T. Kunicki, M. K. Chalos, and R. H. Aster: Studies of platelet conPGE, .8.23.34.42*45 The latter compound, being centrates stored at 22 C and 4 C. Transfusion a potent activator of adenyl cyclase is 13:61, 1973. thought to influence aggregation by stimulat2. Deykin, D., S. Rittenhouse-Simmons, and F. A. Russell: Impaired Ca2+mobilization: A possible ing cyclic AMP formation. Cyclic AMP has cause of platelet dysfunction. Clin. Res. 26:503A, been postulated to inhibit aggregation by 1978. stimulating the removal of cytoplasmic cal3. Feinman, R. D., and T. C. Detwiler: Platelet secrecium.s.ll Membrane fragments of platelets tion induced by divalent cation ionophores. Nature 249: 172, 1974. have been shown to contain a mechanism M. B., and C. Fraser: Human platelet which can concentrate c a l c i ~ m and ~ ~ re* ~ ~ *4. ~Feinstein, ~ secretion and aggregation induced by calcium cently it has been shown that cyclic AMP ionophores: Inhibition by PGE, and dibutyryl can stimulatethe uptake of calcium by platecyclic AMP. J. Gen. Physiol. 66561, 1975. 5 . Gambino, S. R., and H. Schreiber: The measurelet membrane vesicles.I1 The activation of ment of C02content with the autoanalyzer. Am. an intracellular calcium sequestration mechJ. Clin. Pathol. 45:406, 1%6. anism by cyclic AMP exists also in cardiac 6. Gerrard, J. M., J. G. White, and G. H. R. Rao: and skeletal muscle ~ e l l s . ~ Platelets ~ , ~ O stored Effects of the ionophore A23 187 on blood platelets. 11. Influence on ultrastructure. Am. J. at 22 C require a reduced concentration Pathol. 77:151, 1974. range of both PGE, and cyclic AMP to in7. Handin, R. I., and C. R. Valeri: Hemostatic efhibit aggregation induced by ADP, collagen, fectiveness of platelets stored at 22 C. N. End. and ionophore A23 187. We speculate that J. Med. 285538, 1971. 8. Haslam, R. J.: Roles of cyclic nucleotides in platethe increased ease by which platelet aggregalet function. I n : Biochemistry and Pharmacology tion can be inhibited may reflect a more efof Platelets. Ciba Foundation Symposium 35. ficient sequestration of cytoplasmic calcium. Amsterdam, Elsevier, Excerpta Medica, North Holland, 1975, p. 121. The fact that platelets stored at 22 C have ,8*42945

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9. Holmsen, H.: Are platelet shape change, aggregation, and release reaction tangible manifestations of one basic platelet function? In: Platelets in Production, Function, Transfusion and Storage. M.G. Baldini and S. Ebbe, Eds. New York, GNne and Stratton, 1974, p. 207. 10. -, H. J. Day, and E. Storm: Adenine nucleotide metabolism of blood platelets. VI. Subcellular localization of nucleotide pools with different functions in the platelet release reaction. Biochim. Biophys. Acta 186254, 1969. 11. Kaser-Glanzmann, R., M.JakBbovB, J. N. George, and E. F. Liischer: Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3’,5’-cyclic monophosphate and protein kinase. lW7. Biochim. Biophys. Acta -429, 12. Kim, B. K., and M. G. Baldini: The platelet response to hypotonic shock. Its value as an indicator of platelet viability after storage. Transfusion 14:130, 1974. 13. -, M. Steiner, and M. G. Baldini: Response of cyclic nucleotides to stimulation in stored human platelets. Fed. Roc. 37:407, 1978. 14. Kinlough-Rathbone, R. L., M. A. Packham, and J. F. Mustard: Synergism between platelet aggretating agents: The role of the arachidonate pathway. Thromb. Res. 11567, 1977. 15. -, M. A. Packham, H.-J. Reimers, J.-P. Cazenave, and J. F. Mustard: Mechanisms of platelet shape change, aggregation, and release induced by collagen, thrombin, or A23187. J. Lab. Clin. Med. 90:707, 1977. 16. Kunicki, T. J., M.Tuccelli, G. A. Beckerand R. H. Aster: A study of variables affecting the quality of platelets stored at “room temperature”. Transfusion 15:414, 1975. 17. Le Breton, G. C., W. C. Sandler, and H. Feinberg: The effect of 4 0 and chlortetracycline on ADPinduced platelet shape change and aggregation. Thromb. Res. 8477, 1976. 18. Lundberg, A., and S. Murphy: Survival in vivo of human blood platelets frozen without protective additives. Scand. J. Haematol. 9222, 1972. H. T. Meryman, and N. Estwick Response 19. -, of human platelets to hyper- and hypotonic media at 37 and -5 C. Am. J. Physiol. 222 1100, 1972. 20. LLischer, E. F., and P. Massini: Common pathways of membrane reactivity after stimulation of platelets by different agents. In: Biochemistry and Pharmacology of Platelets. Ciba Foundation Symposium 35. Amsterdam, Elsevier, Excerpta Media, North Holland, 1975, p. 5. 21. Massini, P., and E. F. Liischer: Some effects of ionophores for divalent cations on blood platelets. Comparison with the effects of thrombin. Biochim. Biophys. Acta 372:109, 1974. 22. McGill, M..and G. MorofE Unpublished observations.

717

23. Mills, D. C. B., and J. B. Smith: The control of platelet responsiveness by agents that influence cyclic AMP metabolism. Ann. NY Acad. Sci. 201:391, 1972. 24. Moroff, G.: Unpublished observations. 25. Murphy, S., and F. H. Gardner: Platelet storage at 22 C; metabolic, morphologic, and functional studies. J. Clin. Invest. *370, 1971. 26. ,and F. H. Gardner: Platelet storage at 22 C: Role of gas transport across plastic containers in maintenance of viability. Blood 46:209, 1975. 27. , S. N. Sayar, and F. H. Gardner: Storage of platelet concentrates at 22 C. Blood 35549, 1970. 28. Niewiarowski. S.. and D. P. Thomas: Platelet aggregation by ADP and thrombin. Nature 212: 1544,1966. 29. Packham, M. A., M. A. Guccione, P.-L. Chang, and J. F. Mustard: Platelet aggregation and release: Effects of low concentrations of thrombin or collagen. Am. J. Physiol. 22938, 1973. 30. Pickett. W. C., R. L. Jesse, and P. Cohen: Initiation of phospholipase At activity in human platelets by the calcium ion ionophore A23187. Biochim. Biophys. Acta 486:209, 1977. 31. Rittenhouse-Simmons, S., and D. Deykin: The mobilization of arachidonic acid in platelets exposed to thrombin or ionophore A23 187. J. Clin. Invest. 60:495, 1977. 32. Robblee. L. S.. D. Shepro. and F. A. Belamarich: Calcium uptake and associated adenosine triphosphatase activity of isolated platelet membranes. J. Gen. Physiol. 61:462, 1973. 33. Rock, G., and A. Figueredo: Metabolic changes during platelet storage. Transfusion 16571, 1976. 34. Salzman, E. W.: Cyclic AMP and platelet function. N. Engl. J. Med. 2%6:358, 1972. 35. Schwartz, A., M. L. Entman. K. Kaniike, L. K. Lane, W. B. Van Winkle, and E. P. Bornet: The rate of calcium uptake into sarcoplasmic reticulum of cardiac muscle and skeletal muscle. Biochim. Biophys. Acta 42657, 1976. 36. Silver, M.J.. J. B. Smith, C. Ingerman, and J. J. Kocsis: Arachidonic acid-induced human platelet aggregation and prostaglandin formation. Prostaglandins 4:863, 1973. 37. Silvertsen, U.: The influence of storage on ADPinduced platelet aggregation. Scand. J. Haematol. 9215, 1972. 38. Slichter, S.J., and L. A. Harker: Preparation and storage of platelet concentrates. 11. Storage variables influencing platelet viability and function. Br. J. Haematol. 34:403, 1976. 39. Statland, B. E., B. M. Heagan, and J. 0.White: Uptake of calcium by platelet relaxing factor. Nature 223521, 1969. 40. Tada, M., M. A. Kirchberger, D. 1. Repke. and A. M. Katz: The stimulation of calcium trans-

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port in cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependentprotein kinase. J. Biol. Chem. 2496174, 1974. 41. Valeri, C. R., H. Feingold. and L. D. Marchionni: The relation between response to hypotonic stress and W r recovery in vivo of preserved platelets. Transfusion 14331, 1974. 42. Vigdahl, R. L.,N. R. Marquis, and P. A. Tavormina: Platelet aggregation. 11. Adenyl cyclase, prostaglandin El, and calcium. Biochem. Biophys. Res. Comm. 37:409, 1969. 43. White, J. G.: Fine structural alterations induced in platelets by adenosine diphosphate. Blood 31: 604,1968.

44. -, G. H. R. Rao, and J. M.Gerrard: Effects of the ionophore A23187 on blood platelets. I. Influence on aggregation and secretion. Am. J. Pathol. 72135, 1974.

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45. Wolfe, S. M.,and N. R. Shulman: Adenyl cyclase activity in human platelets. Biochem. Biophys. Res. Comm. 35965, 1969. 46. Yoshida, N.,and N. Aoki: Potentiation by collagen or epinephrine of platelet responsiveness to aggregation. The possible role of substance(s) released from platelet membranes. J. Lab. Clin. Med. %9:603. 1977.

Gary Moroff, Ph.D., Research Scientist, American Red Cross Blood Services, Cell Preservation Laboratory, 9312Old Georgetown Road, Bethesda, Maryland, 20014 (reprint requests). Cecilia H. Chang, M.S.,Formerly, Research Technologist, American Red Cross Blood Services, Bethesda, Maryland, 20014.

Aggregation response of human platelets stored at 22 C as platelet-rich plasma.

Aggregation Response of Human Platelets Stored at 22 C as Platelet-Rich Plasma G. MOROFFA N D C. H. CHANG From the American Red Cross Blood Services,...
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