Vol.
THROMBOSIS RESEARCH Printed in the United
THE EFFECT
OFDEXTRAN
States
ON COLLAGEN-INDUCED
6, pp. ,409~420,
1975
Pergamon
Inc.
Press,
PLATELET AGGREGATION
IN VITRO S . Bygdeman and 0.
Department of Clinical
Physiology,
at the Serafimer Hospital, Biochemical
Research,
(Received
Tangen
Karolinska Institutet
Stockholm and Department of
Pharmacia AB , Uppsala,
20.12.1974;
Accepted
Sweden
in revised form 12.3.1975. by Editor B. Blomb'rick)
ABSTRACT Dextran was shown to potentiate platelet aggregation induced by acid soluble collagen in vitro. Both the extent of aggregation and the lag time were influenced by dextmn. In contMst, platelet aggregation induced by suspensions of insoluble collagen was not affected by dextmn. Further experiments using a turbidimetric technique demonstrated that the rate of precipitation of collagen fibres from tropocollagen was enhanced in the presence of dextmn. This effect could be demonstmted both in salt solutions and in plasma. It is proposed that the potentiation by dextran on platelet aggregation induced by acid soluble collagen can be explained by the increased rate of collagen fibre formation in the presence of dextran.
INTRODUCTION Dextran has an antithrombotic by dextran-induced
effect,
which at least partly has been explained
inhibition of the platelet retention and aggregation
tion (1) and inhibition of platelet thrombus formation in viva (2,3). hibitory effect observed
of dextran on platelet retention and aggregation
following
infusions
of dextran,
ted that the effect of dextmn infusions one or more plasma factors necessary than to an effect on the platelets However,
The in-
is mainly
and we therefore originally
sugges-
could be due to an interference for normal platelet responses
themselves
reac-
with
rather
(1).
in recent years it has been suggested
that dextran can directly
modify platelet function in vitro , and that these effects
also might explain
PLATELET
410
the antithrombotic
AGGREGATION
effect of dextmn in vivo. Thus, it has been shown that in-
cubation of human platelet rich plasma (PRP) with dextran in vitro can induce a limited degree of platelet aggregation
(4). Gordon and Evans (5) have re-
ported that when titrated pig and rabbit PRP containing
dextran was continuous-
ly rotated for up to 150 minutes,
followed
an initial potentiation
sive inhibition of the aggregation Small platelet aggregates taining dextran. aggregates
response
to ADP and collagen
were occasionally
It was proposed that the formation of small transient platelet
further aggregating
of these platelets to
stimuli could partially explain the antithrombotic
The mechanisms involved in dextran-induced
in vitro are not understood, condary to a release a facilitation in accordance
although it has been suggested
of adenosine
diphosphate
of the platelet release reaction with some other findings
(51, These hypotheses
situations
in man
of dextran on platelet funcresponse
induced by
preparation to human and rabbit PRP was
In the present investigation,
the mechanism of the
has been studied with respect to type of collagen
of dextran on collagen
use of
and we have therefore
that the platelet aggregation
addition of an acid soluble collagen potentiated by dextran.
effects
(6) or
are not
In view of the extensive
the mechanism of action is not without importance, tion in vitro. We discovered
to be either se-
(ADP) from the platelets
(1,7).
attempted to further evaluate the possible
effect
platelet aggregation
dextran in the prevention of thrombosis in different clinical
potentiation
was observed
seen in rotated samples con-
in moving blood followed by refractoriness
of dextran.
by a progres-
used and effect
fibre formation in vitro. METHODS
Platelet aggregation
was studied using either venous blood obtained from
healthy human volunteers
or arterial blood taken from rabbits through a
catheter inserted in the carotid artery under light pentobarbitone
anaesthesia.
The blood was collected
l/lOth
into siliconised
glass tubes containing
vo-
lume of 3.13 per cent trisodium citrate dihydmte. PRP was prepared 30 minutes after blood collection blood at 2 90 x g for 15 minutes at room temperature. plasma (PPP) the platelet rich plasma was centrifuged tes.
Platelet aggregation
in response
by centrifuging
To obtaine platelet poor at 1200 x g for 20 minu-
to standard amounts of collagen
and
adenosine
diphosphate
according
to the turbidimetric method of Born (81. Three types of collagen
preparations were used,
(ADP) was determined at 33’C
whole
namely acid soluble collagen
or room temperature obtained from Stago
411
PLATELET AGGREGATION
vo1.6,No.5
Laboratories
(France) and Sigma Chemical Co.
(St. Louis) and an insoluble
preparation from Sigma Chemical Co. The collagen
collagen
ratories was used in the experiments
in Figs.
from Stago Lab+
1 and 2. Collagen
Chemical Co. was used for all other experiments.
The soluble
from Sigma collagen
pre-
parations were solved in 0.1 N acetic acid pH 3.5 and diluted with THAMbuffered saline before use. buffered saline according ments THAM-buffered
The insoluble
was suspended
in THAM-
to Evans et al. (9) before use. In control experi-
saline was added instead of dextran.
ADP (Sigma Chemical Co.) and dissolved
collagen
400 ug/ml was prepared as a stock solution
in THAM-buffered
saline.
The solution was frozen in portions,
mixed, and diluted before use. A photometric technique was used to study the rate of collagen tate formation.
Aliquots of collagen
red saline or PPP in absence
were added to cuvettes
or presence
of dextran.
in a Vitatron photometer Model UC 200 connected
precfpi-
containing
The cuvettes
buffe-
were placed
to a Vitatron lin-log
recor-
der. The mixtures were stirred by magnetic stirrers and the temperature in the cuvette maintained at 33’C.
Changes in optical density with time could
thus be recorded and gave a relative measure of the rate of collagen
preci-
pitation. Three dextran fractions with a molecular weight of 40,000, 500,000
70,000 and
were used and referred to in the text as dx 40, dx 70 and dx 500 re-
s pectively . RESULTS The effects shown in Figs. potentiated
of dextran on collagen-induced l-4.
are
Addition of dx 40 and 70 to human titrated PRP markedly
the aggregation
reaction induced by acid soluble collagen
Stago Lab. (Fig. 1). All differences
between the dextmn-containing
and the appropriate controls were significant level.
platelet aggregation
from samples
at least at the one per cent
The potentiation was largely independent of the dextran fraction used,
Similar results were also obtained in a pilot study utilising dx 500. The degree of aggregation
was dependent on temperature,
red also at room temperature. ween addition of collagen decreased
significantly
was independent
Similarly,
but potentiation
occur-
the lag time, that is the time bet-
to the PRP-sample and the onset of the aggregation,
(pd 0.01) as seen in Fig. 2. The decrease
in lag time
of the molecular weight of the dextran preparation and could
be demonstrated both at room temperature and at 33’C.
C 4D”o C
Fig. 1
ID
70
Dx
6
70
cE cDx
d il
22 OC
fSE
.
Effects of dx 40 and 70 in a final concentration of 1.2 % on human platelet aggregation induced by addition of SO.pg/ml of an acid soluble collagen solution. C = control experiment. Normal saline added instead of dextran. Mean values of 5-7 experiments
0
0.05-
O.lO-
1
33Oc
A OD/15set
70
Fiu. 2,
sl c4D”o cDx
40
C Dx
22 OC T
Effect of dx 40 and 70 in a final concentration of 1.2 % on the lag time between addition of 50 ug of acid soluble collagen to PRP and the onset of the platelet aggregation reaction. C = control experiments. Normal saline added instead of dextran. Mean values of 5-7 experimens f SE.
0,
100,
200
300
400,
500
600
set
Lag time,
3 r .
twm’
25
soluble
Fig. 3,’
13
Acid
50
Collagen
200
p’/m’
100
Effect of dx 70 in a final concentration of 1.2% on human platelet aggregation induced by 13-50 pg/ml of acid soluble collagen solution or 100-200 ug/ml of an insoluble collagen preparatian. C = control experiments. Normal saline added instead of dextran. Mean values of 5-7 experiments k SE.
0.05
0.10
0.15
AOD/l!Wc
Fls. 4.
25 PgP
cb&
50
CR
loo 200 pVml
70
CL33 CDX
Suspension
Effect of dextran 70 in a final concentration of 1.2% on lag time between addition of different concentrations of an acid soluble collagen solution or different amounts of insoluble collagen preparation, C = control experiments. Normal saline adde$ instead of dextran. Mean values of 5-7 experiments - SE.
13
cog
Acid soluble
Collagen
PLATELET AGGREGATION
414
A significant
potentiation
of collagen-induced
also be demonstrated when a more effective
Vo1.6,No.5
platelet aggregation
acid soluble collagen
could
preparation
obtained from another manufacturer was used (Sigma Chemical Co.).
In this
series of experiments
and it
different concentrations
of collagen
was used,
could be shown that the potentiation was most marked with small concentrations of collagen
(Figs.
platelet aggregation when an insoluble
3-4).
Only a small change of collagen-induced
could be observed collagen
following
suspension
was used (Figs.
the fact that in control experiments this collagen lar degree of aggregation ved with a collagen
as the acid soluble
suspension
response
were compared.
soluble
collagen
preparation induced a simi-
preparation.
The lag time obser-
when dosages
giving the same
Here, the lag time was not significant-
ly changed (p > 0.05) by preincubation In 11 experiments
3-41, in spite of
was in control experiments markedly shorter
than that found with acid soluble collagen aggregation
incubation with dextran
with dx 40 and 70.
(no included in the figures) a concentration
too small to induce an aggregation
response
of acid
in control ex-
periments was used. After addition of dx 40, 70 and 500 to final plasma concentrations
of 1.2 per cent the same amounts of collagen
ficant aggregation
now induced a signi-
reaction in 9 of the 11 experiments.
Addition of dx 40 and 70 to PRP induced no change or a slight inhibition of ADP-induced potentiation
platelet aggregation
be observed
(0.05 4 p (
0.10).
In no case could a
(Table I). TABLEI
,Effect of Dextran on First Phase of ADP-Induced Platelet Aggregation at Room Temperature. ADP-dose 0.2 pg/ml. In control experiments 0.154 m NaCl was added instead of dextran . Type and concentration of dextran
Change in O.D./lS
No dextran
0.085 + 0.017
dx40
0.068 + 0.018
1.2%
dx 70 1.2 %
set’)
0.069 + 0.019
1) The results are expressed as the arithmetic means f S . D. of 9 measurements.
PLATELET
AGGREGATION
415
TABLEII Effect of Dextran on the Precipitation of Collagen in THAM Buffer-Saline Mixture. Precipitation mixture was 2.2 ml 0.154 M THAM-buffer, pH 7.4, 0.3 ml 0,154’ M ‘NaCl, 0.3 ml 0.154 M NaCl or desired concentrations of dx 7 0 in saline, and 0.2 ml of 2 mg/ml of collagen preparation dissolved in HAC 0.1 N or suspended in saline. Concentration of dx 70 in %
Type of collagen
Increase in O.D. l’ after 10 min after 30 min
Acid soluble
0
0.006 + 0.004
0.032 + 0.021
Acid soluble
1.2
0.053 + 0.025
0.110 + 0.056
Collagen
suspension
0
0.004 + 0.005
0.008 -I 0.009
Collagen
suspension
1.2
0.018 + 0.005
0.028 + 0.011
1) The results are expressed as the arithmetic mean + S. D. of separate experiments.
In Table II is shown that dx 70 significantly acid soluble collagen
in a salt solution.
found when the collagen collagen
suspension
enhanced
precipitation
of
No comparable change in O.D.
was
was used instead of the acid soluble
solution.
The results summarized in Tables III, IV and V demonstMte that the same effect
of dextran was obtained also when added to human or rabbit PPP. The
effect
of dextran was independent on molecular weight in the range of 40,000
to 500,000
Mw. TABLBIII
Effect of Dextran on the Precipitation of Collagen in Human PPP. The experimental conditions were similar to those described in Table II although PPP was used instead of THAM-buffer. Type of collagen
1)
Concentration of dx 70 in %
Increase in O.D. I1 after 10 min
Acid soluble
0
0.0054 + 0.0030
Acid soluble
1.2
0.0300 2 0.0100
Collagen
suspension
0
0.0034
Collagen
suspension
1.2
0.0032 + 0.0006
+ 0.0011
The results are expressed as the arithmetic means 2 S .D. of four separate experiments.
416
PLATELET
AGGREGATION
vo1.6,No.5
TABLEIV Effect of Dextran on the Precipitation of Acid Soluble Collagen Experimental conditions see Table III. Type and concentration of dextran
1) ~~ Increase in O.D. after 10 min after 30 min
No dextran
0.004 + 0.004
0.006 + 0.005
dx40
0.3%
0.018 + 0.021
0.040 2 0.044
II
0.6 %
0.025 + 0.016
0.040 + 0.016
II
1.2 %
0.039 + 0.023
0.053 + 0.024
II
2.0 %
0.039 + 0.009
0.051 + 0.021
0.3%
0.007 + 0.006
0.020 + 0.014
II
0.6 %
0.016 + 0.007
0.031+
,I
1.2 %
0.021:
0.010
0.042 + 0.024
II
2.0 %
0.044 + 0.025
0.076 2 0.019
dx 500
0.3 %
0.025 + 0.021
0.062 + 0.068
1,
0.6 %
0.032 + 0.041
0.057 2 0.030
II
1.2 %
0.054 + 0.051
0.091 : 0.090
II
2.0 %
0.054 -I 0.034
0.091+
dx70
in Rabbit PPP.
0.019
0.059
1) The results are expressed as the arithmetic means -+ S.D. of five separate experiments.
TABLEV Effect of Dextran on Precipitation of Collagen Experimental conditions see Table III.
Type and concentration of dextran
Increased after 10 min
Suspensions
in Rabbit PPP.
in 0. D. ‘) after 30 min
No dextran
0.001 + - 0.002
0.007 f 0.010
dx 40
0.6 %
0.005 2 0.004
0.018 + 0.011
dx 40
2.0 %
0.011+
0.013
0.024 + 0.030
dx 70
0.6 %
0.007 + 0.010
0.035 2 0.050
dx 70
2.0 %
0.011 + 0.008
0.035 + 0.028
dx 500
0.6 %
0.004 + 0.002
0.015 + 0.004
dx 500
2.0 %
0.021 + 0.017
0.044 + 0.047
1) The results are expressed as the arithmetic means + - S.D. five separate experiments.
of
PLATELET
vo1.6,No.5
417
AGGREGATION
DISCUSSION The antithrombotic
action of dextran infusions
several of the many biological tion of ADP-induced rical charge
effects
of dextran as for instance the inhibi-
platelet retention and aggregation,
of the formed elements of blood,
sity , and the recently demonstrated effect 11). An alternative
is most likely related to
explanation
decrease
creased
to a platelet release
platelets
platelet reactivity
infusions
observed,
This aggregation
has been sugges-
of ADP. If transient platelet aggrega-
of dextmn this might explain the desince it is known thgt once aggregated
be explained
of dextran,
do not decrease
of an increased
to
in vitro based on the
amount of ADP in plasma following
incubation
of
removal of the platelets by centrifugation.
in an attempt to reproduce these findings (7), we found that after
preparation of PPP by centrifugation, platelets.
in doses sufficient
by plasma dilution (1). Paterson and Dhall (6) reported that
PRP with dextran and subsequent However,
In many
platelet numbers more than can
dextran could induce a release of ADP from platelets observation
stimuli (12).
speak against this hypothesis.
studies it has been shown that infusions platelet reactivity,
(1, 10 ,
that dextmn in vitro
are at least temporarily refractory to aggregating
In our view several observations decrease
of whole blood visco-
has been proposed by Dhall and Matheson (4)
can induce a small degree of aggregation. tion occurs in vivo following
of the elect-
on fibrin formation and 1ySis
and by Cordon and Evans (5) based on the observation ted to be secondary
increase
In no instances
nucleotides
the ADP in PPP was related to remaining
could we detect any release
of neither adenine
nor serotonin by dextran.
The present results demonstmte that dextran potentiates gation induced by acid soluble collagen.
platelet aggre-
This might be taken as an argument
for the theory that the platelets are modified so as to potentiate their release reaction.
However,
the finding that dextmn influences
also the mode) of collagen Recently,
evidence
not aggregate
fibre formation offers an alternative
has been presented that monodisperse
platelets,
and that microfibrils
gation can take place (13). In addition,
the effect
fibre formation.
lets.
tropocollagen
does
have to be formed before aggreinduced by soluble collagen
de-
Thus, the present results indicate that
of dextran on platelet aggregation
due to an effect on collagen
explanation.
Caen and Legrand (14) and Hugues
(15) have reported that platelet aggregation pend on the collagen
the Mte (and possibly
induced by soluble collagen
is
fibre formation and not to an effect on the plate-
This is also strongly supported by the experiments with collagen
PLATELET
418
suspensions,
Vo1.6,No.5
AGGREGATION
where dextran did not affect the platelet
ly that the initial potentiation
of platelet reactivity
Gordon and Evans (5) in circulating Neither our experimental any conclusions
aggregation.
to collagen
PRP can be explained
It is like-
observed by
in a similar way.
design nor our instrumentation allows us to draw
about the exact nature of the effect of dextran on the colla-
gen fibre fromation.
Therefore,
it is not possible
to state whether it is an
altered rate of fibre formation or a change in fibre structure which is responsible for the potentiated platelet aggregation. dextran decreases
the solubility
of some proteins and that the phenomenon
seems to be due to a steric exclusion It is possible
instance
of the proteins by the dextran molecules,
that this may explain the effect of dextran on collagen
formation observed solubility
Laurent (16) has shown that
in the present study although the effect
of collagen fibrinogen
tration exceeds
has not been investigated
fibre
of dextran on the
and a precipitation
of for
in plasma seems only to occur when the dextran concen-
4-5 per cent.
lymer forming systems.
Dextran is also known to influence
The presence
other po-
of dextran has been shown to accele-
rate fibrin clot formation from fibrin monomers (lo),
and it has also been
shown that the structure of such fibrin is different from control fibrin (11). The two systems,
fibrin monomer
fibrin clot,
fibres are similar in several respects: the initial aggregation,
and tropocollagen
no covalent
bonds are produced during
both monomers are extremely asymmetrical molecules,
and the fibres formed have definite ordered structures. induces an increase
in absorbance
to formation of collagen gen microfibrils
of collagen
That dextran also
suspensions
may just be due
fibres from small amounts of tropocollagen
or colla-
presen In such preparations.
Very little is still known about the mechanism of dextran-induced let aggregation
collagen
in vitro.
However,
plate-
the results obtained in the present study
as well as earlier results (1,7) clearly demonstrate that dextran does not activate
human platelets
stances
like ADP and collagen
responsible
or portentiate platelet reactions fibers,
induced by sub-
which generally are thought to be
for the formation of platelet
aggregates
in viva.
ACKNCWLEDGMENTS This work was supported by a research grant from The Swedish Medical Research Council,
project no. 14X-1019.
PLATELET
vo1.6,No.5
419
AGGREGATION
REFERENCES 1.
BYGDEMAN , S. Prevention
with dextran.
Progr.
Surg.:
and thempy of thrombo-embolic I, 114. 1969.
complications
2.
BERMAN, H. J. and FULTON , G. P. Platelets in the peripheral circulation. latelets. S . A. Johnson, R .W. Monte, J.W. Rebuch and R. C . In: Blood &n-don, Churchill, 1961, P. 7.
3.
ARFORS, K.-E., DHALL, D.P., ENGESET, J., HINT, H., MATHESON, N. A. and TANGEN, 0. In vivo quantitation of platelet activity using biolaser-induced endothelial injury. Bibl. anat.: l0, 505. 1969.
4.
DHALL, D. P. and MATHESON, N.A. In vitro clumping of human by dextran. Thromb. Diath, Haemorrh. : l9, 70, 1968.
5.
GORDON, J. L. and EVANS, R. J. Dextran inhibition of platelet aggregation in vivo and in vitro. IVth Int. Congr. Thromb. Haemost. , Vienna 1973, 411.
6.
PATERSON, N. and DHALL, D. P .. The release of adenosine diphosphate 6th Europ. Conf . Microcircula(ADP) from human platelets by dextran. tion, Aalborg 1970, 324.
7.
, S. and TANGEN, 0. Studies on the antithrombotic effect of dextran. Effect on platelet 5-hydroxytryptamine and adenine nucleotides . 7th Europ. Congr. Microcirculation, Aberdeen 1972. Bibl. anat. : l2, 333. 1973.
8.
BORN, G. V. R. Quantitative platelets. J. Physiol. : 162,
9.
MUSTARD, J.F. and EVANS, G., PACKHAM, M.A., NISHIZAWA, E.E., MURPHY, E. The effect of acetylsalicylic acid on platelet function. J. Exptl Med.: 128, 877. 1968.
10.
of dextmn TANGEN, O., PHELAN III, W. J. and BERMAN, H. J. Influence and other macromolecules on thrombin time and the polymerization of fibrin monomer. XIII Int. Congr. Hematol. Munich 1970. Abstract 144.
11.
TANGEN, 0. , WIK, K. 0. , ALMQVIST, I.A. M. , ARFORS, K.-E. and HINT, H. C. Effect of dextran on the structure and plasmin-induced lysis of human fibrin. Thrombosis Res .: 1, 487. 1972.
12.
O’BRIEN, J.R. Changes in platelet membranes platelet stickiness. Nature: 212, 1057. 1966.
13.
MUGGLI
14.
CAEN, J. and LEGRAND, Y. Plaquettes, collagine concepts of coagulation and hemostasis . Thromb. 1971. Suppl. 48, 167.
platelets
BYGDEMAN
investigation 67. 1962.
into the aggregation
possibly
, R. and BAUMGARTNER , H.R. Collagen gation: Requirement for tropocollagen multimers. Thromb. Haemost. , Vienna 1973. Abstract 53.
of blood
associated
with
induced platelet aggreHIEG 73-82, IVth Congr. et elastine. In current Diath. Haemorrh, :
420
PLATELET
AGGREGATION
15. HUGUES, J. Some remarks concerning collagen, & Platelets in haemostasis. Exptl Biol. Med.: 3, 145. 1968.
v01.6,~0.5
platelet aggregation induced by S. Karger (Ed. 1 Base1 1968.
and other macro16. LAURENT, T. C . The interaction between polysaccharides molecules. The solubility of proteins in the presence of dextran. Biochem. J.: 89, 253. 1963.
THROMBOSIS RESEARCH Printed in the United
THE EFFECT
OFDEXTRAN
States
ON COLLAGEN-INDUCED
6, pp. ,409~420,
1975
Pergamon
Inc.
Press,
PLATELET AGGREGATION
IN VITRO S . Bygdeman and 0.
Department of Clinical
Physiology,
at the Serafimer Hospital, Biochemical
Research,
(Received
Tangen
Karolinska Institutet
Stockholm and Department of
Pharmacia AB , Uppsala,
20.12.1974;
Accepted
Sweden
in revised form 12.3.1975. by Editor B. Blomb'rick)
ABSTRACT Dextran was shown to potentiate platelet aggregation induced by acid soluble collagen in vitro. Both the extent of aggregation and the lag time were influenced by dextmn. In contMst, platelet aggregation induced by suspensions of insoluble collagen was not affected by dextmn. Further experiments using a turbidimetric technique demonstrated that the rate of precipitation of collagen fibres from tropocollagen was enhanced in the presence of dextmn. This effect could be demonstmted both in salt solutions and in plasma. It is proposed that the potentiation by dextran on platelet aggregation induced by acid soluble collagen can be explained by the increased rate of collagen fibre formation in the presence of dextran.
INTRODUCTION Dextran has an antithrombotic by dextran-induced
effect,
which at least partly has been explained
inhibition of the platelet retention and aggregation
tion (1) and inhibition of platelet thrombus formation in viva (2,3). hibitory effect observed
of dextran on platelet retention and aggregation
following
infusions
of dextran,
ted that the effect of dextmn infusions one or more plasma factors necessary than to an effect on the platelets However,
The in-
is mainly
and we therefore originally
sugges-
could be due to an interference for normal platelet responses
themselves
reac-
with
rather
(1).
in recent years it has been suggested
that dextran can directly
modify platelet function in vitro , and that these effects
also might explain
PLATELET
410
the antithrombotic
AGGREGATION
effect of dextmn in vivo. Thus, it has been shown that in-
cubation of human platelet rich plasma (PRP) with dextran in vitro can induce a limited degree of platelet aggregation
(4). Gordon and Evans (5) have re-
ported that when titrated pig and rabbit PRP containing
dextran was continuous-
ly rotated for up to 150 minutes,
followed
an initial potentiation
sive inhibition of the aggregation Small platelet aggregates taining dextran. aggregates
response
to ADP and collagen
were occasionally
It was proposed that the formation of small transient platelet
further aggregating
of these platelets to
stimuli could partially explain the antithrombotic
The mechanisms involved in dextran-induced
in vitro are not understood, condary to a release a facilitation in accordance
although it has been suggested
of adenosine
diphosphate
of the platelet release reaction with some other findings
(51, These hypotheses
situations
in man
of dextran on platelet funcresponse
induced by
preparation to human and rabbit PRP was
In the present investigation,
the mechanism of the
has been studied with respect to type of collagen
of dextran on collagen
use of
and we have therefore
that the platelet aggregation
addition of an acid soluble collagen potentiated by dextran.
effects
(6) or
are not
In view of the extensive
the mechanism of action is not without importance, tion in vitro. We discovered
to be either se-
(ADP) from the platelets
(1,7).
attempted to further evaluate the possible
effect
platelet aggregation
dextran in the prevention of thrombosis in different clinical
potentiation
was observed
seen in rotated samples con-
in moving blood followed by refractoriness
of dextran.
by a progres-
used and effect
fibre formation in vitro. METHODS
Platelet aggregation
was studied using either venous blood obtained from
healthy human volunteers
or arterial blood taken from rabbits through a
catheter inserted in the carotid artery under light pentobarbitone
anaesthesia.
The blood was collected
l/lOth
into siliconised
glass tubes containing
vo-
lume of 3.13 per cent trisodium citrate dihydmte. PRP was prepared 30 minutes after blood collection blood at 2 90 x g for 15 minutes at room temperature. plasma (PPP) the platelet rich plasma was centrifuged tes.
Platelet aggregation
in response
by centrifuging
To obtaine platelet poor at 1200 x g for 20 minu-
to standard amounts of collagen
and
adenosine
diphosphate
according
to the turbidimetric method of Born (81. Three types of collagen
preparations were used,
(ADP) was determined at 33’C
whole
namely acid soluble collagen
or room temperature obtained from Stago
411
PLATELET AGGREGATION
vo1.6,No.5
Laboratories
(France) and Sigma Chemical Co.
(St. Louis) and an insoluble
preparation from Sigma Chemical Co. The collagen
collagen
ratories was used in the experiments
in Figs.
from Stago Lab+
1 and 2. Collagen
Chemical Co. was used for all other experiments.
The soluble
from Sigma collagen
pre-
parations were solved in 0.1 N acetic acid pH 3.5 and diluted with THAMbuffered saline before use. buffered saline according ments THAM-buffered
The insoluble
was suspended
in THAM-
to Evans et al. (9) before use. In control experi-
saline was added instead of dextran.
ADP (Sigma Chemical Co.) and dissolved
collagen
400 ug/ml was prepared as a stock solution
in THAM-buffered
saline.
The solution was frozen in portions,
mixed, and diluted before use. A photometric technique was used to study the rate of collagen tate formation.
Aliquots of collagen
red saline or PPP in absence
were added to cuvettes
or presence
of dextran.
in a Vitatron photometer Model UC 200 connected
precfpi-
containing
The cuvettes
buffe-
were placed
to a Vitatron lin-log
recor-
der. The mixtures were stirred by magnetic stirrers and the temperature in the cuvette maintained at 33’C.
Changes in optical density with time could
thus be recorded and gave a relative measure of the rate of collagen
preci-
pitation. Three dextran fractions with a molecular weight of 40,000, 500,000
70,000 and
were used and referred to in the text as dx 40, dx 70 and dx 500 re-
s pectively . RESULTS The effects shown in Figs. potentiated
of dextran on collagen-induced l-4.
are
Addition of dx 40 and 70 to human titrated PRP markedly
the aggregation
reaction induced by acid soluble collagen
Stago Lab. (Fig. 1). All differences
between the dextmn-containing
and the appropriate controls were significant level.
platelet aggregation
from samples
at least at the one per cent
The potentiation was largely independent of the dextran fraction used,
Similar results were also obtained in a pilot study utilising dx 500. The degree of aggregation
was dependent on temperature,
red also at room temperature. ween addition of collagen decreased
significantly
was independent
Similarly,
but potentiation
occur-
the lag time, that is the time bet-
to the PRP-sample and the onset of the aggregation,
(pd 0.01) as seen in Fig. 2. The decrease
in lag time
of the molecular weight of the dextran preparation and could
be demonstrated both at room temperature and at 33’C.
C 4D”o C
Fig. 1
ID
70
Dx
6
70
cE cDx
d il
22 OC
fSE
.
Effects of dx 40 and 70 in a final concentration of 1.2 % on human platelet aggregation induced by addition of SO.pg/ml of an acid soluble collagen solution. C = control experiment. Normal saline added instead of dextran. Mean values of 5-7 experiments
0
0.05-
O.lO-
1
33Oc
A OD/15set
70
Fiu. 2,
sl c4D”o cDx
40
C Dx
22 OC T
Effect of dx 40 and 70 in a final concentration of 1.2 % on the lag time between addition of 50 ug of acid soluble collagen to PRP and the onset of the platelet aggregation reaction. C = control experiments. Normal saline added instead of dextran. Mean values of 5-7 experimens f SE.
0,
100,
200
300
400,
500
600
set
Lag time,
3 r .
twm’
25
soluble
Fig. 3,’
13
Acid
50
Collagen
200
p’/m’
100
Effect of dx 70 in a final concentration of 1.2% on human platelet aggregation induced by 13-50 pg/ml of acid soluble collagen solution or 100-200 ug/ml of an insoluble collagen preparatian. C = control experiments. Normal saline added instead of dextran. Mean values of 5-7 experiments k SE.
0.05
0.10
0.15
AOD/l!Wc
Fls. 4.
25 PgP
cb&
50
CR
loo 200 pVml
70
CL33 CDX
Suspension
Effect of dextran 70 in a final concentration of 1.2% on lag time between addition of different concentrations of an acid soluble collagen solution or different amounts of insoluble collagen preparation, C = control experiments. Normal saline adde$ instead of dextran. Mean values of 5-7 experiments - SE.
13
cog
Acid soluble
Collagen
PLATELET AGGREGATION
414
A significant
potentiation
of collagen-induced
also be demonstrated when a more effective
Vo1.6,No.5
platelet aggregation
acid soluble collagen
could
preparation
obtained from another manufacturer was used (Sigma Chemical Co.).
In this
series of experiments
and it
different concentrations
of collagen
was used,
could be shown that the potentiation was most marked with small concentrations of collagen
(Figs.
platelet aggregation when an insoluble
3-4).
Only a small change of collagen-induced
could be observed collagen
following
suspension
was used (Figs.
the fact that in control experiments this collagen lar degree of aggregation ved with a collagen
as the acid soluble
suspension
response
were compared.
soluble
collagen
preparation induced a simi-
preparation.
The lag time obser-
when dosages
giving the same
Here, the lag time was not significant-
ly changed (p > 0.05) by preincubation In 11 experiments
3-41, in spite of
was in control experiments markedly shorter
than that found with acid soluble collagen aggregation
incubation with dextran
with dx 40 and 70.
(no included in the figures) a concentration
too small to induce an aggregation
response
of acid
in control ex-
periments was used. After addition of dx 40, 70 and 500 to final plasma concentrations
of 1.2 per cent the same amounts of collagen
ficant aggregation
now induced a signi-
reaction in 9 of the 11 experiments.
Addition of dx 40 and 70 to PRP induced no change or a slight inhibition of ADP-induced potentiation
platelet aggregation
be observed
(0.05 4 p (
0.10).
In no case could a
(Table I). TABLEI
,Effect of Dextran on First Phase of ADP-Induced Platelet Aggregation at Room Temperature. ADP-dose 0.2 pg/ml. In control experiments 0.154 m NaCl was added instead of dextran . Type and concentration of dextran
Change in O.D./lS
No dextran
0.085 + 0.017
dx40
0.068 + 0.018
1.2%
dx 70 1.2 %
set’)
0.069 + 0.019
1) The results are expressed as the arithmetic means f S . D. of 9 measurements.
PLATELET
AGGREGATION
415
TABLEII Effect of Dextran on the Precipitation of Collagen in THAM Buffer-Saline Mixture. Precipitation mixture was 2.2 ml 0.154 M THAM-buffer, pH 7.4, 0.3 ml 0,154’ M ‘NaCl, 0.3 ml 0.154 M NaCl or desired concentrations of dx 7 0 in saline, and 0.2 ml of 2 mg/ml of collagen preparation dissolved in HAC 0.1 N or suspended in saline. Concentration of dx 70 in %
Type of collagen
Increase in O.D. l’ after 10 min after 30 min
Acid soluble
0
0.006 + 0.004
0.032 + 0.021
Acid soluble
1.2
0.053 + 0.025
0.110 + 0.056
Collagen
suspension
0
0.004 + 0.005
0.008 -I 0.009
Collagen
suspension
1.2
0.018 + 0.005
0.028 + 0.011
1) The results are expressed as the arithmetic mean + S. D. of separate experiments.
In Table II is shown that dx 70 significantly acid soluble collagen
in a salt solution.
found when the collagen collagen
suspension
enhanced
precipitation
of
No comparable change in O.D.
was
was used instead of the acid soluble
solution.
The results summarized in Tables III, IV and V demonstMte that the same effect
of dextran was obtained also when added to human or rabbit PPP. The
effect
of dextran was independent on molecular weight in the range of 40,000
to 500,000
Mw. TABLBIII
Effect of Dextran on the Precipitation of Collagen in Human PPP. The experimental conditions were similar to those described in Table II although PPP was used instead of THAM-buffer. Type of collagen
1)
Concentration of dx 70 in %
Increase in O.D. I1 after 10 min
Acid soluble
0
0.0054 + 0.0030
Acid soluble
1.2
0.0300 2 0.0100
Collagen
suspension
0
0.0034
Collagen
suspension
1.2
0.0032 + 0.0006
+ 0.0011
The results are expressed as the arithmetic means 2 S .D. of four separate experiments.
416
PLATELET
AGGREGATION
vo1.6,No.5
TABLEIV Effect of Dextran on the Precipitation of Acid Soluble Collagen Experimental conditions see Table III. Type and concentration of dextran
1) ~~ Increase in O.D. after 10 min after 30 min
No dextran
0.004 + 0.004
0.006 + 0.005
dx40
0.3%
0.018 + 0.021
0.040 2 0.044
II
0.6 %
0.025 + 0.016
0.040 + 0.016
II
1.2 %
0.039 + 0.023
0.053 + 0.024
II
2.0 %
0.039 + 0.009
0.051 + 0.021
0.3%
0.007 + 0.006
0.020 + 0.014
II
0.6 %
0.016 + 0.007
0.031+
,I
1.2 %
0.021:
0.010
0.042 + 0.024
II
2.0 %
0.044 + 0.025
0.076 2 0.019
dx 500
0.3 %
0.025 + 0.021
0.062 + 0.068
1,
0.6 %
0.032 + 0.041
0.057 2 0.030
II
1.2 %
0.054 + 0.051
0.091 : 0.090
II
2.0 %
0.054 -I 0.034
0.091+
dx70
in Rabbit PPP.
0.019
0.059
1) The results are expressed as the arithmetic means -+ S.D. of five separate experiments.
TABLEV Effect of Dextran on Precipitation of Collagen Experimental conditions see Table III.
Type and concentration of dextran
Increased after 10 min
Suspensions
in Rabbit PPP.
in 0. D. ‘) after 30 min
No dextran
0.001 + - 0.002
0.007 f 0.010
dx 40
0.6 %
0.005 2 0.004
0.018 + 0.011
dx 40
2.0 %
0.011+
0.013
0.024 + 0.030
dx 70
0.6 %
0.007 + 0.010
0.035 2 0.050
dx 70
2.0 %
0.011 + 0.008
0.035 + 0.028
dx 500
0.6 %
0.004 + 0.002
0.015 + 0.004
dx 500
2.0 %
0.021 + 0.017
0.044 + 0.047
1) The results are expressed as the arithmetic means + - S.D. five separate experiments.
of
PLATELET
vo1.6,No.5
417
AGGREGATION
DISCUSSION The antithrombotic
action of dextran infusions
several of the many biological tion of ADP-induced rical charge
effects
of dextran as for instance the inhibi-
platelet retention and aggregation,
of the formed elements of blood,
sity , and the recently demonstrated effect 11). An alternative
is most likely related to
explanation
decrease
creased
to a platelet release
platelets
platelet reactivity
infusions
observed,
This aggregation
has been sugges-
of ADP. If transient platelet aggrega-
of dextmn this might explain the desince it is known thgt once aggregated
be explained
of dextran,
do not decrease
of an increased
to
in vitro based on the
amount of ADP in plasma following
incubation
of
removal of the platelets by centrifugation.
in an attempt to reproduce these findings (7), we found that after
preparation of PPP by centrifugation, platelets.
in doses sufficient
by plasma dilution (1). Paterson and Dhall (6) reported that
PRP with dextran and subsequent However,
In many
platelet numbers more than can
dextran could induce a release of ADP from platelets observation
stimuli (12).
speak against this hypothesis.
studies it has been shown that infusions platelet reactivity,
(1, 10 ,
that dextmn in vitro
are at least temporarily refractory to aggregating
In our view several observations decrease
of whole blood visco-
has been proposed by Dhall and Matheson (4)
can induce a small degree of aggregation. tion occurs in vivo following
of the elect-
on fibrin formation and 1ySis
and by Cordon and Evans (5) based on the observation ted to be secondary
increase
In no instances
nucleotides
the ADP in PPP was related to remaining
could we detect any release
of neither adenine
nor serotonin by dextran.
The present results demonstmte that dextran potentiates gation induced by acid soluble collagen.
platelet aggre-
This might be taken as an argument
for the theory that the platelets are modified so as to potentiate their release reaction.
However,
the finding that dextmn influences
also the mode) of collagen Recently,
evidence
not aggregate
fibre formation offers an alternative
has been presented that monodisperse
platelets,
and that microfibrils
gation can take place (13). In addition,
the effect
fibre formation.
lets.
tropocollagen
does
have to be formed before aggreinduced by soluble collagen
de-
Thus, the present results indicate that
of dextran on platelet aggregation
due to an effect on collagen
explanation.
Caen and Legrand (14) and Hugues
(15) have reported that platelet aggregation pend on the collagen
the Mte (and possibly
induced by soluble collagen
is
fibre formation and not to an effect on the plate-
This is also strongly supported by the experiments with collagen
PLATELET
418
suspensions,
Vo1.6,No.5
AGGREGATION
where dextran did not affect the platelet
ly that the initial potentiation
of platelet reactivity
Gordon and Evans (5) in circulating Neither our experimental any conclusions
aggregation.
to collagen
PRP can be explained
It is like-
observed by
in a similar way.
design nor our instrumentation allows us to draw
about the exact nature of the effect of dextran on the colla-
gen fibre fromation.
Therefore,
it is not possible
to state whether it is an
altered rate of fibre formation or a change in fibre structure which is responsible for the potentiated platelet aggregation. dextran decreases
the solubility
of some proteins and that the phenomenon
seems to be due to a steric exclusion It is possible
instance
of the proteins by the dextran molecules,
that this may explain the effect of dextran on collagen
formation observed solubility
Laurent (16) has shown that
in the present study although the effect
of collagen fibrinogen
tration exceeds
has not been investigated
fibre
of dextran on the
and a precipitation
of for
in plasma seems only to occur when the dextran concen-
4-5 per cent.
lymer forming systems.
Dextran is also known to influence
The presence
other po-
of dextran has been shown to accele-
rate fibrin clot formation from fibrin monomers (lo),
and it has also been
shown that the structure of such fibrin is different from control fibrin (11). The two systems,
fibrin monomer
fibrin clot,
fibres are similar in several respects: the initial aggregation,
and tropocollagen
no covalent
bonds are produced during
both monomers are extremely asymmetrical molecules,
and the fibres formed have definite ordered structures. induces an increase
in absorbance
to formation of collagen gen microfibrils
of collagen
That dextran also
suspensions
may just be due
fibres from small amounts of tropocollagen
or colla-
presen In such preparations.
Very little is still known about the mechanism of dextran-induced let aggregation
collagen
in vitro.
However,
plate-
the results obtained in the present study
as well as earlier results (1,7) clearly demonstrate that dextran does not activate
human platelets
stances
like ADP and collagen
responsible
or portentiate platelet reactions fibers,
induced by sub-
which generally are thought to be
for the formation of platelet
aggregates
in viva.
ACKNCWLEDGMENTS This work was supported by a research grant from The Swedish Medical Research Council,
project no. 14X-1019.
PLATELET
vo1.6,No.5
419
AGGREGATION
REFERENCES 1.
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Progr.
Surg.:
and thempy of thrombo-embolic I, 114. 1969.
complications
2.
BERMAN, H. J. and FULTON , G. P. Platelets in the peripheral circulation. latelets. S . A. Johnson, R .W. Monte, J.W. Rebuch and R. C . In: Blood &n-don, Churchill, 1961, P. 7.
3.
ARFORS, K.-E., DHALL, D.P., ENGESET, J., HINT, H., MATHESON, N. A. and TANGEN, 0. In vivo quantitation of platelet activity using biolaser-induced endothelial injury. Bibl. anat.: l0, 505. 1969.
4.
DHALL, D. P. and MATHESON, N.A. In vitro clumping of human by dextran. Thromb. Diath, Haemorrh. : l9, 70, 1968.
5.
GORDON, J. L. and EVANS, R. J. Dextran inhibition of platelet aggregation in vivo and in vitro. IVth Int. Congr. Thromb. Haemost. , Vienna 1973, 411.
6.
PATERSON, N. and DHALL, D. P .. The release of adenosine diphosphate 6th Europ. Conf . Microcircula(ADP) from human platelets by dextran. tion, Aalborg 1970, 324.
7.
, S. and TANGEN, 0. Studies on the antithrombotic effect of dextran. Effect on platelet 5-hydroxytryptamine and adenine nucleotides . 7th Europ. Congr. Microcirculation, Aberdeen 1972. Bibl. anat. : l2, 333. 1973.
8.
BORN, G. V. R. Quantitative platelets. J. Physiol. : 162,
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MUSTARD, J.F. and EVANS, G., PACKHAM, M.A., NISHIZAWA, E.E., MURPHY, E. The effect of acetylsalicylic acid on platelet function. J. Exptl Med.: 128, 877. 1968.
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of dextmn TANGEN, O., PHELAN III, W. J. and BERMAN, H. J. Influence and other macromolecules on thrombin time and the polymerization of fibrin monomer. XIII Int. Congr. Hematol. Munich 1970. Abstract 144.
11.
TANGEN, 0. , WIK, K. 0. , ALMQVIST, I.A. M. , ARFORS, K.-E. and HINT, H. C. Effect of dextran on the structure and plasmin-induced lysis of human fibrin. Thrombosis Res .: 1, 487. 1972.
12.
O’BRIEN, J.R. Changes in platelet membranes platelet stickiness. Nature: 212, 1057. 1966.
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MUGGLI
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CAEN, J. and LEGRAND, Y. Plaquettes, collagine concepts of coagulation and hemostasis . Thromb. 1971. Suppl. 48, 167.
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