Increased Thrombin Sensitivity of Human Platelets After Storage at 4 C L. S. ROBBLEE,D. SHEPRO, J. J. VECCHIONEA N D C. R. VALERI From the Department of Biology, Boston University, Boston University Medical Center, and the Naval Blood Research Laboratory, Boston. Massachusetts
In vitro function of fresh platelets and platelets stored for 24 hours at 4 C and 22 C was studied using the release of “C-5-hydroxytryptamine (5-HT), adenine nucleotides and calcium following stimulation with thrombin. Storage of platelets did not affect “C-S-HT uptake capability, adenine nucleotide content or adenine nucleotide release. Platelets stored in 4 C vaned from fresh platelets and 22 C-stored platelets with respect to 5-HTand calcium release. For measurement of 5-HT release, different thrombin-to-platelet concentrations were used. At thrombin-to-platelet concentrations of 0.013 to 0.02 U/lOa platelets, 4 C-stored platelets released more 5-HT than did fresh platelets. Differences between 4 C-stored platelets and 22 C stored platelets were observed over a wider range of thrombin-to-platelets concentrations. The 4 C-stored platelets released significantly greater amounts of 5-HT at thrombin-toplatelet Concentrations of 0.008-0.02 Ulnil. Calcium release, measured with the calcium electrode, was studied at high (1.0 Vml) and low (0.06 U/ml) thrombin concentrations and at a platelet concentration of 8 x 10” plateletslml. At the high thrombin concentration, the amount of calriuni released was the same for all platelets studied. At the low thrombin concentration, platelets stored at 4 C released a greater amount of calcium than did either fresh platelets or platelets stored at 22 C. The results ohtained with different thrombinto-platelet concentrations suggest that there is only a narrow range of thrombin concentrations per platelet at which the subtle differences between platelet populations may be detected. The range may reflect the threshold concentration of thrombin that is required to initiate a release reaction. The apparently lower thrombin threshold required by 4 C platelets to undergo a release reaction may be a factor contributing to their reduced circulation and superior hemostatic effectiveness.
INCREASED INTEREST in liquid storage of human platelet concentrates in recent years has stimulated research to determine how best to ensure satisfactory posttransfusion survival and function of preserved platelets. Platelets are thought to be hemostatically more effective after 4 C storage than after
Materials and Methods Preparation of Platelet Concentrutes
Approximately 450 ml of human blood was drawn into a standard triple pack* containing
Received for publication February 6. 1978: accepted March 5 . 1978.
Transfusion January-February I979
storage at 22 C, but platelets stored at 4 C have a reduced survival time in the circulation compared with platelets stored at 22 C.4*24 Studies have focused on the changes in platelet metabolism, morphology and in v i m function after storage in an attempt to define alterations that might explain the observed differences in survival and hemostatic capability. Adenine nucleotide metabolism is impaired at both 4 C and 22 C, although the impairment appears to be greater at 22 C.6*11*13 Platelets stored at 4 C exhibit a loss of microtubules with a concomitant loss of discoid shape ,25*26 increased responsiveness in v i m to aggregating agents ,8,9*22 a reduction in electrophoretic mobility2 and increased sensitivity to hypotonic stress.2,10 At both 4 C and 22 C, proteolysis of platelet myosin’ and alteration of membrane glycoproteins7 occur. Speculation that release of intracellular platelet constituents such as ADP (adenosine diphosphate), serotonin, calcium and heparin-neutralizing molecules may be important in both formation and propagation of a platelet plug influenced the design of this study to determine whether the storage conditions of platelet concentrates might influence the degree of thrombininduced release of serotonin, adenine nucleotides and calcium.
* Fenwal Laboratories, Deerfield. IL.
45
Volume 19 Number I
46
ROBBLEE ET AL.
63 ml of citrate-phosphate-dextrose (CPD) anticoagulant. The blood was centrifuged at 4470 x g in an RC-3 refrigerated centrifuge for 2.5 minutes at 22 & 2 C to produce platelet-rich plasma (PRP). The PRP was then expressed into an integrally attached satellite bag and centrifuged at 4470 x g for five minutes at 22 2 C to concentrate the platelets. Supernatant platelet-poor plasma was removed so that 25 to 30 ml of plasma remained. The platelet concentrate was left undisturbed at room temperature (22 to 25 C) for 60 minutes, following which the platelets were manually resuspended. On a few occasions 10 per cent (V/V) ACD (acid-citratedextrose, NIH, Formula A) was added to the platelet concentrate to facilitate resuspension of the platelets. The recovery of platelets in the platelet concentrate was about 65 per cent of the platelets present in the whole blood. Plateletpoor plasma was added to the resuspended concentrate to obtain a final plasma volume of 75 to 80 ml. An aliquot of about 25 ml was transferred to each of three 150 ml polyvinyl chloride (PL-146) TA-3 transfer packs.* One aliquot was stored at 22 & 2 C and was studied within four hours of collection; this is referred to as fresh platelet concentrate. The second aliquot was stored in a constanttemperature chamber at 22 & 2 C and was constantly agitated on a rotator revolving eight times per minute during 24 hours of storage. The third aliquot was stored at 4 2 C without agitation for 24 hours, and was allowed to reach room temperature before study. Platelet counts were measured in the Technicon Auto-Counter,t and they ranged between 1.0 and 1.5 x 10Vpl. Platelet counts decreased by about 10 per cent during storage at both 4 C and 22 C, and the pH ranged between 6.5 and 7.0 in all the platelet concentrates. Preparation of Platelets for In Vitro Testing
In all instances, the platelet concentrates were treated with 10% (V/V) ACD immediately before centrifugation at 2000 X g at 22 C for 15 minutes. The platelet pellet was rinsed three times with 10 ml of buffered saline containing 0.9% NaCl and 0.5% glucose in 0.2 M Tris-HC1, pH 7.4, and then allowed to remain undisturbed in 5 ml of buffered saline for 20 to 30 minutes before resuspension with a Pasteur pipet. Leaving the platelet pellet undisturbed in buffered saline was found to facilitate the subsequent resuspension. The platelet concentration in this suspension was adjusted to 5 to 7 x 10“ platelets/ t Technicon Instruments Corp., Tarrytown, NY.
Transfusion
January-February 1919
ml by the addition of buffered saline. Platelet counts were performed by phase microscopy. Labeling of Platelets With ‘‘C-5-hydroxytryptamine ( "C-5-HT)
Platelets were labeled with ’“C-5-HTSby incubating a 20 ml aliquot of the suspension with a final concentration of 1.8 x 10-o M “C-5-HT at 37 C for 60 minutes in a Dubnoff metabolic incubator (75 strokes/minute). Following this incubation, the platelet suspension was allowed to return to room temperature before further manipulation. To determine the I4C-5-HT uptake, a 0.5 ml aliquot of platelets was centrifuged in a Fisher Model 59 Microfuge at 7000 x g for two minutes, and the radioactivity of the supernatant fluid was compared to that of the platelet suspension. Prior to platelet release experiments, the labeled platelets were centrifuged at 2000 x g for ten minutes at 2 2 2 2 C and resuspended in fresh buffered saline by the same procedure used in preparing the initial suspension. This final suspension of labeled platelets was divided into two aliquots and the platelet counts adjusted to 3 x lo8 and 5 x 10” platelets/ ml by the addition of buffered saline. Release of ’“C-5-HT
Two 0.5 ml aliquots were taken from each labeled platelet suspension. Each aliquot was placed in a 1 ml polyethylene centrifuge tube, and 0.02 mi of thrombin was added to provide a final thrombin concentration ranging from 0.02 to 0.1 Ulml of platelet suspension. After one minute incubation with thrombin at 22 to 25 C, the suspension was centrifuged at 7000 x g for two minutes in a Fisher Model 59 microfuge. Duplicate aliquots of the supernatant solutions were placed immediately in scintillation vials and mixed with ScintiverseP for measurement of 14C radioactivity. The release of “C-5-HT, expressed as the per cent of the radioactivity bound by the platelets was calculated as foll0ws:l4 cpm of supernatant from treated platelets- cpm of supernatant from untreated platelets % Release = cprn of platelet suspensioncpm of supernatant from untreated platelets ~~
-
$ Hydroxytryptamine binoxalate, 27.7 Cilmole, New
England Nuclear, Boston, MA. 9: Fisher Chemical Co., Medford, MA.
Volume 19 Number I
Release of Adenine Nucleotides In some experiments, adenine nucleotide release was determined for comparison with 14C5-HT release. In these experiments, an additional 0.2 ml aliquot of the supernatant fluid from a 14C-5-HT sample was mixed with an equal volume of ice-cold 10% perchloric acid and placed in an ice bath for one to four hours. The total adenine nucleotide content of the deproteinized perchloric acid extract was determined from the absorbance at 260 nm corrected for turbidity by subtracting the absorbance at 310 nm.*' The net optical density was compared to a standard curve of adenosine triphosphate prepared in Tris-buffered saline containing 5% perchloric acid. The release of adenine nucleotides is expressed as the per cent of the total adenine nucleotide content of the platelet suspension. Relecise of Cnlcium The thrombin-induced release of calcium was monitored continuously using a calcium ion electrode" and Model PHM 64 pH mete+' to follow the time course of the change in extracellular calcium concentration which results from the release of calcium from platelets.12.'9 Platelets were first separated from platelet concentrates as described above and were suspended in buffered saline to a platelet concentration of 1.6 x lo9 platelets/ml. This suspension was mixed with an equal volume of buffered saline containing M CaCI, to give a final calcium concentration M and a final platelet concentration of 5 x of 8 x 10" platelets/ml. The calcium electrode was immersed in a two ml aliquot of the suspension in a five ml polyethylene beaker; and 0.02 ml of thrombin was added to provide final thrombin concentrations ranging from 0.03 to 1 .O U/ml. The suspension was stirred continuously with a magnetic stirrer. The response of the electrode in millivolts (mV) was recorded on a chart recorder. The amount of calcium released from the platelets was calculated using the following modification of the Nernst equation:
where E = mV readings (initial and final); F = Faraday constant; R = gas constant; and pCa = -log (Ca++). Chemicds
All chemicals were reagent grade. Thrombin" was prepared as a stock solution of 1000 U/ml "
#
47
THROMBIN SENSITIVITY OF PLATELETS
Radiometer. Copenhagen. Parke-Davis Topical Thrombin.
FIG. I . The effect of platelet number and thrombin concentration on the release of IT-5-HT. Final thrombin concentrations of 0.4 U/ml ( ' . . . . ) and 0.06 U/ml (- - -).
in 0.02 M Tris-HCI buffer, pH 7.0, dialyzed overnight against the same buffer, and stored frozen (-20 C) in 0.2 ml aliquots. Aliquots were thawed and diluted serially with buffered saline immediately before use.
Results Uptake of 14C-5-HT
The ability of platelets to take up I4C-5-HT from the suspending medium was not significantly different whether the platelets were fresh or stored. The mean percent uptake in eight experiments was fresh platelets, 86.5 5 . I , platelets stored at 4 C 84.3 7.1; and platelets stored at 22 C 87.0 6.2.
*
*
Release of I4C-5-HT In initial release experiments, we observed that the extent of thrombin-induced release of 14-C-5-HTwas dependent on both thrombin concentration and platelet number (Fig. I ) . Increasing the thrombin concentration produced an increased release of I4C-5-HT, but increasing the platelet number resulted in a reduced release because of the lower thrombin concentration per platelet. Therefore, the release of I4C-5-HT by fresh and stored 'platelets was studied at two platelet concentrations, 3 x IO"/ml and 5 x lo"/ ml, and with thrombin concentrations ranging from 0.02 to 0.1 U/ml. Particular care was taken to maintain the same platelet concentrations for all experiments. Table I shows the per cent release of 14C-5-HT (mean & SD) of fresh platelets and platelets after 24 hours of storage at 4 C and 22 C. Between 7 and l l per cent of the labeled 5-HT appeared in the supernatant fluid of unstimulated control platelets, with no differences observed between the fresh and stored platelets. The
Translusion January-February 1979
ROBBLEE ET AL. Table 1. Per Cent Release of 14C-5-Hydroxytryptarnine
t values (p)
Series
A. 3 x lOVml
Thrombin (Units ml)
0.02 0.03 0.04 0.06 0.08 0.10 Unstimulatedt
B. 5 x 10Yml
0.02 0.03 0.04 0.06 0.08 0.10 Unstimulated
22 C vs Fresh
Fresh
4 C. 24 hr
22 C. 24 hr
4 C vs Fresh
9.1 f 5.1' 15.8f 7.7 2 4 . 5 6.4 ~ 43.8f 6.6 50.6 f 14.0 62.1 f 9.2 10.8f 7.5
9.8f 5.1 25.4 f 13.0 33.6 f 9.0 57.7 f 15.4 61.4f 14.1 68.2f 11.3 10.9 f 7.3
7.4 f 5.6 18.4f 12.9 21.2 f 5.2 44.1 f 15.0 47.4 f 8.5 55.9 f 8.5 9.0f 6.4
1.479(>0.1) 2.10 (0.05) 1.891(0.05) 1.422(>0.1) 0.991(>0.1)
1.079(>0.1) 3.19 (0.1) 0.04(>0.1) 0.51(>0.1) 1.16(>0.1)
2.5 f 2.1 12.8f 14.2 13.4f 9.4 33.9 f 18.7 43.0f 14.2 49.5 f 10.8 10.6 f 8.7
3.8f 4.2 16.2 f 16.0 25.2 f 12.9 4 7 . 414.2 ~ 58.3 k 5.6 64.3f 10.5 7.1 f 5.5
2.2 f 2.1 6.8f 7.1 11.5 f 8.3 31.2 f 16.4 43.1 k 10.0 46.8 f 9.0 8.9 f 7.8
0.432(>0.1) 1.854(>0.1) 1.647(>0.1) 2.639( 0.05). Comparison of the data obtained from fresh platelets and platelets stored at 22 C indicated
that the release from the 22 C-stored platelets was slightly lower than that from fresh platelets, but the difference was not statistically significant. The differences between the release response of platelets stored at 4 C and platelets stored at 22 C were highly significant at all thrombin concentrations above 0.03 U/ml and for both platelet concentrations (p < 0.05). The dose response curve for 5 X 10Vml of 4 Cstored platelets were nearly identical to the dose response curves for 3 x 10Vml of fresh platelets or platelets stored at 22 C (Fig. 2). Adenine Nucleotide Contcnt und Releusc
The total adenine nucleotide content of the stored platelets was not significantly different from that of fresh platelets (fresh platelets: 52.6 k 12.3 nmoles/lODplatelets; platelets stored at 4 C: 44.7 ? 6.5 nmoles/lOBplatelets; platelets stored at 22 C: 42.2 & 8.1 nmoles/lODplatelets; n = 4; p > 0. I ) . Adenine nucleotide release, measured at a platelet concentration of 5 x 10" platelets/ml, was essentially the same for both fresh and stored platelets over the range of thrombin concentrations tested (Table 2). Releuse of Culcium
.
.
.
l h r m w n h . lulMlll
FIG. 2. The effect of platelet number and thrombin concentration on the release of 14C-5-HTby fresh plateand platelets stored at 4 C).( or 22 C (A). lets ,).( Platelet concentrations are 3 x IOU platelets/ml (- - -) and 5 x IOR platelets/ml (. . . . .). The final thrombin concentrations are indicated on the abscissa.
The calcium electrode provided a continuous recording of the change in extracellular calcium concentration during the course of its release from thrombin-stimulated platelets. Representative tracings are shown of the time-course of calcium release from fresh platelets at different thrombin concentrations (Fig. 3). The amount of calcium released was directly related to thrombin
Volume 19 Number I
49
THROMBIN SENSITIVITY OF PLATELETS
Table 2. Thrombin-Induced Release of Adenine Nucleotides Storage Conditions Thrombin (Ulml) 0 0.01 0.015 0.02 0.03 0.04 0.06 0.08 0.10
Fresh 19.2 2 3 3 4 ) ' 28.8 (1) 28.9 2 4.7(3) 26.3 2 6.6(2) 36.7 ? 6.6(3) 32.0 ? 3.4(2) 50.1 2 4.0(3) 45.2 2 5.5(2) 52.5 (1)
t values (p) Fresh vs 4 C
4 C. 24 hr
22 C, 24 hr
15.52 2.3 (5) 21.5 2 2.5 (2) 24.8 ? 9.4 (4) 25.9 2 2.1 (2) 35.7 ? 6.6 (4) 46.8 ? 11.6(2) 50.5 ? 7.0 (5) 56.5 2 6.2 (2) 53.1 2 0.6 (2)
20.9? 1.8 (5) 20.4 2 3.0 (2) 24.3 ? 2.1 (4) 22.5 2 2.2 (2) 31.4 ? 3.0 (4) 32.0 ? 4.9 (2) 48.9 ? 4.2 (5) 54.4 2 10.1(2) 54.4 2 4.1 (2)
.174(p
* 0.1)
.102(p
* 0.1)
1.37 (p > 0.1)
Results as the per cent adenine nucleotides found in the supernatant 2 SE of the mean. Numbers in parentheses refer to the number of experi-
ments performed with platelets from different donors. The variation between duplicate reactions within a single experiment was less than 22%.
concentration, increasing as the thrombin concentration was increased. A typical experiment comparing fresh and stored platelets at high and low thrombin concentrations i s illustrated in Fig. 4. A t the higher thrombin concentration o f 1 U!ml, the release curves were similar for both fresh and stored platelets. However, when the thrombin concentration was low, (0.06 U/ml), the release curve for platelets stored at 4 C was higher than those for fresh platelets or platelets stored at 22 C.
It is in the release of I4C-5-HT and calcium that the greatest differences were observed between fresh and stored platelets. The most significant difference in 5-HT release between fresh platelets and those stored at 4 C was observed with a platelet concentration of 5 x lo8 platelets/ml and thrombin concentrations of 0.08 to 0.1 U/ml. This combination of platelet and thrombin concentrations gives a thrombin-to-platelet ratio of 0.016 to 0.02 U/108 platelets. When the platelet concentration was 3 x lO*/ml
Discussion
Liquid storage of platelets for 24 hours at 4 C or 22 C did not influence I4C-5-HT uptake capacity. Our data showed that total adenine nucleotide content was not reduced after 24 hours of storage at 4 C or 22 C, a finding similar to that of Murphy and Gardner.I5 Thrombin-induced release of adenine nucleotides from stored platelets was similar to that of fresh platelets. Other investigators have reported that collagen-induced ADP release was greatly reduced after 72 hours of platelet storage at 4 C or 22 C, with storage at 22 C producing the greatest reduction.6 The variations in results from different laboratories may be a reflection of the quality of the platelets used and the agent used to induce the release reaction. In addition, the method used in this study to measure adenine nucleotides does not distinguish between the various nucleotides.
I t .
I
10 soc
w
.
.
.
.
.
.
.
.
.
.
.
.
.
limo
FIG.3. Time progress curves of calcium release from fresh platelets as recorded by the calcium ion electrode. At the time indicated by the arrow, 0.02 ml thrombin is added to 2.0 rnl platelet suspension (8 x lonplatelets/ml) to provide the following final thrornbin concentrations (Unitslml): a) 1 .O; b) 0.66; c) 0.50; d) 0.25; e ) 0.125; f ) 0.06; g) 0.03.
50
ROBBLEE ET AL.
I
limo
FIG.4. Time progress curves of calcium release from fresh and stored platelets as recorded by the calcium ion electrode: a) fresh platelets; b) 4 C-stored platelets; c) 22 C-stored platelets. At the time indicated by the arrow, 0.02 ml thrombin is added to 2.0 ml platelet suspension (8 x loRplatelets/ml): A. 1.0 U/ml; B. 0.06 U/ml.
the greatest difference between fresh and 4 C-stored platelets occurred at thrombin concentrations between 0.04 and 0.06 U/ml. This combination of platelet and thrombin concentrations yields a thrombin-to-platelet ratio of 0.013 to 0.02 U/108platelets. In both series of experiments, the platelets stored at 4 C released significantly more 5-HT than did fresh platelets when the thrombin-to-platelet ratio was the order of 0.013 to 0.02 U/lOBplatelets, even though this ratio was obtained by different combinations of thrombin and platelet concentrations. Comparing the 4 C and 22 Cstored platelets in the same way, there is a similar yet wider range of thrombin-toplatelet ratios at which 4 C-stored platelets release more 5-HT than do 22 C-stored
Transfusion J~uary-Febmpry1979
platelets, 0.008 to 0.033 U/10* platelets. The similar thrombin-to-platelet ratios may indicate that there is only a narrow range of thrombin concentrations per platelet at which subtle differences between platelet populations may be detected. This range of thrombin concentrations may approximate the threshold concentration of thrombin that has been demonstrated to be required to initiate a release The increased release of “C-5-HT at low thrombin-to-platelet concentrations by 4 Cstored platelets suggests that these platelets are more sensitive to thrombin than are fresh platelets or 22 C-stored platelets. The upward displacement of both dose response curves of 4 C-stored platelets in Figure 2 indicates that these platelets required less thrombin per platelet for a release response equivalent to that of fresh platelets or platelets stored at 22 C for 24 hours. The amount of calcium released from thrombin-treated fresh or stored platelets is dependent upon the thrombin concentration, confirmingprevious ~ b s e r v a t i o n s . ~ * ~ ~ * ~ Because calcium release is also dependent upon platelet concentration, and ultimately, the thrombin-to-plateletconcentration, care was taken in the present experiments to maintain a constant platelet number when measuring calcium release as it was for measuring 5-HT and adenine nucleotide release. Platelets that were stored at 4 C released more calcium at low thrombin concentration than did fresh platelets or those stored at 22 C. This observation is consistent with the results obtained for 14C5-HT release. The greater release of 5-HT and calcium by 4 C-stored platelets is probably not the result of an increased fragility of these platelets. In other studies only two to four per cent of the total lactate dehydrogenase activity appeared in the supernatant fluid of thrombin-treated platelets and this activity was independent of thrombin concentration or previous treatment of the platelets. It is also unlikely that the increased
Volume 19 Number I
THROMBIN SENSITIVITY OF PLATELETS
sensitivity of 4 C-stored platelets is due to ADP potentiation of the thrombin-induced release reactionlB because these platelets were not observed to leak or release substantially different amounts of adenine nucleotides. The absence of difference between the 4 C-stored platelets and fresh or 22 C-stored platelets with regard to adenine nucleotide release is difficult to explain in view of the generally accepted idea that adenine nucleotides, serotonin and calcium are stored in the same granules. It may be that different mechanisms of release exist for the different substances or that one or the other are selectively released at different levels of stimulus. The greater responsiveness of 4 C-stored platelets to low concentrations of thrombin may be a factor contributing to their reduced circulation and superior hemostatic effectiveness3 Recent studies indicate that a threshold level of thrombin is necessary for initiation of the thrombin release react i ~ n , ~and J ~that this thrombin threshold may be related to the number and type of thrombin receptor sites that are occupied on the platelet^.^^*^^^*^ Platelets with a low thrombin threshold may respond to a thrombin stimulus level in the circulation that would not affect platelets with a higher thrombin requirement. Consequently they might aggregate and be removed from the circulation at a faster rate or participate more readily in hemostasis. Intrinsic alterations of the platelets may produce the differences in thrombin sensitivity after storage at 4 C. A multitude of parameters may be affected in diverse ways by different storage conditions. If the platelet secretory response is considered to be an early and necessary event in hemostasis, there are three general areas where a slight alteration of the platelets may influence their hemostatic effectiveness. One is an alteration of surface catalytic or receptor sites which affect the initial stimulus-receptorinteraction. The apparent decrease in thrombin threshold concentration of platelets
51
after storage at 4 C may result from a change in the number or type of thrombin-binding sites resulting in a change in thrombinturnover time. A decrease in turnover time would make more thrombin available to interact with successive populations of platelets and increase the effective thrombin-toplatelet ratio. A second type of platelet alteration might be in the transmission mechanism which translates the external message to the interior of the cell. A third might be the intracellular mechanism involved in bringing about the release of granular contents. The last two of these events are subject to regulation by the internal environment of the cell, including ion fluxes and concentration, cyclic nucleotide concentration, and polymerization-depolymerization equilibria of structural proteins. Obviously, these interactions are complex, and very little is known about the actual sequence of events leading from stimulus to response. Further study of platelets after liquid storage may provide some indication of the mechanisms involved in the reactions of fresh platelets. The use of a wide range of closely-spaced thrombin concentrations, coupled with different platelet concentrations, seems to be a useful way to demonstrate differences in platelet responsiveness that might otherwise by obscured. Acknowledgments This work was supported by the U.S.Navy (Naval Medical Research and Development Command Research Task No. M0095-PN.001-0040) and by the National Institute of Health Grant HL-16714-04. The opinions or assertions contained herein are those of the authors, and are not to be construed as official or reflecting the views of the Navy Department or Naval Service at large. We wish to thank Dr. Jonathan L. Costa, NIH, for his comments and suggestions related to this article, and to Laurel Sutton for her assistance in preparing this manuscript.
References I . Abramowitz, J., A. Stracher, and T. C. Detwiler: Proteolysis of myosin during platelet storage. J. Clin. Invest. 53:1493, 1974. 2. Ando, Y . , M. Steiner, and M. Baldini: Effect of chilling on membrane related functions of platelets. Transfusion 14453, 1974.
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ROBBLEE ET AL.
M. Hamid, and D. N. Calvert: Storage of platelet concentrates at 4°C; use of refrigerated platelet concentrates in the treatment of hemorrhage in thrombocytopenic patients. I n : Platelets; Production, Function, Transfusion and Storage, M. G. Baldini and S. Ebbe, Eds. New York, Grune and Stratton, 1974, p. 361. Becker, G. A., M. Tuccelli, T. Kunicki, M. K. Chalos, and R. H. Aster: Studies of platelet concentrates stored at 22 C and 4 C. Transfusion 13:61, 1973. Detwiler, T. C., and R. D. Feinman: Kinetics of the thrombin-induced release of calcium (11) by platelets. Biochemistry 12: 282, 1973. Filip, D. J., J. D. Eckstein, and C. A. Sibley: The effect of platelet concentrate storage temperature on adenine nucleotide metabolism. Blood 45749, 1975. George, J. N.: Platelet membrane glycoproteins: alteration during storage of human platelet concentrates. Thrombosis Res. 8:719, 1976. Kattlove, H. E., and B. Alexander: The effect of cold on platelets. I. Cold-induced platelet aggregation. Blood 3839, 1971. -, B. Alexander, and F. White: The effect of cold on platelets. 11. Platelet function after shortterm storage at cold temperatures. Blood 40:
3. Aster, R. H., G. A. Becker,
4.
5.
6.
7.
8.
9.
688, 1972. 10. Kim, B. K.. and M. G. Baldini: The osmotic platelet response as an indicator of platelet viabilityduringstorage. Fed. Proc. 29:717, 1970. 11. , and M. G. Baldini: Glycolytic intermediates and adenine nucleotides of human platelets. 11. Effect of short-term storage at 4 C. Transfusion 12: 1, 1972. 12. Kornstein, L. B., L. S. Robblee and D. Shepro: Thrombin-platelet interactions investigated with the calcium electrode. Thrombosis Res. 11:471, 1977. 13. Kotelba-Witkowska, B., H. Holmsen, and E. H. Muer: Storage of human platelets: effects on metabolically active ATP and on the release reaction. Br. J. Haematol. 22:429, 1972. 14. Massini, P., and E. F. Luscher: Some effects of ionophores for divalent cations on blood platelets: Comparison with the effects of thrombin. Biochim. Biophys. Acta 372:109, 1974. 15. Murphy, S., and F. H. Gardner: Platelet storage at 22°C. Metabolic, morphologic and functional studies. J. Clin. Invest. 50:370, 1971. 16. Packham, M. A.. M. A. Guccione. P.-L. Chang,
17.
18.
19.
20.
21.
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and J. F. Mustard: Platelet aggregation and release: effects of low concentrations of thrombin and collagen. Am. J. Physiol. 22538, 1973. Phillips, D. R.: Thrombin interaction with human platelets. Potentiation of thrombin-induced aggregation and release by inactivated thrombin. Thromb. Diath. Haemorrh. 32:207, 1974. -,and P. Agin: Thrombin substrates and the proteolytic site of thrombin action on humanplatelet plasma membranes. Biochim. Biophys. Acta. 352:218, 1974. Robblee, L. S., L. B. Kornstein, and D. Shepro: Calcium electrode. A method for the continuous monitoring of the platelet release reaction. Thrombosis and Haemostasis 37:407, 1977. , and D. Shepro: The effect of external calcium and lanthanum on platelet calcium content andon the release reaction. Bioch. Biophys. Acta 436:448. 1976. Salganicoff, L., P. A. Hebda, J. Yandrasitz, and M. H. Fukami: Subcellular fractionation of pig platelets. Biochim. Biophys. Acta 385:394,
-
1975. 22. Shively, J. A., C. L. Gott, and D. S. deJongh:
23.
24.
25.
26.
The effect of storage on adhesion and aggregation of platelets. Vox Sang. 18:204. 1970. Tollefsen, D. M.,J. R. Feagler, and P. W. Majerus: The binding of thrombin to the surface of human platelets. J. Biol. Chem. 249:2646, 1974. Valeri, C. R.: Hemostatic effectiveness of liquidpreserved and previously frozen human platelets. N. Engl. J. Med. 290:353. 1974. White, J. G., and W. Krivit: An ult~astructural basis for the shape changes induced in platelets by chilling. Blood 30:625, 1967. Zucker, M. B., and J. Borrelli: Reversible alterations in platelet morphology produced by anticoagulants and by cold. Blood 9:602, 1954.
Lois S. Robblee, Ph.D., Research Associate, EIC, 67 Chapel Street, Newton, Massachusetts.
David Shepro, Ph.D., Professor, Boston University, Microvascular Research, 2 Cummington Street, Boston, Massachusetts 02215. J . J . Vecchione, M.D., Research Medical Officer, Naval Blood Research Laboratory, 615 Albany Street, Boston, Massachusetts 021 18. C. Robert Valeri, M.D., Officer-in-Charge, Naval Blood Research Laboratory.
In vitro function of fresh platelets and platelets stored for 24 hours at 4 C and 22 C was studied using the release of “C-5-hydroxytryptamine (5-HT), adenine nucleotides and calcium following stimulation with thrombin. Storage of platelets did not affect “C-S-HT uptake capability, adenine nucleotide content or adenine nucleotide release. Platelets stored in 4 C vaned from fresh platelets and 22 C-stored platelets with respect to 5-HTand calcium release. For measurement of 5-HT release, different thrombin-to-platelet concentrations were used. At thrombin-to-platelet concentrations of 0.013 to 0.02 U/lOa platelets, 4 C-stored platelets released more 5-HT than did fresh platelets. Differences between 4 C-stored platelets and 22 C stored platelets were observed over a wider range of thrombin-to-platelets concentrations. The 4 C-stored platelets released significantly greater amounts of 5-HT at thrombin-toplatelet Concentrations of 0.008-0.02 Ulnil. Calcium release, measured with the calcium electrode, was studied at high (1.0 Vml) and low (0.06 U/ml) thrombin concentrations and at a platelet concentration of 8 x 10” plateletslml. At the high thrombin concentration, the amount of calriuni released was the same for all platelets studied. At the low thrombin concentration, platelets stored at 4 C released a greater amount of calcium than did either fresh platelets or platelets stored at 22 C. The results ohtained with different thrombinto-platelet concentrations suggest that there is only a narrow range of thrombin concentrations per platelet at which the subtle differences between platelet populations may be detected. The range may reflect the threshold concentration of thrombin that is required to initiate a release reaction. The apparently lower thrombin threshold required by 4 C platelets to undergo a release reaction may be a factor contributing to their reduced circulation and superior hemostatic effectiveness.
INCREASED INTEREST in liquid storage of human platelet concentrates in recent years has stimulated research to determine how best to ensure satisfactory posttransfusion survival and function of preserved platelets. Platelets are thought to be hemostatically more effective after 4 C storage than after
Materials and Methods Preparation of Platelet Concentrutes
Approximately 450 ml of human blood was drawn into a standard triple pack* containing
Received for publication February 6. 1978: accepted March 5 . 1978.
Transfusion January-February I979
storage at 22 C, but platelets stored at 4 C have a reduced survival time in the circulation compared with platelets stored at 22 C.4*24 Studies have focused on the changes in platelet metabolism, morphology and in v i m function after storage in an attempt to define alterations that might explain the observed differences in survival and hemostatic capability. Adenine nucleotide metabolism is impaired at both 4 C and 22 C, although the impairment appears to be greater at 22 C.6*11*13 Platelets stored at 4 C exhibit a loss of microtubules with a concomitant loss of discoid shape ,25*26 increased responsiveness in v i m to aggregating agents ,8,9*22 a reduction in electrophoretic mobility2 and increased sensitivity to hypotonic stress.2,10 At both 4 C and 22 C, proteolysis of platelet myosin’ and alteration of membrane glycoproteins7 occur. Speculation that release of intracellular platelet constituents such as ADP (adenosine diphosphate), serotonin, calcium and heparin-neutralizing molecules may be important in both formation and propagation of a platelet plug influenced the design of this study to determine whether the storage conditions of platelet concentrates might influence the degree of thrombininduced release of serotonin, adenine nucleotides and calcium.
* Fenwal Laboratories, Deerfield. IL.
45
Volume 19 Number I
46
ROBBLEE ET AL.
63 ml of citrate-phosphate-dextrose (CPD) anticoagulant. The blood was centrifuged at 4470 x g in an RC-3 refrigerated centrifuge for 2.5 minutes at 22 & 2 C to produce platelet-rich plasma (PRP). The PRP was then expressed into an integrally attached satellite bag and centrifuged at 4470 x g for five minutes at 22 2 C to concentrate the platelets. Supernatant platelet-poor plasma was removed so that 25 to 30 ml of plasma remained. The platelet concentrate was left undisturbed at room temperature (22 to 25 C) for 60 minutes, following which the platelets were manually resuspended. On a few occasions 10 per cent (V/V) ACD (acid-citratedextrose, NIH, Formula A) was added to the platelet concentrate to facilitate resuspension of the platelets. The recovery of platelets in the platelet concentrate was about 65 per cent of the platelets present in the whole blood. Plateletpoor plasma was added to the resuspended concentrate to obtain a final plasma volume of 75 to 80 ml. An aliquot of about 25 ml was transferred to each of three 150 ml polyvinyl chloride (PL-146) TA-3 transfer packs.* One aliquot was stored at 22 & 2 C and was studied within four hours of collection; this is referred to as fresh platelet concentrate. The second aliquot was stored in a constanttemperature chamber at 22 & 2 C and was constantly agitated on a rotator revolving eight times per minute during 24 hours of storage. The third aliquot was stored at 4 2 C without agitation for 24 hours, and was allowed to reach room temperature before study. Platelet counts were measured in the Technicon Auto-Counter,t and they ranged between 1.0 and 1.5 x 10Vpl. Platelet counts decreased by about 10 per cent during storage at both 4 C and 22 C, and the pH ranged between 6.5 and 7.0 in all the platelet concentrates. Preparation of Platelets for In Vitro Testing
In all instances, the platelet concentrates were treated with 10% (V/V) ACD immediately before centrifugation at 2000 X g at 22 C for 15 minutes. The platelet pellet was rinsed three times with 10 ml of buffered saline containing 0.9% NaCl and 0.5% glucose in 0.2 M Tris-HC1, pH 7.4, and then allowed to remain undisturbed in 5 ml of buffered saline for 20 to 30 minutes before resuspension with a Pasteur pipet. Leaving the platelet pellet undisturbed in buffered saline was found to facilitate the subsequent resuspension. The platelet concentration in this suspension was adjusted to 5 to 7 x 10“ platelets/ t Technicon Instruments Corp., Tarrytown, NY.
Transfusion
January-February 1919
ml by the addition of buffered saline. Platelet counts were performed by phase microscopy. Labeling of Platelets With ‘‘C-5-hydroxytryptamine ( "C-5-HT)
Platelets were labeled with ’“C-5-HTSby incubating a 20 ml aliquot of the suspension with a final concentration of 1.8 x 10-o M “C-5-HT at 37 C for 60 minutes in a Dubnoff metabolic incubator (75 strokes/minute). Following this incubation, the platelet suspension was allowed to return to room temperature before further manipulation. To determine the I4C-5-HT uptake, a 0.5 ml aliquot of platelets was centrifuged in a Fisher Model 59 Microfuge at 7000 x g for two minutes, and the radioactivity of the supernatant fluid was compared to that of the platelet suspension. Prior to platelet release experiments, the labeled platelets were centrifuged at 2000 x g for ten minutes at 2 2 2 2 C and resuspended in fresh buffered saline by the same procedure used in preparing the initial suspension. This final suspension of labeled platelets was divided into two aliquots and the platelet counts adjusted to 3 x lo8 and 5 x 10” platelets/ ml by the addition of buffered saline. Release of ’“C-5-HT
Two 0.5 ml aliquots were taken from each labeled platelet suspension. Each aliquot was placed in a 1 ml polyethylene centrifuge tube, and 0.02 mi of thrombin was added to provide a final thrombin concentration ranging from 0.02 to 0.1 Ulml of platelet suspension. After one minute incubation with thrombin at 22 to 25 C, the suspension was centrifuged at 7000 x g for two minutes in a Fisher Model 59 microfuge. Duplicate aliquots of the supernatant solutions were placed immediately in scintillation vials and mixed with ScintiverseP for measurement of 14C radioactivity. The release of “C-5-HT, expressed as the per cent of the radioactivity bound by the platelets was calculated as foll0ws:l4 cpm of supernatant from treated platelets- cpm of supernatant from untreated platelets % Release = cprn of platelet suspensioncpm of supernatant from untreated platelets ~~
-
$ Hydroxytryptamine binoxalate, 27.7 Cilmole, New
England Nuclear, Boston, MA. 9: Fisher Chemical Co., Medford, MA.
Volume 19 Number I
Release of Adenine Nucleotides In some experiments, adenine nucleotide release was determined for comparison with 14C5-HT release. In these experiments, an additional 0.2 ml aliquot of the supernatant fluid from a 14C-5-HT sample was mixed with an equal volume of ice-cold 10% perchloric acid and placed in an ice bath for one to four hours. The total adenine nucleotide content of the deproteinized perchloric acid extract was determined from the absorbance at 260 nm corrected for turbidity by subtracting the absorbance at 310 nm.*' The net optical density was compared to a standard curve of adenosine triphosphate prepared in Tris-buffered saline containing 5% perchloric acid. The release of adenine nucleotides is expressed as the per cent of the total adenine nucleotide content of the platelet suspension. Relecise of Cnlcium The thrombin-induced release of calcium was monitored continuously using a calcium ion electrode" and Model PHM 64 pH mete+' to follow the time course of the change in extracellular calcium concentration which results from the release of calcium from platelets.12.'9 Platelets were first separated from platelet concentrates as described above and were suspended in buffered saline to a platelet concentration of 1.6 x lo9 platelets/ml. This suspension was mixed with an equal volume of buffered saline containing M CaCI, to give a final calcium concentration M and a final platelet concentration of 5 x of 8 x 10" platelets/ml. The calcium electrode was immersed in a two ml aliquot of the suspension in a five ml polyethylene beaker; and 0.02 ml of thrombin was added to provide final thrombin concentrations ranging from 0.03 to 1 .O U/ml. The suspension was stirred continuously with a magnetic stirrer. The response of the electrode in millivolts (mV) was recorded on a chart recorder. The amount of calcium released from the platelets was calculated using the following modification of the Nernst equation:
where E = mV readings (initial and final); F = Faraday constant; R = gas constant; and pCa = -log (Ca++). Chemicds
All chemicals were reagent grade. Thrombin" was prepared as a stock solution of 1000 U/ml "
#
47
THROMBIN SENSITIVITY OF PLATELETS
Radiometer. Copenhagen. Parke-Davis Topical Thrombin.
FIG. I . The effect of platelet number and thrombin concentration on the release of IT-5-HT. Final thrombin concentrations of 0.4 U/ml ( ' . . . . ) and 0.06 U/ml (- - -).
in 0.02 M Tris-HCI buffer, pH 7.0, dialyzed overnight against the same buffer, and stored frozen (-20 C) in 0.2 ml aliquots. Aliquots were thawed and diluted serially with buffered saline immediately before use.
Results Uptake of 14C-5-HT
The ability of platelets to take up I4C-5-HT from the suspending medium was not significantly different whether the platelets were fresh or stored. The mean percent uptake in eight experiments was fresh platelets, 86.5 5 . I , platelets stored at 4 C 84.3 7.1; and platelets stored at 22 C 87.0 6.2.
*
*
Release of I4C-5-HT In initial release experiments, we observed that the extent of thrombin-induced release of 14-C-5-HTwas dependent on both thrombin concentration and platelet number (Fig. I ) . Increasing the thrombin concentration produced an increased release of I4C-5-HT, but increasing the platelet number resulted in a reduced release because of the lower thrombin concentration per platelet. Therefore, the release of I4C-5-HT by fresh and stored 'platelets was studied at two platelet concentrations, 3 x IO"/ml and 5 x lo"/ ml, and with thrombin concentrations ranging from 0.02 to 0.1 U/ml. Particular care was taken to maintain the same platelet concentrations for all experiments. Table I shows the per cent release of 14C-5-HT (mean & SD) of fresh platelets and platelets after 24 hours of storage at 4 C and 22 C. Between 7 and l l per cent of the labeled 5-HT appeared in the supernatant fluid of unstimulated control platelets, with no differences observed between the fresh and stored platelets. The
Translusion January-February 1979
ROBBLEE ET AL. Table 1. Per Cent Release of 14C-5-Hydroxytryptarnine
t values (p)
Series
A. 3 x lOVml
Thrombin (Units ml)
0.02 0.03 0.04 0.06 0.08 0.10 Unstimulatedt
B. 5 x 10Yml
0.02 0.03 0.04 0.06 0.08 0.10 Unstimulated
22 C vs Fresh
Fresh
4 C. 24 hr
22 C. 24 hr
4 C vs Fresh
9.1 f 5.1' 15.8f 7.7 2 4 . 5 6.4 ~ 43.8f 6.6 50.6 f 14.0 62.1 f 9.2 10.8f 7.5
9.8f 5.1 25.4 f 13.0 33.6 f 9.0 57.7 f 15.4 61.4f 14.1 68.2f 11.3 10.9 f 7.3
7.4 f 5.6 18.4f 12.9 21.2 f 5.2 44.1 f 15.0 47.4 f 8.5 55.9 f 8.5 9.0f 6.4
1.479(>0.1) 2.10 (0.05) 1.891(0.05) 1.422(>0.1) 0.991(>0.1)
1.079(>0.1) 3.19 (0.1) 0.04(>0.1) 0.51(>0.1) 1.16(>0.1)
2.5 f 2.1 12.8f 14.2 13.4f 9.4 33.9 f 18.7 43.0f 14.2 49.5 f 10.8 10.6 f 8.7
3.8f 4.2 16.2 f 16.0 25.2 f 12.9 4 7 . 414.2 ~ 58.3 k 5.6 64.3f 10.5 7.1 f 5.5
2.2 f 2.1 6.8f 7.1 11.5 f 8.3 31.2 f 16.4 43.1 k 10.0 46.8 f 9.0 8.9 f 7.8
0.432(>0.1) 1.854(>0.1) 1.647(>0.1) 2.639( 0.05). Comparison of the data obtained from fresh platelets and platelets stored at 22 C indicated
that the release from the 22 C-stored platelets was slightly lower than that from fresh platelets, but the difference was not statistically significant. The differences between the release response of platelets stored at 4 C and platelets stored at 22 C were highly significant at all thrombin concentrations above 0.03 U/ml and for both platelet concentrations (p < 0.05). The dose response curve for 5 X 10Vml of 4 Cstored platelets were nearly identical to the dose response curves for 3 x 10Vml of fresh platelets or platelets stored at 22 C (Fig. 2). Adenine Nucleotide Contcnt und Releusc
The total adenine nucleotide content of the stored platelets was not significantly different from that of fresh platelets (fresh platelets: 52.6 k 12.3 nmoles/lODplatelets; platelets stored at 4 C: 44.7 ? 6.5 nmoles/lOBplatelets; platelets stored at 22 C: 42.2 & 8.1 nmoles/lODplatelets; n = 4; p > 0. I ) . Adenine nucleotide release, measured at a platelet concentration of 5 x 10" platelets/ml, was essentially the same for both fresh and stored platelets over the range of thrombin concentrations tested (Table 2). Releuse of Culcium
.
.
.
l h r m w n h . lulMlll
FIG. 2. The effect of platelet number and thrombin concentration on the release of 14C-5-HTby fresh plateand platelets stored at 4 C).( or 22 C (A). lets ,).( Platelet concentrations are 3 x IOU platelets/ml (- - -) and 5 x IOR platelets/ml (. . . . .). The final thrombin concentrations are indicated on the abscissa.
The calcium electrode provided a continuous recording of the change in extracellular calcium concentration during the course of its release from thrombin-stimulated platelets. Representative tracings are shown of the time-course of calcium release from fresh platelets at different thrombin concentrations (Fig. 3). The amount of calcium released was directly related to thrombin
Volume 19 Number I
49
THROMBIN SENSITIVITY OF PLATELETS
Table 2. Thrombin-Induced Release of Adenine Nucleotides Storage Conditions Thrombin (Ulml) 0 0.01 0.015 0.02 0.03 0.04 0.06 0.08 0.10
Fresh 19.2 2 3 3 4 ) ' 28.8 (1) 28.9 2 4.7(3) 26.3 2 6.6(2) 36.7 ? 6.6(3) 32.0 ? 3.4(2) 50.1 2 4.0(3) 45.2 2 5.5(2) 52.5 (1)
t values (p) Fresh vs 4 C
4 C. 24 hr
22 C, 24 hr
15.52 2.3 (5) 21.5 2 2.5 (2) 24.8 ? 9.4 (4) 25.9 2 2.1 (2) 35.7 ? 6.6 (4) 46.8 ? 11.6(2) 50.5 ? 7.0 (5) 56.5 2 6.2 (2) 53.1 2 0.6 (2)
20.9? 1.8 (5) 20.4 2 3.0 (2) 24.3 ? 2.1 (4) 22.5 2 2.2 (2) 31.4 ? 3.0 (4) 32.0 ? 4.9 (2) 48.9 ? 4.2 (5) 54.4 2 10.1(2) 54.4 2 4.1 (2)
.174(p
* 0.1)
.102(p
* 0.1)
1.37 (p > 0.1)
Results as the per cent adenine nucleotides found in the supernatant 2 SE of the mean. Numbers in parentheses refer to the number of experi-
ments performed with platelets from different donors. The variation between duplicate reactions within a single experiment was less than 22%.
concentration, increasing as the thrombin concentration was increased. A typical experiment comparing fresh and stored platelets at high and low thrombin concentrations i s illustrated in Fig. 4. A t the higher thrombin concentration o f 1 U!ml, the release curves were similar for both fresh and stored platelets. However, when the thrombin concentration was low, (0.06 U/ml), the release curve for platelets stored at 4 C was higher than those for fresh platelets or platelets stored at 22 C.
It is in the release of I4C-5-HT and calcium that the greatest differences were observed between fresh and stored platelets. The most significant difference in 5-HT release between fresh platelets and those stored at 4 C was observed with a platelet concentration of 5 x lo8 platelets/ml and thrombin concentrations of 0.08 to 0.1 U/ml. This combination of platelet and thrombin concentrations gives a thrombin-to-platelet ratio of 0.016 to 0.02 U/108 platelets. When the platelet concentration was 3 x lO*/ml
Discussion
Liquid storage of platelets for 24 hours at 4 C or 22 C did not influence I4C-5-HT uptake capacity. Our data showed that total adenine nucleotide content was not reduced after 24 hours of storage at 4 C or 22 C, a finding similar to that of Murphy and Gardner.I5 Thrombin-induced release of adenine nucleotides from stored platelets was similar to that of fresh platelets. Other investigators have reported that collagen-induced ADP release was greatly reduced after 72 hours of platelet storage at 4 C or 22 C, with storage at 22 C producing the greatest reduction.6 The variations in results from different laboratories may be a reflection of the quality of the platelets used and the agent used to induce the release reaction. In addition, the method used in this study to measure adenine nucleotides does not distinguish between the various nucleotides.
I t .
I
10 soc
w
.
.
.
.
.
.
.
.
.
.
.
.
.
limo
FIG.3. Time progress curves of calcium release from fresh platelets as recorded by the calcium ion electrode. At the time indicated by the arrow, 0.02 ml thrombin is added to 2.0 rnl platelet suspension (8 x lonplatelets/ml) to provide the following final thrornbin concentrations (Unitslml): a) 1 .O; b) 0.66; c) 0.50; d) 0.25; e ) 0.125; f ) 0.06; g) 0.03.
50
ROBBLEE ET AL.
I
limo
FIG.4. Time progress curves of calcium release from fresh and stored platelets as recorded by the calcium ion electrode: a) fresh platelets; b) 4 C-stored platelets; c) 22 C-stored platelets. At the time indicated by the arrow, 0.02 ml thrombin is added to 2.0 ml platelet suspension (8 x loRplatelets/ml): A. 1.0 U/ml; B. 0.06 U/ml.
the greatest difference between fresh and 4 C-stored platelets occurred at thrombin concentrations between 0.04 and 0.06 U/ml. This combination of platelet and thrombin concentrations yields a thrombin-to-platelet ratio of 0.013 to 0.02 U/108platelets. In both series of experiments, the platelets stored at 4 C released significantly more 5-HT than did fresh platelets when the thrombin-to-platelet ratio was the order of 0.013 to 0.02 U/lOBplatelets, even though this ratio was obtained by different combinations of thrombin and platelet concentrations. Comparing the 4 C and 22 Cstored platelets in the same way, there is a similar yet wider range of thrombin-toplatelet ratios at which 4 C-stored platelets release more 5-HT than do 22 C-stored
Transfusion J~uary-Febmpry1979
platelets, 0.008 to 0.033 U/10* platelets. The similar thrombin-to-platelet ratios may indicate that there is only a narrow range of thrombin concentrations per platelet at which subtle differences between platelet populations may be detected. This range of thrombin concentrations may approximate the threshold concentration of thrombin that has been demonstrated to be required to initiate a release The increased release of “C-5-HT at low thrombin-to-platelet concentrations by 4 Cstored platelets suggests that these platelets are more sensitive to thrombin than are fresh platelets or 22 C-stored platelets. The upward displacement of both dose response curves of 4 C-stored platelets in Figure 2 indicates that these platelets required less thrombin per platelet for a release response equivalent to that of fresh platelets or platelets stored at 22 C for 24 hours. The amount of calcium released from thrombin-treated fresh or stored platelets is dependent upon the thrombin concentration, confirmingprevious ~ b s e r v a t i o n s . ~ * ~ ~ * ~ Because calcium release is also dependent upon platelet concentration, and ultimately, the thrombin-to-plateletconcentration, care was taken in the present experiments to maintain a constant platelet number when measuring calcium release as it was for measuring 5-HT and adenine nucleotide release. Platelets that were stored at 4 C released more calcium at low thrombin concentration than did fresh platelets or those stored at 22 C. This observation is consistent with the results obtained for 14C5-HT release. The greater release of 5-HT and calcium by 4 C-stored platelets is probably not the result of an increased fragility of these platelets. In other studies only two to four per cent of the total lactate dehydrogenase activity appeared in the supernatant fluid of thrombin-treated platelets and this activity was independent of thrombin concentration or previous treatment of the platelets. It is also unlikely that the increased
Volume 19 Number I
THROMBIN SENSITIVITY OF PLATELETS
sensitivity of 4 C-stored platelets is due to ADP potentiation of the thrombin-induced release reactionlB because these platelets were not observed to leak or release substantially different amounts of adenine nucleotides. The absence of difference between the 4 C-stored platelets and fresh or 22 C-stored platelets with regard to adenine nucleotide release is difficult to explain in view of the generally accepted idea that adenine nucleotides, serotonin and calcium are stored in the same granules. It may be that different mechanisms of release exist for the different substances or that one or the other are selectively released at different levels of stimulus. The greater responsiveness of 4 C-stored platelets to low concentrations of thrombin may be a factor contributing to their reduced circulation and superior hemostatic effectiveness3 Recent studies indicate that a threshold level of thrombin is necessary for initiation of the thrombin release react i ~ n , ~and J ~that this thrombin threshold may be related to the number and type of thrombin receptor sites that are occupied on the platelet^.^^*^^^*^ Platelets with a low thrombin threshold may respond to a thrombin stimulus level in the circulation that would not affect platelets with a higher thrombin requirement. Consequently they might aggregate and be removed from the circulation at a faster rate or participate more readily in hemostasis. Intrinsic alterations of the platelets may produce the differences in thrombin sensitivity after storage at 4 C. A multitude of parameters may be affected in diverse ways by different storage conditions. If the platelet secretory response is considered to be an early and necessary event in hemostasis, there are three general areas where a slight alteration of the platelets may influence their hemostatic effectiveness. One is an alteration of surface catalytic or receptor sites which affect the initial stimulus-receptorinteraction. The apparent decrease in thrombin threshold concentration of platelets
51
after storage at 4 C may result from a change in the number or type of thrombin-binding sites resulting in a change in thrombinturnover time. A decrease in turnover time would make more thrombin available to interact with successive populations of platelets and increase the effective thrombin-toplatelet ratio. A second type of platelet alteration might be in the transmission mechanism which translates the external message to the interior of the cell. A third might be the intracellular mechanism involved in bringing about the release of granular contents. The last two of these events are subject to regulation by the internal environment of the cell, including ion fluxes and concentration, cyclic nucleotide concentration, and polymerization-depolymerization equilibria of structural proteins. Obviously, these interactions are complex, and very little is known about the actual sequence of events leading from stimulus to response. Further study of platelets after liquid storage may provide some indication of the mechanisms involved in the reactions of fresh platelets. The use of a wide range of closely-spaced thrombin concentrations, coupled with different platelet concentrations, seems to be a useful way to demonstrate differences in platelet responsiveness that might otherwise by obscured. Acknowledgments This work was supported by the U.S.Navy (Naval Medical Research and Development Command Research Task No. M0095-PN.001-0040) and by the National Institute of Health Grant HL-16714-04. The opinions or assertions contained herein are those of the authors, and are not to be construed as official or reflecting the views of the Navy Department or Naval Service at large. We wish to thank Dr. Jonathan L. Costa, NIH, for his comments and suggestions related to this article, and to Laurel Sutton for her assistance in preparing this manuscript.
References I . Abramowitz, J., A. Stracher, and T. C. Detwiler: Proteolysis of myosin during platelet storage. J. Clin. Invest. 53:1493, 1974. 2. Ando, Y . , M. Steiner, and M. Baldini: Effect of chilling on membrane related functions of platelets. Transfusion 14453, 1974.
52
ROBBLEE ET AL.
M. Hamid, and D. N. Calvert: Storage of platelet concentrates at 4°C; use of refrigerated platelet concentrates in the treatment of hemorrhage in thrombocytopenic patients. I n : Platelets; Production, Function, Transfusion and Storage, M. G. Baldini and S. Ebbe, Eds. New York, Grune and Stratton, 1974, p. 361. Becker, G. A., M. Tuccelli, T. Kunicki, M. K. Chalos, and R. H. Aster: Studies of platelet concentrates stored at 22 C and 4 C. Transfusion 13:61, 1973. Detwiler, T. C., and R. D. Feinman: Kinetics of the thrombin-induced release of calcium (11) by platelets. Biochemistry 12: 282, 1973. Filip, D. J., J. D. Eckstein, and C. A. Sibley: The effect of platelet concentrate storage temperature on adenine nucleotide metabolism. Blood 45749, 1975. George, J. N.: Platelet membrane glycoproteins: alteration during storage of human platelet concentrates. Thrombosis Res. 8:719, 1976. Kattlove, H. E., and B. Alexander: The effect of cold on platelets. I. Cold-induced platelet aggregation. Blood 3839, 1971. -, B. Alexander, and F. White: The effect of cold on platelets. 11. Platelet function after shortterm storage at cold temperatures. Blood 40:
3. Aster, R. H., G. A. Becker,
4.
5.
6.
7.
8.
9.
688, 1972. 10. Kim, B. K.. and M. G. Baldini: The osmotic platelet response as an indicator of platelet viabilityduringstorage. Fed. Proc. 29:717, 1970. 11. , and M. G. Baldini: Glycolytic intermediates and adenine nucleotides of human platelets. 11. Effect of short-term storage at 4 C. Transfusion 12: 1, 1972. 12. Kornstein, L. B., L. S. Robblee and D. Shepro: Thrombin-platelet interactions investigated with the calcium electrode. Thrombosis Res. 11:471, 1977. 13. Kotelba-Witkowska, B., H. Holmsen, and E. H. Muer: Storage of human platelets: effects on metabolically active ATP and on the release reaction. Br. J. Haematol. 22:429, 1972. 14. Massini, P., and E. F. Luscher: Some effects of ionophores for divalent cations on blood platelets: Comparison with the effects of thrombin. Biochim. Biophys. Acta 372:109, 1974. 15. Murphy, S., and F. H. Gardner: Platelet storage at 22°C. Metabolic, morphologic and functional studies. J. Clin. Invest. 50:370, 1971. 16. Packham, M. A.. M. A. Guccione. P.-L. Chang,
17.
18.
19.
20.
21.
Transfusion January-February 1979
and J. F. Mustard: Platelet aggregation and release: effects of low concentrations of thrombin and collagen. Am. J. Physiol. 22538, 1973. Phillips, D. R.: Thrombin interaction with human platelets. Potentiation of thrombin-induced aggregation and release by inactivated thrombin. Thromb. Diath. Haemorrh. 32:207, 1974. -,and P. Agin: Thrombin substrates and the proteolytic site of thrombin action on humanplatelet plasma membranes. Biochim. Biophys. Acta. 352:218, 1974. Robblee, L. S., L. B. Kornstein, and D. Shepro: Calcium electrode. A method for the continuous monitoring of the platelet release reaction. Thrombosis and Haemostasis 37:407, 1977. , and D. Shepro: The effect of external calcium and lanthanum on platelet calcium content andon the release reaction. Bioch. Biophys. Acta 436:448. 1976. Salganicoff, L., P. A. Hebda, J. Yandrasitz, and M. H. Fukami: Subcellular fractionation of pig platelets. Biochim. Biophys. Acta 385:394,
-
1975. 22. Shively, J. A., C. L. Gott, and D. S. deJongh:
23.
24.
25.
26.
The effect of storage on adhesion and aggregation of platelets. Vox Sang. 18:204. 1970. Tollefsen, D. M.,J. R. Feagler, and P. W. Majerus: The binding of thrombin to the surface of human platelets. J. Biol. Chem. 249:2646, 1974. Valeri, C. R.: Hemostatic effectiveness of liquidpreserved and previously frozen human platelets. N. Engl. J. Med. 290:353. 1974. White, J. G., and W. Krivit: An ult~astructural basis for the shape changes induced in platelets by chilling. Blood 30:625, 1967. Zucker, M. B., and J. Borrelli: Reversible alterations in platelet morphology produced by anticoagulants and by cold. Blood 9:602, 1954.
Lois S. Robblee, Ph.D., Research Associate, EIC, 67 Chapel Street, Newton, Massachusetts.
David Shepro, Ph.D., Professor, Boston University, Microvascular Research, 2 Cummington Street, Boston, Massachusetts 02215. J . J . Vecchione, M.D., Research Medical Officer, Naval Blood Research Laboratory, 615 Albany Street, Boston, Massachusetts 021 18. C. Robert Valeri, M.D., Officer-in-Charge, Naval Blood Research Laboratory.
