THROMBOSIS RESE,\RCH Printed in the United States
~01. 6, pp. 1j9-h68, 1975 Pergamon Press, Inc.
INVESTIGATIONS ON THE PRESERVATION OF HUIAN PLATELETS I, THE EFFECT OF GLYCEROL ON PLATELET METABOLISM AND FUNCTION K.Undeutsch, H.Reuter, and R.Gross Medizinische Universitatsklinik Koln, Germany
(Received 20.2.1975.
Accepted by Editor M.I. Barnhart)
ABSTRACT Because of the favourable effect of glycerol as a cryoprotective agent for the preservation of erythrocytes in the frozen state, the effect of glycerol on metabolism and functional capacity of platelets was examined to determine if this substance could also be used for the preservation of platelets. Platelet functions in the presence and absence of glycerol were studied by means of the ADP- and adrenaline-induced aggregation (BORN-test). In the presence of glycerol, higher ATP-levels were found in the platelets than in the controls, thus indicating a membrane stabilizing effect of glycerol. A maximal stabilization was obtained with 0.1 - 0.5 M glycerol. The membrane stabilizing effect of 0.3 M glycerol was paralleled by a 54% inhibition of ADPinduced aggregation and a 70% inhibition of adrenaline-induced aggregation. The spreading capacity of the platelets, which depends on an intact metabolism, decreased with increasing glycerol concentration. A 20% inhibition of the spreading capacity was obtained with 1.0 M glycerol. Platelet sediments prepared from platelet-rich plasma in the presence of glycerol could not be resuspended as readily as sediments prepared from pure platelet-rich plasma in the absence of glycerol. This work was supplied by the SFB 68 of the DEUTSCHE FORSCHUNGSGEMEINSCHAFT.
459
460
PRESERVATION HUMAJi PLATELETS
Vo1.6,No.6
For the therapy of bleeding disorders resulting from platelet defects, an adequate supply of functionally intact platelets is necessary. Therefore during the last years increasing interest was directed to preservation methods. Methods used for the preservation of whole blood e.g., storing at 4OC, are not practicable for the preservation of platelets. In titrated whole blood as well as in titrated platelet-rich plasma spontaneous aggregates form within a short period of time. Also by addition of substances active in platelet metabolism, such as adenine, glucose and nicotinamide, platelets can not be prevented from aggregation. More recently, by addition of PGE1 directly after blood withdrawal, spontaneous aggregation could be inhibited for a short period of time. Long term preservation, however could not be performed by these methods. Investigations on the preservation of erythrocytes in the solid and liquid state (below and above the freezing point] have shown that optimal results can be obtained by the use of glycerol (1, 2). As some investigators could show, glycerol seems to be useful for the preservation of platelets also (3, 4) * Concerning the preservation of platelets it was of special interest to examine the effect of glycerol on metabolism and functional activity of platelets. Therefore platelet functions in the presence and absence of glycerol were studied by means of the spreading test and by means of the ADP- and adrenalineinduced aggregation. The spreading capacity of platelets has proved to be the most sensitive indicator of platelet metabolism as could be demonstrated by the effect on spreading of inhibitors of glycolysis and oxidative metabolism (5, 6). MATERIALS AND METHODS 1.
Preparation of platelet-rich plasma
Platelet-rich plasma (PRP) was prepared using EDTA and citrate as anticoagulants as described in this journal, ~01.3, 307-325 (1973). Preparation of EDTA-plasma was carried out at
Vo1.6,So.6
PRESERVXTION
HUMX?J PLXTELETS
4OC, of titrated
plasma
at room temperature.
2. Determination
of the spreading
capacity
capacity 0.5 ml ciFor the determination of the spreadin trated PRP containing 278000 platelets/mm 8 (obtained by mixing PRP and PPP) was diluted by 9.5 ml physiological saline solution. Using EDTA-plasma, 0.45 ml PRP were mixed with 0.05 ml 2x10-2 M CaC12-solution and the mixture diluted by 9.5 ml physiological saline solution. An aliquote of the diluted sample (0.5 ml) was transferred to a siliconized glass slide and this placed in a moist chamber for 60 minutes. Then the slides were rinsed by dipping 10 times in a solution of 3.8% trisodiumcitrate in saline. After fixation of the samples with glutardialdehyde for 15 minutes the slides were rinsed in destilled water and airdried. The percentage of spreaded forms was determined using a phase contrast microscope. 3. Determination method
of platelet
aggregation
by the photometric
Aggregation was induced in titrated plasma by addition of 0.1 ml 40 E_IMADP and adrenalin respectively to 0.9 ml PRP containing 278000 platelets/mm3. The course of aggregation was registered and the increase of transmission per minute in the linear part of the aggregation curve (ctgti) and the maximum change of transmission were determined. For further details see this journal 2, 307-325 (1973). 4. Determination
of substrates
10 ml platelet-rich EDTA-plasma was centrifuged at 5500-g and the sediments resuspended in 2 ml WARBURG-buffer (6.055 g TRIS, 1.07 g MgEDTA, 0.336 g Kcl, 5.84 g NaCl, 1.8 g glucose, 30 ml M/15 SOERENSEN-phosphate buffer, pH 7.4 ad 1000 ml). After one hour incubation at 37oC, 2 ml cold perchloric acid (6%) were added, the samples refrigerated at 4OC for 30 minutes, and then centrifuged at 5500-g. Three ml of the supernatant after addition of 1 ml saturated K2C03-solution were stored in the refrigerator, centrifuged and the supernatant used for the determination of the substrates ATP, ADP, AMP, lactate, and pyruvate according to the test instructions of BIOCHEMICA BOEHRINGER. For testing the oxygen consumption and the formation of CO2 platelet sediments were resuspended in WARBURG-buffer, the suspension introduced into the WARBURGvessels and incubated for 1 hour at 370C. 5.
Determination
of enzymes
Platelet sediments from 10 ml PRP were resuspended in 4.5 ml 5~10~~ M TRA-buffer, pH 7.6 and 0.5 ml of a saturated digitonin solution. After lysis of the platelets the cytolysate was centrifuged at 5500-g and the supernatant used for the analysis of enzymes according to the test instructions of BIOCHEMICA BOEHRINGER.
$61
PRESERVATION
462
H-WAN
Vo1.6,No.6
PLATELETS
RESULTS
1. Effect of glycerol on platelet metabolism Platelet-rich plasma was incubated in the presence of 0.1, 0.5,
and 1.0 M glycerol for 3 hours at 370C and ATP, ADP, AMP,
lactate, pyruvate, and oxygen-consumption of the platelets were determined. The results in part are summarized in table I. Highest ATP-values were obtained in the presence of 0.1 and 0.5 M glycerol. Parallel to the elevated ATP-values oxygenconsumption increased in the presence of 0.1 and 0.5 M glycerol, while lactate formation decreased with increasing glycerol concentration. Tab. I ATP, lactate, and oxygen-consumption in platelets, incubated in the presence of 0.01, 0.1, 0.5, and 1.0 M glycerol (3 h, 37OC, mean values of 5 testpersons)
control
0.01 M
ATP
100%
102%
Lactate
100%
92%
02-Consumption
100%
98%
0.1 M
0.5 M
1.0 M
111%
108%
104%
80%
81%
66%
116%
115%
95%
The enzymatic capacity of the platelets was not affected significantly by concentrations as high as 1 M glycerol during 24 hours incubation at room temperature. 2. Effect of glycerol on the spreading capacity of platelets Spreading tests were performed in the presence of 0.1 M (0.92%), 0.3 M (2.76%), 0.5 M (4.6%), 0.7 M (6.44%), and 1.0 M (9.2%) glycerol. As is shown in figure 1 with increasing concentrations of glycerol the percentage of spreaded forms decreased. 1
M glycerol inhibited the spreading capacity of the plate-
lets to about
20%.
PRESERV.XTI03 HUYU
THE EFFECT OF GLYCEROL SPREADING OF PLATELETS
363
PL_XTELETS
ON THE
20_
cnonspreaded
0.5
0.3
0,’
0.7
Fig.
platelets
1.0 m glycerol
1
The effect of 0.1 - 1.0 M glycerol on the spreading capacity of the platelets
3.
Effect of glycerol on the resuspension ments obtained from EDTA- and titrated Platelet
plasma
sediments
obtained
by centrifuging
platelet-poor
with
plasma without
cause of the formation
from titrated
a significant
presence
resuspension spreading
EDTA-plasma
and absence
dose-dependant
aggregates.
sediments
of glycerol.
Glycerol
(figures 2-4).
further
in
loss beAddition
in resuspen-
obtained
The spreading
of the platelets
manner
platelet
could be resuspended
was decreased.
capacity
platelet-rich
did not cause an increase
sion. On the other hand, platelet platelet-rich
sedi-
1000-g can not be resuspended
of irreversible
of 0.5 - 1.0 M glycerol
of platelet plasma
from
readily
in the
capacity
decreased
after resuspension
after
the in a
464
PRESERVXTIOX
HUNAN
vo1.6,?io.6
PLATELETS
%
60
a Fig. 2
a Fig. 3
b
b
a Fig. 4
b
The effect of resuspension on the spreading of platelets in EDTA-plasma (black columns = % spreaded forms) Fig. 2 Fig. 3 Fig. 4
Spreading capacity in the absence of glycerol Spr. cap. in the presence of 0.5 M glycerol Spr. cap. in the presence of 1.0 M glycerol
a) Before resuspension
4. The effect of glycerol
b) After resuspension
on the aggregation
of platelets
% aggregation I aggregation induced by 4Omp adrenalin
El aggregation induced by L0mj.l ADP
control
0,lm
02-n
0,3m
Fig. 5 Inhibition
of adrenalin- and ADP-induced glycerol
aggregation
by
PRESERVXTIOX
vo1.6,?;0.6
Aggregation
of platelets
plasma by adrenalin
induced
in titrated
and ADP respectively
cerol in a dose-dependant
$63
HUXAJi PLATELETS
manner
platelet-rich
is inhibited
by gly-
(figure 5).
DISCUSSIOS Preservation -196OC during
of erythrocytes
at temperatures
the last years has become
preserving
the functional
has proved
to be a suitable
of -80 to
a routine method.
state of the erythrocytes, cryoprotective
others polyvinylpyrrolidone,
dextranes,
For
glycerol
substance.
Among
dimethylsulfoxide
(DMSO) and glycerol
have been tested for their cryoprotective
effect on platelets
(7, 8, 9). It could be shown, that DMSO
has a maximal
protective
effect
as compared
On the other hand the possibly
stances.
DMSO have prevented
the general
rest to investigate capacity
toxic side effects
use of this compound
Therefore
field of cell preservation.
The ATP-content
of
in the
it was of special
the effect of glycerol
of platelets
to the other sub-
inte-
on the functional
(10, 11, 12).
of platelets
cator of their metabolic
has been found to be an indi-
state and of their membrane
stabili-
ty* A high ATP-content of the platelets depends on an intact oxydative metabolism. Intracellular ATP can be provided in part by glycolysis. brane induces
Activation
the destruction
lity of the membrane and consequently lic activity. of glycerol Optimal
Our experiments
stabilization
sufficient glycerol,
cerol in plasma.
that in the presence
membranes
is obtained
As preliminary effect
experiments is obtained
to an approximate
0.3 M glycerol
curves obtained
by 0.1
of aggregation
to the membrane
have shown,
a
by at least 0.3 M
10% solution
aggregation
in the presence
by
stabilizing
inhibits ADP-induced
tion by 54%, and adrenalin-induced aggregation
and loss of metabo-
Thus the 54% inhibition
corresponding
the permeabi-
are found than in the controls.
of platelet
cryoprotective
mem-
ADP and other substances,
have shown,
can be attributed
effect of glycerol.
in the platelet
increases
about aggregation
higher ATP-levels
0.3 M glycerol
of ATP,
for endogeneous
brings
to 0.5 M glycerol.
of ATPases
of gly-
aggrega-
by 70%. The
of 0.3 M glycerol
Vo1.6,No.6
PRESERVXTIOY HU!LkS PLATELETS
b66
can still be evaluated. Thus it is not necessary to separate platelets from the preservation medium and to resuspend the platelet sediments in platelet-poor plasma, as required, if DMSO, PVP, or dextranes are used as cryoprotective substances. Investigations of the spreading capacity of the platelets which depends on an intact metabolism have shown that by increasing glycerol concentrations (0.1 - 1.0 M) this platelet function decreases (20% decrease
in the presence
of 1.0 M gly-
cerol). Extensive statistical evaluation of the experiments concerning the spreading capacity of the platelets at different glycerol concentrations by means of the BARTLETT test, variance analysis, and the DUNCAN test have shown that with 5 test persons the difference in glycerol concentration must be 0.3 M for significant differences to occur. It can be expected that by an increasing number of test persons significant differences in spreading capacity will already occur at concentration differences of less than 0.3 M glycerol. The results of the resuspension experiments in the presence of glycerol are in agreement with reports of other authors (13, 14, 15). In EDTA-plasma resuspension of platelets could easily be performed, while in titrated plasma aggregates formed. Platelet sediments obtained from PRP in the presence of glycerol seem to be more sensitive in regard to resuspension than those obtained from pure PRP. Probably platelets are damaged by glycerol in a dose-dependant manner. After resuspension the spreading capacity of glycerol-injured platelets is inhibited to a higher extent than that of platelets which have not been treated with glycerol. BREDDIN et al (16) have stated that washed platelets have a reduced functional capacity. The reduced spreading capacity of glycerol-treated platelets indicates an intracellular effect of glycerol. Not clear is if glycerol permeates all platelets, but inhibits the spreading capacity of those platelets only whose metabolism has been impaired by sedimentation and resuspension, or if glycerol permeates only platelets with an impaired metabolism which consequently
are inhibited
in their spreading
capacity.
PRESERVXTIOS
VOl.d,SO.6
As TIBBLING
A67
PLXTELETS
(17) could show, the amount of glycerol
rated by platelets these findings permeates
HUMX?
depends
on the number
it seems reasonable
incorpo-
of platelets.
to assume
By
that glycerol
all platelets.
Furthermore
glycerol
after permeation
brane could cause an insensitiveness exogeneous
the spreading
capacity
rol concentration lets become
Despite
stimulation.
of the platelets
would decrease, against
mem-
against
of an intact energy metabolism
of the platelets
insensitive
of the platelet
with increasing
because
glyce-
more and more plate-
exogeneous
stimulation
of
spreading.
REFERENCES
1.
TULLIS,J.L.,KETCHEL,M.M.,PYLE,H.M.,PENNEL,R.B.,GIBSON,J.G., TINCH,R.J.,and DRISOOLL,S.G.: Studies on the In Vivo Survival of Glycerolized and Frozen Human Red Blood Cells. J.Amer.med.Ass.: 168, 399, 1958.
2.
HURN,B.A.L.: Storage York, 1968, p. 58.
3.
and BARR,M.A.: BALLINGER,W.F., WEISE,A.J.,JACKSON,L.G., In Vivo and In Vitro Survival of Glycerolized Frozen Platelets. J.Amer.med.Ass.: 179, 148, 1962.
4.
COHEN,P. and GARDNER,F.H.: Platelet Preservation. IV. Preservation of Human Platelet Concentrates by Controlled Slow Freezing in a Glycerol Medium. New Engl.J.Med.5 274, 1400, 1966.
5.
BREDDIN,K. and BURCK,K.H.: Zur Klinik der Thrombozytenfunktionsstorungen unter besonderer Berticksichtigung der Ausbreitungsfahigkeit der Thrombozyten an silikonisierten Glasflachen. Thrombos.Diathes.haemorrh.(Stuttg.): 2, 525, 1963.
6.
BREDDIN,K. and LANGBEIN,H.: Uber den EinfluJ3 verschiedener Stoffwechselhemmer auf die Thrombozytenfunktion. Thrombos. Diathes.haemorrh.(Stuttg.): 10, 29, 1963.
7.
DJERASSI,I. Hemorrhage: 1965.
8.
GARDNER,F.H.: 2, 42, 1968.
of Blood. Academic
Press,
London-New
and FARBER,S.: Control and Prevention of Platelet Transfusion. Cancer Res.: 25, 1499, Platelet
Transfusion
Problems.
Cryobiology:
465
PRESERVXTION HU?LqN PLXTELETS
vo1.6,?1:0.6
9.
STREIFF,F.,ALEXANDRE,P.,STOLTZ,J.-F., and GENETET,B.: Conservation et transfusion de concentrds plaquettaires congeles d -800 en DMSO. Etude technique, biologique et clinique. Ann.Biol.Clin.: -28, 295, 1970.
10.
CRONBERG,S.,ROBERTSON,B.,NILSON,I.M., and NILEHN,J.-E.: Suppressive Effect of Dextran on Platelet adhesiveness. Thrombos.Diathes.haemorrh.(Stuttg.): -16, 384, 1968.
11.
SCHNEIDER,J.A. and SANBAR,S.S.: Plasma Expanders and Platelet Adhesiveness. Clin.Res.: 2, 456, 1968
12.
REUTER,H. and GROSS,R.: Preservation of Human Platelets at Low Temperature and Methods for the Estimation of the Functional Efficiency of Preserved Platelets. Proc. 12th Conqr. int. Sot. Blood Transf .,Moscow 1969. Bibl.haemat.: x,-376, 1971.
13.
GARDNER,F.H.,HOWELL,D., and HIRSCH,E.O.: Platelet Transfusions Utilizing Plastic Equipment. J.Lab.clin.Med.: 43, 196, 1954
14.
AAS,K. and GARDNER,F.H.: Survival of Blood Platelets Labelled with Chromium51. J.Clin.Invest.: 37, 1257, 1958.
15.
ASTER,R.H. and JANDL,J.H.: Platelet Sequestration in Man. I. Methods. J.Clin.Invest.: 43, 843, 1964.
16.
BREDDIN,K.,FRITZSCHE,W.,and SPIELMANN,W.: Zur Frage der Thrombozytenkonservierung. Klin.Wschr.: 42, 180, 1964.
17.
TIBBLING,G.: Glycerol Uptake in Leucocytes and Thrombocytes. Scand.J.clin.Lab.Invest.: 2, 185, 1970.
~01. 6, pp. 1j9-h68, 1975 Pergamon Press, Inc.
INVESTIGATIONS ON THE PRESERVATION OF HUIAN PLATELETS I, THE EFFECT OF GLYCEROL ON PLATELET METABOLISM AND FUNCTION K.Undeutsch, H.Reuter, and R.Gross Medizinische Universitatsklinik Koln, Germany
(Received 20.2.1975.
Accepted by Editor M.I. Barnhart)
ABSTRACT Because of the favourable effect of glycerol as a cryoprotective agent for the preservation of erythrocytes in the frozen state, the effect of glycerol on metabolism and functional capacity of platelets was examined to determine if this substance could also be used for the preservation of platelets. Platelet functions in the presence and absence of glycerol were studied by means of the ADP- and adrenaline-induced aggregation (BORN-test). In the presence of glycerol, higher ATP-levels were found in the platelets than in the controls, thus indicating a membrane stabilizing effect of glycerol. A maximal stabilization was obtained with 0.1 - 0.5 M glycerol. The membrane stabilizing effect of 0.3 M glycerol was paralleled by a 54% inhibition of ADPinduced aggregation and a 70% inhibition of adrenaline-induced aggregation. The spreading capacity of the platelets, which depends on an intact metabolism, decreased with increasing glycerol concentration. A 20% inhibition of the spreading capacity was obtained with 1.0 M glycerol. Platelet sediments prepared from platelet-rich plasma in the presence of glycerol could not be resuspended as readily as sediments prepared from pure platelet-rich plasma in the absence of glycerol. This work was supplied by the SFB 68 of the DEUTSCHE FORSCHUNGSGEMEINSCHAFT.
459
460
PRESERVATION HUMAJi PLATELETS
Vo1.6,No.6
For the therapy of bleeding disorders resulting from platelet defects, an adequate supply of functionally intact platelets is necessary. Therefore during the last years increasing interest was directed to preservation methods. Methods used for the preservation of whole blood e.g., storing at 4OC, are not practicable for the preservation of platelets. In titrated whole blood as well as in titrated platelet-rich plasma spontaneous aggregates form within a short period of time. Also by addition of substances active in platelet metabolism, such as adenine, glucose and nicotinamide, platelets can not be prevented from aggregation. More recently, by addition of PGE1 directly after blood withdrawal, spontaneous aggregation could be inhibited for a short period of time. Long term preservation, however could not be performed by these methods. Investigations on the preservation of erythrocytes in the solid and liquid state (below and above the freezing point] have shown that optimal results can be obtained by the use of glycerol (1, 2). As some investigators could show, glycerol seems to be useful for the preservation of platelets also (3, 4) * Concerning the preservation of platelets it was of special interest to examine the effect of glycerol on metabolism and functional activity of platelets. Therefore platelet functions in the presence and absence of glycerol were studied by means of the spreading test and by means of the ADP- and adrenalineinduced aggregation. The spreading capacity of platelets has proved to be the most sensitive indicator of platelet metabolism as could be demonstrated by the effect on spreading of inhibitors of glycolysis and oxidative metabolism (5, 6). MATERIALS AND METHODS 1.
Preparation of platelet-rich plasma
Platelet-rich plasma (PRP) was prepared using EDTA and citrate as anticoagulants as described in this journal, ~01.3, 307-325 (1973). Preparation of EDTA-plasma was carried out at
Vo1.6,So.6
PRESERVXTION
HUMX?J PLXTELETS
4OC, of titrated
plasma
at room temperature.
2. Determination
of the spreading
capacity
capacity 0.5 ml ciFor the determination of the spreadin trated PRP containing 278000 platelets/mm 8 (obtained by mixing PRP and PPP) was diluted by 9.5 ml physiological saline solution. Using EDTA-plasma, 0.45 ml PRP were mixed with 0.05 ml 2x10-2 M CaC12-solution and the mixture diluted by 9.5 ml physiological saline solution. An aliquote of the diluted sample (0.5 ml) was transferred to a siliconized glass slide and this placed in a moist chamber for 60 minutes. Then the slides were rinsed by dipping 10 times in a solution of 3.8% trisodiumcitrate in saline. After fixation of the samples with glutardialdehyde for 15 minutes the slides were rinsed in destilled water and airdried. The percentage of spreaded forms was determined using a phase contrast microscope. 3. Determination method
of platelet
aggregation
by the photometric
Aggregation was induced in titrated plasma by addition of 0.1 ml 40 E_IMADP and adrenalin respectively to 0.9 ml PRP containing 278000 platelets/mm3. The course of aggregation was registered and the increase of transmission per minute in the linear part of the aggregation curve (ctgti) and the maximum change of transmission were determined. For further details see this journal 2, 307-325 (1973). 4. Determination
of substrates
10 ml platelet-rich EDTA-plasma was centrifuged at 5500-g and the sediments resuspended in 2 ml WARBURG-buffer (6.055 g TRIS, 1.07 g MgEDTA, 0.336 g Kcl, 5.84 g NaCl, 1.8 g glucose, 30 ml M/15 SOERENSEN-phosphate buffer, pH 7.4 ad 1000 ml). After one hour incubation at 37oC, 2 ml cold perchloric acid (6%) were added, the samples refrigerated at 4OC for 30 minutes, and then centrifuged at 5500-g. Three ml of the supernatant after addition of 1 ml saturated K2C03-solution were stored in the refrigerator, centrifuged and the supernatant used for the determination of the substrates ATP, ADP, AMP, lactate, and pyruvate according to the test instructions of BIOCHEMICA BOEHRINGER. For testing the oxygen consumption and the formation of CO2 platelet sediments were resuspended in WARBURG-buffer, the suspension introduced into the WARBURGvessels and incubated for 1 hour at 370C. 5.
Determination
of enzymes
Platelet sediments from 10 ml PRP were resuspended in 4.5 ml 5~10~~ M TRA-buffer, pH 7.6 and 0.5 ml of a saturated digitonin solution. After lysis of the platelets the cytolysate was centrifuged at 5500-g and the supernatant used for the analysis of enzymes according to the test instructions of BIOCHEMICA BOEHRINGER.
$61
PRESERVATION
462
H-WAN
Vo1.6,No.6
PLATELETS
RESULTS
1. Effect of glycerol on platelet metabolism Platelet-rich plasma was incubated in the presence of 0.1, 0.5,
and 1.0 M glycerol for 3 hours at 370C and ATP, ADP, AMP,
lactate, pyruvate, and oxygen-consumption of the platelets were determined. The results in part are summarized in table I. Highest ATP-values were obtained in the presence of 0.1 and 0.5 M glycerol. Parallel to the elevated ATP-values oxygenconsumption increased in the presence of 0.1 and 0.5 M glycerol, while lactate formation decreased with increasing glycerol concentration. Tab. I ATP, lactate, and oxygen-consumption in platelets, incubated in the presence of 0.01, 0.1, 0.5, and 1.0 M glycerol (3 h, 37OC, mean values of 5 testpersons)
control
0.01 M
ATP
100%
102%
Lactate
100%
92%
02-Consumption
100%
98%
0.1 M
0.5 M
1.0 M
111%
108%
104%
80%
81%
66%
116%
115%
95%
The enzymatic capacity of the platelets was not affected significantly by concentrations as high as 1 M glycerol during 24 hours incubation at room temperature. 2. Effect of glycerol on the spreading capacity of platelets Spreading tests were performed in the presence of 0.1 M (0.92%), 0.3 M (2.76%), 0.5 M (4.6%), 0.7 M (6.44%), and 1.0 M (9.2%) glycerol. As is shown in figure 1 with increasing concentrations of glycerol the percentage of spreaded forms decreased. 1
M glycerol inhibited the spreading capacity of the plate-
lets to about
20%.
PRESERV.XTI03 HUYU
THE EFFECT OF GLYCEROL SPREADING OF PLATELETS
363
PL_XTELETS
ON THE
20_
cnonspreaded
0.5
0.3
0,’
0.7
Fig.
platelets
1.0 m glycerol
1
The effect of 0.1 - 1.0 M glycerol on the spreading capacity of the platelets
3.
Effect of glycerol on the resuspension ments obtained from EDTA- and titrated Platelet
plasma
sediments
obtained
by centrifuging
platelet-poor
with
plasma without
cause of the formation
from titrated
a significant
presence
resuspension spreading
EDTA-plasma
and absence
dose-dependant
aggregates.
sediments
of glycerol.
Glycerol
(figures 2-4).
further
in
loss beAddition
in resuspen-
obtained
The spreading
of the platelets
manner
platelet
could be resuspended
was decreased.
capacity
platelet-rich
did not cause an increase
sion. On the other hand, platelet platelet-rich
sedi-
1000-g can not be resuspended
of irreversible
of 0.5 - 1.0 M glycerol
of platelet plasma
from
readily
in the
capacity
decreased
after resuspension
after
the in a
464
PRESERVXTIOX
HUNAN
vo1.6,?io.6
PLATELETS
%
60
a Fig. 2
a Fig. 3
b
b
a Fig. 4
b
The effect of resuspension on the spreading of platelets in EDTA-plasma (black columns = % spreaded forms) Fig. 2 Fig. 3 Fig. 4
Spreading capacity in the absence of glycerol Spr. cap. in the presence of 0.5 M glycerol Spr. cap. in the presence of 1.0 M glycerol
a) Before resuspension
4. The effect of glycerol
b) After resuspension
on the aggregation
of platelets
% aggregation I aggregation induced by 4Omp adrenalin
El aggregation induced by L0mj.l ADP
control
0,lm
02-n
0,3m
Fig. 5 Inhibition
of adrenalin- and ADP-induced glycerol
aggregation
by
PRESERVXTIOX
vo1.6,?;0.6
Aggregation
of platelets
plasma by adrenalin
induced
in titrated
and ADP respectively
cerol in a dose-dependant
$63
HUXAJi PLATELETS
manner
platelet-rich
is inhibited
by gly-
(figure 5).
DISCUSSIOS Preservation -196OC during
of erythrocytes
at temperatures
the last years has become
preserving
the functional
has proved
to be a suitable
of -80 to
a routine method.
state of the erythrocytes, cryoprotective
others polyvinylpyrrolidone,
dextranes,
For
glycerol
substance.
Among
dimethylsulfoxide
(DMSO) and glycerol
have been tested for their cryoprotective
effect on platelets
(7, 8, 9). It could be shown, that DMSO
has a maximal
protective
effect
as compared
On the other hand the possibly
stances.
DMSO have prevented
the general
rest to investigate capacity
toxic side effects
use of this compound
Therefore
field of cell preservation.
The ATP-content
of
in the
it was of special
the effect of glycerol
of platelets
to the other sub-
inte-
on the functional
(10, 11, 12).
of platelets
cator of their metabolic
has been found to be an indi-
state and of their membrane
stabili-
ty* A high ATP-content of the platelets depends on an intact oxydative metabolism. Intracellular ATP can be provided in part by glycolysis. brane induces
Activation
the destruction
lity of the membrane and consequently lic activity. of glycerol Optimal
Our experiments
stabilization
sufficient glycerol,
cerol in plasma.
that in the presence
membranes
is obtained
As preliminary effect
experiments is obtained
to an approximate
0.3 M glycerol
curves obtained
by 0.1
of aggregation
to the membrane
have shown,
a
by at least 0.3 M
10% solution
aggregation
in the presence
by
stabilizing
inhibits ADP-induced
tion by 54%, and adrenalin-induced aggregation
and loss of metabo-
Thus the 54% inhibition
corresponding
the permeabi-
are found than in the controls.
of platelet
cryoprotective
mem-
ADP and other substances,
have shown,
can be attributed
effect of glycerol.
in the platelet
increases
about aggregation
higher ATP-levels
0.3 M glycerol
of ATP,
for endogeneous
brings
to 0.5 M glycerol.
of ATPases
of gly-
aggrega-
by 70%. The
of 0.3 M glycerol
Vo1.6,No.6
PRESERVXTIOY HU!LkS PLATELETS
b66
can still be evaluated. Thus it is not necessary to separate platelets from the preservation medium and to resuspend the platelet sediments in platelet-poor plasma, as required, if DMSO, PVP, or dextranes are used as cryoprotective substances. Investigations of the spreading capacity of the platelets which depends on an intact metabolism have shown that by increasing glycerol concentrations (0.1 - 1.0 M) this platelet function decreases (20% decrease
in the presence
of 1.0 M gly-
cerol). Extensive statistical evaluation of the experiments concerning the spreading capacity of the platelets at different glycerol concentrations by means of the BARTLETT test, variance analysis, and the DUNCAN test have shown that with 5 test persons the difference in glycerol concentration must be 0.3 M for significant differences to occur. It can be expected that by an increasing number of test persons significant differences in spreading capacity will already occur at concentration differences of less than 0.3 M glycerol. The results of the resuspension experiments in the presence of glycerol are in agreement with reports of other authors (13, 14, 15). In EDTA-plasma resuspension of platelets could easily be performed, while in titrated plasma aggregates formed. Platelet sediments obtained from PRP in the presence of glycerol seem to be more sensitive in regard to resuspension than those obtained from pure PRP. Probably platelets are damaged by glycerol in a dose-dependant manner. After resuspension the spreading capacity of glycerol-injured platelets is inhibited to a higher extent than that of platelets which have not been treated with glycerol. BREDDIN et al (16) have stated that washed platelets have a reduced functional capacity. The reduced spreading capacity of glycerol-treated platelets indicates an intracellular effect of glycerol. Not clear is if glycerol permeates all platelets, but inhibits the spreading capacity of those platelets only whose metabolism has been impaired by sedimentation and resuspension, or if glycerol permeates only platelets with an impaired metabolism which consequently
are inhibited
in their spreading
capacity.
PRESERVXTIOS
VOl.d,SO.6
As TIBBLING
A67
PLXTELETS
(17) could show, the amount of glycerol
rated by platelets these findings permeates
HUMX?
depends
on the number
it seems reasonable
incorpo-
of platelets.
to assume
By
that glycerol
all platelets.
Furthermore
glycerol
after permeation
brane could cause an insensitiveness exogeneous
the spreading
capacity
rol concentration lets become
Despite
stimulation.
of the platelets
would decrease, against
mem-
against
of an intact energy metabolism
of the platelets
insensitive
of the platelet
with increasing
because
glyce-
more and more plate-
exogeneous
stimulation
of
spreading.
REFERENCES
1.
TULLIS,J.L.,KETCHEL,M.M.,PYLE,H.M.,PENNEL,R.B.,GIBSON,J.G., TINCH,R.J.,and DRISOOLL,S.G.: Studies on the In Vivo Survival of Glycerolized and Frozen Human Red Blood Cells. J.Amer.med.Ass.: 168, 399, 1958.
2.
HURN,B.A.L.: Storage York, 1968, p. 58.
3.
and BARR,M.A.: BALLINGER,W.F., WEISE,A.J.,JACKSON,L.G., In Vivo and In Vitro Survival of Glycerolized Frozen Platelets. J.Amer.med.Ass.: 179, 148, 1962.
4.
COHEN,P. and GARDNER,F.H.: Platelet Preservation. IV. Preservation of Human Platelet Concentrates by Controlled Slow Freezing in a Glycerol Medium. New Engl.J.Med.5 274, 1400, 1966.
5.
BREDDIN,K. and BURCK,K.H.: Zur Klinik der Thrombozytenfunktionsstorungen unter besonderer Berticksichtigung der Ausbreitungsfahigkeit der Thrombozyten an silikonisierten Glasflachen. Thrombos.Diathes.haemorrh.(Stuttg.): 2, 525, 1963.
6.
BREDDIN,K. and LANGBEIN,H.: Uber den EinfluJ3 verschiedener Stoffwechselhemmer auf die Thrombozytenfunktion. Thrombos. Diathes.haemorrh.(Stuttg.): 10, 29, 1963.
7.
DJERASSI,I. Hemorrhage: 1965.
8.
GARDNER,F.H.: 2, 42, 1968.
of Blood. Academic
Press,
London-New
and FARBER,S.: Control and Prevention of Platelet Transfusion. Cancer Res.: 25, 1499, Platelet
Transfusion
Problems.
Cryobiology:
465
PRESERVXTION HU?LqN PLXTELETS
vo1.6,?1:0.6
9.
STREIFF,F.,ALEXANDRE,P.,STOLTZ,J.-F., and GENETET,B.: Conservation et transfusion de concentrds plaquettaires congeles d -800 en DMSO. Etude technique, biologique et clinique. Ann.Biol.Clin.: -28, 295, 1970.
10.
CRONBERG,S.,ROBERTSON,B.,NILSON,I.M., and NILEHN,J.-E.: Suppressive Effect of Dextran on Platelet adhesiveness. Thrombos.Diathes.haemorrh.(Stuttg.): -16, 384, 1968.
11.
SCHNEIDER,J.A. and SANBAR,S.S.: Plasma Expanders and Platelet Adhesiveness. Clin.Res.: 2, 456, 1968
12.
REUTER,H. and GROSS,R.: Preservation of Human Platelets at Low Temperature and Methods for the Estimation of the Functional Efficiency of Preserved Platelets. Proc. 12th Conqr. int. Sot. Blood Transf .,Moscow 1969. Bibl.haemat.: x,-376, 1971.
13.
GARDNER,F.H.,HOWELL,D., and HIRSCH,E.O.: Platelet Transfusions Utilizing Plastic Equipment. J.Lab.clin.Med.: 43, 196, 1954
14.
AAS,K. and GARDNER,F.H.: Survival of Blood Platelets Labelled with Chromium51. J.Clin.Invest.: 37, 1257, 1958.
15.
ASTER,R.H. and JANDL,J.H.: Platelet Sequestration in Man. I. Methods. J.Clin.Invest.: 43, 843, 1964.
16.
BREDDIN,K.,FRITZSCHE,W.,and SPIELMANN,W.: Zur Frage der Thrombozytenkonservierung. Klin.Wschr.: 42, 180, 1964.
17.
TIBBLING,G.: Glycerol Uptake in Leucocytes and Thrombocytes. Scand.J.clin.Lab.Invest.: 2, 185, 1970.
