THRONBOSIS
Printed
RESEARCH in the United
EFFECT
Eaelene
Vol. States
OF IONOPHORB A23,187 WASHED RABBIT
435-449, 7, pp. Pergamon Press,
197j Inc.
0N T~~~~-BEG~~LATEB PLATELETS
A. Chahil, L. Kinlough-Rathbone, H.-J. Reimers and J.F. Mustard
M.A.
Packham,
Department of Pathology, H&aster University, Hamilton, University of Toronto, and Department of Biochemistry, Toronto, Ontario, Canada. in revised form 16.7.1975. (Received 17.6.1975; Accepted by Editor S. Niewiarowski)
ABSTRACT The ionophore A23,187 causes platelets to release their To determine whether granule contents and aggregate. aggregation is a direct effect of A23,187 or is secondary to the release reaction, washed rabbit platelets were treated with thrombin (0.45 U/ml) to cause release of 99X of their granule contents. These degranulated platelets were recovered as disc-shaped platelets which aggregatsdupon the addition of ADP in the presepce of fibrinogen, were unresponsive to further additions of thrombin and adhered to collagen. A23,187 caused shape change and aggregation of the thrombin-degranulated platelets in the presence of CaS+ (2 mM) + Mg++ (1 mM) or Ca++ alone; only shape change occurred in the presence of Mg++ alone, EGTA or EDTA. A23,187 caused aggregation in the absence of fibrinogen, but the presence of fibrinogen increased the extent of aggregation. or caffeine inhibited aggregation of PGgl, theophylline thrombin-degranulated platelets by A23,187 but had no effect on platelet shape change. AMP, adenosine or apyrase had no effect. Thus, A23.187 causes platelet shape change and aggregation independent of ADP release. Ionophore-induced aggregation depends on external calcium but shape change does not depend on external Ca++ or Mg++. Shape change may be caused by A23,187-induced modulation of the intracellular distribution of Ca++ or Ca++ and Mg++.
INTRODUCTION Antibiotics have been described which are capable of transporting calcium and magnesium across membranes (1,2). 435
EFFECT
OF IONOPHOFLE ON PUTELsE'
Foreman et al. (3) and Cochrane 6 Douglas (4) demonstrated that isolated mast cells could be induced to secrete histamine upon exposure to a calcium ionophore provided that calcium was present in the mast cell suspending medium. Feinman & Detwiler (5) demonstrated that human platelet secretion could be induced by the calcium ionophoresA23,187 and X537A and that aggregation was associated with the release of platelet adenine nucleotldes. Yuen and Macey (6) studied the effects of A23,187 on platelet aggregation, but did not consider the contribution of the platelet release reaction in their experiments. Massini and Liischer (7) have also demonstrated platelet aggregation with calcium ionophores and have attributed the aggregation to the release reaction induced by the ionophore secondary-to its making calcium available in the platelet cytoplasm. White et al. (8) also studied the effects of A23,187 on platelet aggregation and secretion and have similarly concluded that an increase in the cytoplasmic concentration of calcium ions from intracellular sites triggers platelet contraction and release. MGrer et al. (9) have studied th e effects of A23,187 on the platelet release reaction and metabolism. However, none of these studies has been independent of the release reaction and direct proof for the role of calcium in platelet aggregation has not been forthcoming. Changes In intracellular calcium as a result of an influx of calcium from the surrounding medium, mobilization from intracellular sites of sequestration, or a combination of these events have been postulated to be responsible for the aggregation that occurs upon exposure of platelets to ADP (10) or the release and aggregation that occur upon addition of thrombin or collagen (11). In the studies reported here we have attempted to elucidate the role of calcium in platelet aggregation using the calcium We have determined whether the ionophore lonophore A23,187. A23,187 can induce shape change and aggregation of platelets which have been depleted of their amine storage granule contents Thus we have been able so that no releasable ATP or ADP remains. to study effects of the ionophore that are not mediated by the release reaction.
MATERIALS
AND
METHODS
creatine phosphate, creatine phosphoADP, AMP, adenosine, kinase, soya bean trypsin inhibitor (SBTI), Hepes (N-2-hydroxy-2-ethane-sulfonic acid) and bovine tendon ethyl-piperazine-N' collagen were obtained from Sigma Chemical Co., St. Louis, MO. TAMe [(p-tosyl-L-arglnine methyl ester (HCl)] was obtained from Heparin was Mann Research Labs, Inc., New York 6, New York. from Connaught Laboratories, Toronto, Ontario, Canada. Pig Batch 74-21) was obtained from Novo Fabrik, plasmin (4.17-NE/mg, Copenhagen, Denmark.
vo1.7,xo.3
EFFECT
OF IONOPHORE
ON PJ.ATELETS
437
EGTA [ethylene glycol bis (amino ethyl)]-tetraacetic acid was from Koch-Light Labs Ltd., Colnbrook, Bucks, England. Prostaglandin El (PGEl) was a gift from the Upjohn Co., Kalamazoo, caffeine was from Eastman Organic Chemicals, Rochester Michigan, from K h K Laboratories Inc., Plain3, New York and theophylline Aspirin was from Matheson, Coleman 6 Bell, East view, New York. Rutherford, New Jersey. The calcium ionophore (A23,187, M.k'. ift of the Eli Lilly Company, Indianapolis, Indiana. ;z;;tz;;n"le C (5-hydroxytryptamine-3 -creatinine sulphate-14C, approximately 50 pCi/umole) was from Amersham/Searle, Arlington Illinois. heights, Bovine thrombin was obtained from Parke, Davis h Co., Detroit, Hichigan. Human fibrinogen (Kabi, Grade L. Stockholm, Sweden) was treated with DFP before use to remove procoagulant activity (12). Apyrase was prepared by the method of Molnar h Lorand (13). Plost soluble reagents were dissolved in Tyrode solution and adjusted to pii 7.35. Pig plasmin was dissolved in unbuffered saline. A23,187 was dissolved in dimethylsulfoxide (DMSO). PGEl solutions vere prepared as described previously (14). Collagen suspensions in Tyrode solution were prepared as previously described (15). Suspensions of washed rabbit platelets were prepared according to the method of Ardlie et al. (16). They were labeled with 14C-serotonin in the first washing fluid (17). After two washes these platelets were suspended in Tyrode solution containing 0.35% albumin (Pentex, Miles Laboratories, Kankakee, Illinois) and apyrase at a concentration capable of converting 0.25 uM ATP to APIP within 120 seconds at 37'C. The platelet count was adjusted to 1 to 1.2 million per mm3. The suspensions were divided into two equal parts and stored at 37°C in a water bath. One part was treated with thrombin (final concentration 0.45 units per ml) to degranulate the platelets and the other part (control) submitted to exactly the same procedures except that Tyrode solution was used instead of thrombin. After one minute, samples were removed for determination of the platelet release reaction by measurement of the amount of 14C in the suspending medium (15). At this time, plasmin (final concentration 0.025%) was added to both the control and thrombintreated platelets. PGEl (10 uil final concentration) was added at the same time. PGEl restored the disc shape of the platelets and prevented plasmin from causing the release reaction (16). Plasmin reduced the extent of fibrin formation that occurred with the thrombin-treated platelets so that they could be recovered more easily. In nearly all experiments, a 30 minute incubation period with plasmin and PGEl was required. After 30 minutes incubation at 37°C sam les of the suspensions were taken for determination of 18 C in the suspending medium. TAMe and SBTI were added (final concentrations of 1 mM and 0.025% respectively). The platelets were centrifuged at 1,200 x g and resuspended in fresh, calcium-free Tyrode-albumin
EFFECT
438
QF IONOPHORE
ON PUTELETS
containing heparin (50 units per ml) and "heparin cofactor" (100 microlitres of rabbit serum per ml of suspension) to neutralize any thrombin still remaining vith the platelets. The suspensions vere centrifuged at 1,200 x g and the platelets resuspended in Tyrode-albumin containing apyrase and 5 mM Hepes to maintain the pH of the medium at fbout 7.3. The platelet count was adjusted to 750,000 per mm .
Aggregation
Studies
For aggregation studies, 1 ml samples of suspension were placed in a cuvette in a turbidimetric device (19). Additions of A23,187 (or of DMSO in control samples) were made with a 10 microlitre pipette. Other additions were made in 0.1 ml volumes. When aggregation induced by ADP was to be studied, fibrinogen (final concentration 0.1%) was added before the ADP. All concentrations given are the final concentrations in the suspension.
Experiments (CP/CPK)
with
creatine
phosphate
and
creatine
phosphokinase
In some experiments this enzyme system was used to convert released ADP to ATP. Creatine phosphate (10 mM) containing 100 units per ml of creatine phosphokinase was prepared a.t the start of the experiment and 0.1 ml of the mixture was added to the platelet suspension one minute before the addition of the aggregating or release-inducing stimulus. Lactate dehydrogenase Bergmeyer et al. (20).
was
measured
by
the method
of
The luciferin-luciferase assay for ATP was done using the method of Kalbhen and Koch (21) as described by Jenkins et al. ADP The lover limit of this assay for ATP was 0.1 uM. (17). was converted to ATP for assay (17).
RESULTS Thrombin
treatment
When washed rabbit platelets were exposed to thrombf; (0.45 Cunits per ml) they released 99.7 f 2.5 percent of their After 30 minserotonin content within one minute (Table 1). of the radioactivity remained in the utes, 76.9 + 2.2 percent indicating that approximately 23 platelet suspending medium, 14C-serotonin had been taken up again by percent of the released since released adenine nucleotides are the platelets. Hovever, it seemed likely that the extent of not taken up by platelets, to deplete the the initial release reaction was sufficient Further stimulation granules of releasable adenine nucleotides.
EFFECT
Vo1.7,No.3
OF IONOPHORE
ON PLATELETS
439
with 5 units per ml of thrombin or with collagen did not result in the release of detectable ATP or ADP determined by the During the luciferin-luciferase assay (CO.1 uM ATP or ADP). first treatment with 0.45 units per ml of thrombin there was a slight increase in the amount of lactic dehydrogenase in the suspending medium (from 0.7 to 1.4 percent) (Table 1) indicating Control that a slight amount of platelet lysis had occurred. platelets exposed to the same procedures (except that thrombin treatment was omitted) did not release their granule contents, nor was there any increase in LDH in the suspending fluid. TABLE Effect of Treatment with Thrombin of Radioactivity and Loss of LDH
Platelets with
or Tyrode Solution on Release from Washed Rabbit Platelets
Time of Exposure to Treatment
Treated
1
Supernatant Radioactivity LDH (4 of Total in Suspension)
Thrombin (0.45 units/ml)
1 min. 30 min.
99.7 76.9
?: 2.5 + 2.2
0.75 1.4
f 0.1 f 0.2
Tyrode Solution
1 min. 30 min.
2.8 3.1
+ 0.4 2 0.4
0.6 0.6
2 0.1 2 0.1
Mean
? S.E.M.
Aggregation
of
10 experiments.
Responses
of Degranulated
Platelets
ADP Both control platelets and platelets pretreated with thrombin aggregated upon the addition of ADP. (Fibrinogen was added to the suspensions before ADP.) The thrombin-treated platelets aggregated more extensively than the control platelets (Figure la). Thrombin Degranulated platelets did not aggregate or change from their disc shape as Indicated by the oscillations In light transmission through a stirred suspension upon the addition of a high concentration of thrombin (0.45 units per ml) (Figure lb) and did not release any further serotonin or ATP and ADP. Collagen The addition of collagen to degranulated platelets always induced shape change and in some cases, collagen induced an increase in light transmission through the suspension, although
440
much
less
EFFECT
OF IONOPHORE
in
controls
than
the
ON PUTELETS
(Figure
lc).
LIGHT TRANSMISSION lb
la
DEGRANULATED
CONTROL
DEGRANULATED
AbP
t THROMGIN 2
4
6
6
4
2
0
MINUTES
MINUTES LIGHT TRANSMISSION
CONTROL
1C
Id
f
I
DEGRANULATED
CONTROL
Jt
DEGRANULATED
A
COLLAGEN 2
4 MINUTES
6
0
FIG.
23167 2
4 MINUTES
6
1
and control Effect of aggregating agents on thrombin-degranulated df fibrinogen, (b) platelets (a) ADP 0.83 IlH, In presence thrombin 0.45 D/ml, (c) collagen, (d) A23,187, 0.1 PM.
EFFECT
vo1.7,?l’o.3
OF IO?jOPHORE ON PLATELETS
4icl
Microscopic examination of the platelet suspensions which showed increased light transmission following the addition of collagen showed platelets adherent to the collagen fibres in the suspensions and platelets adhering to those already adherent to the Although the addition collagen but no free platelet aggregates. of CP/CPK inhibited the increase in light transmission of the control suspension it had little effect on the response of the suspension of degranulated platelets to collagen. A23,187 The addition of A23,187 at a final concentration of 0.1 u?I caused aggregation of both thrombin-degranulated and control platelets (Figure Id). The aggregation induced by this concentration of ionophore was associated with the release of serotonin from the control platelets. These platelets had retained their amine storage granule contents during the preparation procedure and their aggregation could therefore be attributed, at least in ADP. liowever, the ionophore did not cause the part, to released release of 14C, ATP or ADP from the degranulated platelets during the aggregation reaction. The extent of LDH loss from platelets upon exposure to A23,187 was 3.8 + 1.3% of the total in the suspension. The small amount of lysis indicated by LDH loss could not result in the loss of sufficient metabolic pool adenine nucleotides to cause the extent of aggregation seen with A23,187. The presence of fibrinogen in both control suspensions and suspensions of thrombin-degranulated platelets was associated with more extensive aggregation upon the addition of A23,187 than was observed when fibrinogen was not present in the platelet suspending medium (Figure 2).
LIGHT TRANSMISSION
I
NO
FIBRINOGEN
FIBRINOGEN
FIG. 2. DEGRANULATED
I
I OEGRANULATED
A
CONTROL
L
0
2 MINUTES
4
I
0
2 MINUTES
4
Effect of fibrinogen (0.01%) on aggregation induced by 0.1 uM A23,187 with thrombin-degranulated or control platelets. Fibrinogen was added 1 minute before A23,187 in the experiments shown at the right. One of 3 experiments shown.
442
EFFECT
OF IONOPHORE
Effect of calcium or magnesium degranulated platelets
ON
PL4TELET.S
on aggregation
of
Vo1.7,No.3
thrombin-
Figure 3 shows that degranulated platelets aggregated upon the addition of ADP in the presence of added fibrinogen when they were suspended in Tyrode-albumin containing 2 mM calcium and 1 mM magnesium (A), or 2 mM calcium (B). There was less exten sive aggregation when the medium contained only 1 mM magnesium (C) Irrespective of the calcium or magnesium content of the suspending medium, thrombin-degranulated platelets did not aggregate, nor did they change their shape upon exposure to units per ml of thrombin (Figure 3).
0.45
LIGHT TRANSMISSION
r-A.B.C -7 THROMBIN
-\
Lc
ti
f
COLLAGEN
6
2
4
6
0
MINUTES
4
6
MINUTES
FIG. Effect of medium on platelets Suspending B: Ca++ shown.
2
3
++ divalent cation content (Ca Mg++) of suspending aggregation of thrombin-degra:ulated or control by ADP (0.83 PM), thrombin (0.45 U/ml), or collagen. media contained: A: Ca++ (2 mkf) and Mg++ (1 mM); (2 mM) and C: One of three experiments Mg++ (1 mM).
It was possible to induce increase in light transmission
a change of platelet through suspensions
shape and of thrombin-
Vo1.7,No.3
EFFECT
OF IONOPHORE
443
ON PLATELETS
degranulated platelets with high concentrations of collagen when alone (B) were both calcium and magnesium (A), or when calcium However, when there was in the suspending fluid (Figure 3). only magnesium present in the medium (C), platelets did not change their shape updn exposure to collagen and there was no increase in light transmission. Extensive aggregation of thrombin-degranulated platelets in a occurred upon the addition of A23,187 (0.1 PM) to platelets There was medium with calcium and magnesium (A) (Figure 4). less extensive aggregation in the presence of calcium alone (B) and there was no aggregation in the presence of magnesium alone In all cases,the ionophore caused an initial (C) (Figure 4). change in platelet shape as indicated by a change in the amplitude of the oscillations of light transmission.
LIGHT TRANSMISSION
FIG.
4
Effect of divalent cation content (Ca++, Mg++) of suspending medium on aggregation of thrombindegranulated platelets induced by addition of 0.1 PM A23,187. Suspending media contained: A: Ca++ (2 mM) and Fig++ (1 mM), B: Ca++ (2 mM), and C: Mg++ (1 mM). One of 3 experiments shown. A 23187/
0
5
i MINUTES
Inhibitors of ted platelets
ionophore-induced
6
aggregation
of
thrombin-degranula-
The addition of EGTA to suspensions of degranulated platelets in media containing calcium (2 mM) and magnesium (1 mM) inhibited aggregation induced by 0.1 pM A23.187 (Table 2).
444
EFFECT
OF IONOPHORE
TABLE Effect of Inhibitors duced by A23,107
on Platelet
No.
Tyrode EGTA
EDTA
Change
Shape Change
and
Aggregation
+
100.0 97.2
+ + +
1 mM 2 mM 5 mM
3 3 3
+ + +
80.6 3.9
7
+
87.2
2
+
100.0
4
+
94.4
3
+
100.0
7 7
+ +
21.1
4 4 4
+ + +
100.0 9.9 7.4
3 .3
+ +
95.7
2 2
+ +
100.0 100.0
Units/ml
*
5 mM
Adenosine
0.01
mM
0.01 mM 0.001 mM
Theophylline
Caffeine
Aspirin
0.1 mM 1 mM 5 mM
1 mM 5 mM 0.05 mH 0.1 mM
In-
Platelet Aggregation+ (X of Control)
3 3 3
Apyrase
PGEl
Vo1.7,No.3
1 mM 2 mM 5 mM
CP/CPK 1 mM/lO
AMP
PLATELETS
2
Shape
of Experiments
Agent
ON
38.3 4.4 97.2
2.9
6.2
Platelets were exposed to inhibitors for one minute before the +The maximum change in light transaddition of 0.1 UM A23,187. mission through the platelet suspension when Tyrode solution was Maxadded 1 minute before A23,187 was assigned a value of 100%. imum change in light transmission through the samples with inhibitors was expressed as a percentage of this control value. *See Materials and Methods. The extent of inhibition depended upon the concentration of EGTA; aggregation was completely inhibited by 5 mM EGTA but a concentration of 1 mM EGTA did not modify aggregation. EDTA (5 mM) was also an effective inhibitor of A23,187-induced aggregation,
vo1.7,xo.3
EFFECT
OF IONOF'HORE
ON PLATELETS
445
The ionobut was also slightly inhibitory at 2 mM (Table 2). phore caused platelet shape change in the presence of the highest concentrations of the chelating agents (Table 2). phosphokinase Addition of the creatine phosphate :creatine enzyme system or apyrase to thrombin-degranulated, washed rabbit platelets had little effect on the aggregation induced A23,187 (Table 2) whereas it completely inhibited aggregation caused by 8 uM ADP.
by
AMP did not affect aggregation of thrombin-degranulated platelets induced by A23,187 even when it was used at a concentration of 5 mM (Table 2). PGEl at concentrations of 1 and 10 uM inhibited aggregation of thrombin-degranulated platelets induced Adenosine by A23,187 but did not modify platelet shape change. (10 uM) had no effect on aggregation caused by the ionophore. Both theophylline (1 or 5 mM) and caffeine (5 mM) caused inhibition of A23,187-induced aggregation when added one minute Theophylline was more inhibitory than before the ionophore. None of these agents inhibited the ionophorea caffeine (Table 2). induced shape change. Ionophore-induced shape change and aggregation of thrombindegranulated platelets was not modified by a one minute preincubation of the platelets with aspirin (0.1 mbl).
DISCUSSION A number of investigators have demonstrated that divalent release reaction and platecation ionophores cause the platelet None of these studies has establet aggregation (5,6,7,8,9). whether the ionophore A23,187 will induce platelished however, let shape change and aggregation independent of the release To investigate ionophore-induced aggregation in the reaction. absence of the release reaction a method was developed to deplete rabbit platelets of all of their amine storage granule contents of serotonin, ATP and ADP. This was accomplished by treating suspensions of washed platelets with thrombin in the presence of plasmin, recovering the platelets and resuspending them in fresh medium. These platelets aggregated normally upon the addition of ADP provided fibrinogen were in the medium. The necessity for fibrinogen indicates that plasmin treatment has removed the fibrinogen which usually remains associated with rabbit platelets during washing procedures (16). These degranulated platelets did not undergo shape change or aggregation upon the addition of thrombin, nor did they release ATP or ADP. Although there was an increase in light transmission upon the addition of collagen these changes appeared to be predominantly due to the adherence of platelets to the collagen fibres. These changes were not mediated through the release of ADP because CP/CPK or apyrase did not affect them. The concentrations of CP/CPK or apyrase used are sufficient to change the extent of platelet aggregation induced by thrombin or collagen with normal rabbit platelets (22). in light The collagen-induced increase transmission of degranulated platelets did not occur when the Platelets were suspended in a medium containing magnesium but no
446
EFFECT
calcium. It seems the collagen-induced to collagen. This external calcimm. of normal platelets (23).
OF IONOPHORE
ON PLATELETS
Vo1.7,No.3
likely that in a calcium-containing medium changes are caused by platelet adherence adherence, thefefore, is dependent upon It has been shown previously that adherence to collagen also is calcium dependent
The addition of the ionophore A23,187 to these thrombindegranulated rabbit platelets induced shape change and aggregation but no detectable release of serotonin, ATP or ADP. These effects of the ionophore were not mediated by ADP because the CP/CPK system or apyrase was not inhibitory nor were AMP or adenosine. At the concentrations used, A23,187 did not cause appreciable lysis because there was only slight loss of LDti. It seems reasonable to conclude that platelet shape change and aggregation induced by A23,187 are independent of the release of platelet granule ADP or loss of cytoplasmic ADP. White et al. (8) have reported that platelets from human patients with storage pool deficiency are aggregated by A23,187. Although they discuss this as evidence that release of calcium stored in dense bodies is not required for ionophore-induced aggregation it could also be interpreted to mean that the release of ADP is not required for ionophore-induced aggregation of human platelets. The observations of White et al. (8) with storage pool deficient platelets therefore are in agreement with our observations with thrombin-degranulated rabbit platelets. The effect of the ionophore in inducing shape change and aggregation is similar to that of ADP: the ionophore induces shape change independent of external calcium and magnesium in the suspending medium. For aggregation to occur when the degranulated platelets are exposed to the ionophore, calcium is required in the suspending medium. Although A23,187 could induce aggregation of degranulated platelets without the presence of fibrinogen in the suspending medium, the presence of fibrinogen potentiated ionophore-induced aggregation. It should be pointed out that these platelets have been exposed to plasmin which removes the fibrinogen that is normally adsorbed to the surface of washed rabbit platelets. The results with the ionophore indicate either that it does not require fibrinogen to induce aggregation or that it causes the release or loss of platelet fibrinogen that was not accessible to plasmin during the degranulation procedure. In these experiments ionophore-induced platelet shape change occurred in the presence of EDTA or EGTA but aggregation did not White et al. (8) made similar observations with human occur. It is unlikely that the platelets in the presence of EDTA. change of platelet shape in the presence of these strong chelating agents was mediated by the transport of external calcium across it seems more likely that the ionophore can exert the membrane: or on intracellular calcium and/or an effect either on membrane thereby influencing the reaction which is involved in magnesium, This is in agreement with the suggestion platelet shape change. the effect of calcium put forward by White et al. (8) concerning Studies with inhibitors support on platelet contractile protein.
Vol.7
,No.3
EFFECT
OF IONOPHORE
447
ON PLATELETS
the interpretation that A23,187-induced aggregation of thrombinagents which degranulated platelets is not due to released ADP: inhibited ADP-induced aggregation (CP/CPK, apyrase, adenosine, and AMP) had no detectable effect on the ionophore-induced aggreIonophore-induced aggregation cannot be attributed to gation. the formation of the endoperoxide intermediates of prostaglandin synthesis (26) because aspirin had no effect on aggregation inseveral compounds which increased In contrast, duced by A23,187. and theophylline) all platelet cyclic AMP levels (PGEl, caffeine Adenosine which causes inhibited ionophore-induced aggregation. only a slight increase in platelet cyclic AMP levels (24,25) was It may be that the action of PGEl is related to ineffective. the reputed effect of cyclic AMP on calcium availability within the platelets (8). Although White et al. (8) were studying human platelets in a system in which ionophore-induced aggregation and release could not be investigated separately, they obtained similar inhibitory effects with PGEl and theophylline and also observed that adenosine and aspirin were not inhibitory. They interpreted their results with agents that increase cyclic of AMP levels as indicating that "CAMP may block the response platelets by preventing an increase in the cytoplasmic concenIt seems likely that an effect of tration of calcium ions." cyclic AMP on platelet calcium may be the basis for its inhibition of aggregation induced either by ADP or the ionophore, both of which can cause aggregation independent of the release reaction.
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REED, P.W. and LARDY, H.A. phore. J. Biol. Chem. 247,
2.
PRESSMAN, B.C. Properties cation selectivity. Fed.
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FOREMAN, J.C., MONGAR, J.L. and GOMPERTS, B.D. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process. Nature 245, 249, 1973.
4.
COCHRANE, D.E. and DOUGLAS, W.W. Calcium-induced extrusion of secretory granules (exocytosis) in mast cells exposed to 48180 or the ionophores A23,187 and X537A. Proc. Nat. Acad. Sci. 71, 408, 1974.
5.
FEINMAN, R.D. and DETWILER, T.C. Platelet secretion induced by divalent cation ionophores. Nature 249, 1972, 1974.
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YUEN, M. and MACEY, R. Platelet aggregation induced by a calcium ionophore. Fed. Proc. 33, 269 (Abs.), 1974. .. MASSINI, P. and LUSCHER, E.F. Some effects of ionophores for divalent cations on blood platelets. Comparison with the effects of thrombin. Biochim. et Biophys. Acta 372, 109, 1974.
7.
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A divalent A23,187: 6970, 1972.
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iono-
range
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M.A. and MUSTARD, KINLOUGH-RATHBONE, R.L., PACKHAM, The effect of prostaglandin El on platelet function vitro and in vivo. Brit. J. Haemat. 19, 559, 1970.
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M.A. and MUSTARD, J.F. Adenosine diARDLIE, N.G., PACKHAM, phosphate-induced platelet aggregation in suspensions of washed rabbit platelets. Brit. J. Haemat. 19, 7, 1970.
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JENKINS, C.S.P., PACKHAM, M.A., KINLOUGH-RATHBONE, R.L. and Interactions of polylysine with platelets. MUSTARD, J.F. Blood 37, 395, 1971.
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MUSTARD, J.F., HEGARDT, B., ROWSELL, H.C. and MacMILLAN, R.L. Effect of adenosine nucleotides on platelet aggregation and clotting time. J. Lab. Clin. Med. 64, 548, 1964.
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Lactic dehydroBERGMEYER, H.U., BERNT, E. and HESS, B. in Enzymatic Analysis. H.U. Bergmeyer genase. In: Methods (ed.) New York, Academic Press. 1965, p. 736.
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Methodische Untersuchungen zur KALBHEN, D.A. and KOCH, H.J. quantitativenMikrobestimmung von ATP in biologischem Material mit dem Firefly-Enzymsystem. 2. Klin. Chem. 5, 299, 1967.
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PACKHAM, M.A., GuCCIONE, M.A., CHANG, P.-L. and MUSTARD, effects of low Platelet aggregation and release: J.F. Amer. J. Physiol. centrations of thrombin or collagen. 225, 38, 1973.
con-
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23.
M.A. and MUSTARD, CAZENAVE, J.-P., PACKHAM, of platelets to a collagen-coated surface: a quantitative method. J. Lab. Clin. Med.
J.F. Adherence development of 82, 978, 1973.
24.
Interactions of the HASLAM, R.J. of blood platelets with adenylate 6, 333, 1973.
25.
MILLS, D.C.B. and SMITH, J.B. The influence on platelet aggregation of drugs that affect the accumulation of adenosine 3': 5'-cyclic monophosphate in platelets. Biochem. J. 121, 185, 1971.
26.
SMITH, J.B., INGERMAN, C., KOCSIS, J.J. and SILVER, M.J. Formation of an intermediate in prostaglandin biosynthesis and its association with the platelet release reaction. 53, 1468, 1974. J. Clin. Invest.
pharmacological receptors cyclase. Series Haemat.
Printed
RESEARCH in the United
EFFECT
Eaelene
Vol. States
OF IONOPHORB A23,187 WASHED RABBIT
435-449, 7, pp. Pergamon Press,
197j Inc.
0N T~~~~-BEG~~LATEB PLATELETS
A. Chahil, L. Kinlough-Rathbone, H.-J. Reimers and J.F. Mustard
M.A.
Packham,
Department of Pathology, H&aster University, Hamilton, University of Toronto, and Department of Biochemistry, Toronto, Ontario, Canada. in revised form 16.7.1975. (Received 17.6.1975; Accepted by Editor S. Niewiarowski)
ABSTRACT The ionophore A23,187 causes platelets to release their To determine whether granule contents and aggregate. aggregation is a direct effect of A23,187 or is secondary to the release reaction, washed rabbit platelets were treated with thrombin (0.45 U/ml) to cause release of 99X of their granule contents. These degranulated platelets were recovered as disc-shaped platelets which aggregatsdupon the addition of ADP in the presepce of fibrinogen, were unresponsive to further additions of thrombin and adhered to collagen. A23,187 caused shape change and aggregation of the thrombin-degranulated platelets in the presence of CaS+ (2 mM) + Mg++ (1 mM) or Ca++ alone; only shape change occurred in the presence of Mg++ alone, EGTA or EDTA. A23,187 caused aggregation in the absence of fibrinogen, but the presence of fibrinogen increased the extent of aggregation. or caffeine inhibited aggregation of PGgl, theophylline thrombin-degranulated platelets by A23,187 but had no effect on platelet shape change. AMP, adenosine or apyrase had no effect. Thus, A23.187 causes platelet shape change and aggregation independent of ADP release. Ionophore-induced aggregation depends on external calcium but shape change does not depend on external Ca++ or Mg++. Shape change may be caused by A23,187-induced modulation of the intracellular distribution of Ca++ or Ca++ and Mg++.
INTRODUCTION Antibiotics have been described which are capable of transporting calcium and magnesium across membranes (1,2). 435
EFFECT
OF IONOPHOFLE ON PUTELsE'
Foreman et al. (3) and Cochrane 6 Douglas (4) demonstrated that isolated mast cells could be induced to secrete histamine upon exposure to a calcium ionophore provided that calcium was present in the mast cell suspending medium. Feinman & Detwiler (5) demonstrated that human platelet secretion could be induced by the calcium ionophoresA23,187 and X537A and that aggregation was associated with the release of platelet adenine nucleotldes. Yuen and Macey (6) studied the effects of A23,187 on platelet aggregation, but did not consider the contribution of the platelet release reaction in their experiments. Massini and Liischer (7) have also demonstrated platelet aggregation with calcium ionophores and have attributed the aggregation to the release reaction induced by the ionophore secondary-to its making calcium available in the platelet cytoplasm. White et al. (8) also studied the effects of A23,187 on platelet aggregation and secretion and have similarly concluded that an increase in the cytoplasmic concentration of calcium ions from intracellular sites triggers platelet contraction and release. MGrer et al. (9) have studied th e effects of A23,187 on the platelet release reaction and metabolism. However, none of these studies has been independent of the release reaction and direct proof for the role of calcium in platelet aggregation has not been forthcoming. Changes In intracellular calcium as a result of an influx of calcium from the surrounding medium, mobilization from intracellular sites of sequestration, or a combination of these events have been postulated to be responsible for the aggregation that occurs upon exposure of platelets to ADP (10) or the release and aggregation that occur upon addition of thrombin or collagen (11). In the studies reported here we have attempted to elucidate the role of calcium in platelet aggregation using the calcium We have determined whether the ionophore lonophore A23,187. A23,187 can induce shape change and aggregation of platelets which have been depleted of their amine storage granule contents Thus we have been able so that no releasable ATP or ADP remains. to study effects of the ionophore that are not mediated by the release reaction.
MATERIALS
AND
METHODS
creatine phosphate, creatine phosphoADP, AMP, adenosine, kinase, soya bean trypsin inhibitor (SBTI), Hepes (N-2-hydroxy-2-ethane-sulfonic acid) and bovine tendon ethyl-piperazine-N' collagen were obtained from Sigma Chemical Co., St. Louis, MO. TAMe [(p-tosyl-L-arglnine methyl ester (HCl)] was obtained from Heparin was Mann Research Labs, Inc., New York 6, New York. from Connaught Laboratories, Toronto, Ontario, Canada. Pig Batch 74-21) was obtained from Novo Fabrik, plasmin (4.17-NE/mg, Copenhagen, Denmark.
vo1.7,xo.3
EFFECT
OF IONOPHORE
ON PJ.ATELETS
437
EGTA [ethylene glycol bis (amino ethyl)]-tetraacetic acid was from Koch-Light Labs Ltd., Colnbrook, Bucks, England. Prostaglandin El (PGEl) was a gift from the Upjohn Co., Kalamazoo, caffeine was from Eastman Organic Chemicals, Rochester Michigan, from K h K Laboratories Inc., Plain3, New York and theophylline Aspirin was from Matheson, Coleman 6 Bell, East view, New York. Rutherford, New Jersey. The calcium ionophore (A23,187, M.k'. ift of the Eli Lilly Company, Indianapolis, Indiana. ;z;;tz;;n"le C (5-hydroxytryptamine-3 -creatinine sulphate-14C, approximately 50 pCi/umole) was from Amersham/Searle, Arlington Illinois. heights, Bovine thrombin was obtained from Parke, Davis h Co., Detroit, Hichigan. Human fibrinogen (Kabi, Grade L. Stockholm, Sweden) was treated with DFP before use to remove procoagulant activity (12). Apyrase was prepared by the method of Molnar h Lorand (13). Plost soluble reagents were dissolved in Tyrode solution and adjusted to pii 7.35. Pig plasmin was dissolved in unbuffered saline. A23,187 was dissolved in dimethylsulfoxide (DMSO). PGEl solutions vere prepared as described previously (14). Collagen suspensions in Tyrode solution were prepared as previously described (15). Suspensions of washed rabbit platelets were prepared according to the method of Ardlie et al. (16). They were labeled with 14C-serotonin in the first washing fluid (17). After two washes these platelets were suspended in Tyrode solution containing 0.35% albumin (Pentex, Miles Laboratories, Kankakee, Illinois) and apyrase at a concentration capable of converting 0.25 uM ATP to APIP within 120 seconds at 37'C. The platelet count was adjusted to 1 to 1.2 million per mm3. The suspensions were divided into two equal parts and stored at 37°C in a water bath. One part was treated with thrombin (final concentration 0.45 units per ml) to degranulate the platelets and the other part (control) submitted to exactly the same procedures except that Tyrode solution was used instead of thrombin. After one minute, samples were removed for determination of the platelet release reaction by measurement of the amount of 14C in the suspending medium (15). At this time, plasmin (final concentration 0.025%) was added to both the control and thrombintreated platelets. PGEl (10 uil final concentration) was added at the same time. PGEl restored the disc shape of the platelets and prevented plasmin from causing the release reaction (16). Plasmin reduced the extent of fibrin formation that occurred with the thrombin-treated platelets so that they could be recovered more easily. In nearly all experiments, a 30 minute incubation period with plasmin and PGEl was required. After 30 minutes incubation at 37°C sam les of the suspensions were taken for determination of 18 C in the suspending medium. TAMe and SBTI were added (final concentrations of 1 mM and 0.025% respectively). The platelets were centrifuged at 1,200 x g and resuspended in fresh, calcium-free Tyrode-albumin
EFFECT
438
QF IONOPHORE
ON PUTELETS
containing heparin (50 units per ml) and "heparin cofactor" (100 microlitres of rabbit serum per ml of suspension) to neutralize any thrombin still remaining vith the platelets. The suspensions vere centrifuged at 1,200 x g and the platelets resuspended in Tyrode-albumin containing apyrase and 5 mM Hepes to maintain the pH of the medium at fbout 7.3. The platelet count was adjusted to 750,000 per mm .
Aggregation
Studies
For aggregation studies, 1 ml samples of suspension were placed in a cuvette in a turbidimetric device (19). Additions of A23,187 (or of DMSO in control samples) were made with a 10 microlitre pipette. Other additions were made in 0.1 ml volumes. When aggregation induced by ADP was to be studied, fibrinogen (final concentration 0.1%) was added before the ADP. All concentrations given are the final concentrations in the suspension.
Experiments (CP/CPK)
with
creatine
phosphate
and
creatine
phosphokinase
In some experiments this enzyme system was used to convert released ADP to ATP. Creatine phosphate (10 mM) containing 100 units per ml of creatine phosphokinase was prepared a.t the start of the experiment and 0.1 ml of the mixture was added to the platelet suspension one minute before the addition of the aggregating or release-inducing stimulus. Lactate dehydrogenase Bergmeyer et al. (20).
was
measured
by
the method
of
The luciferin-luciferase assay for ATP was done using the method of Kalbhen and Koch (21) as described by Jenkins et al. ADP The lover limit of this assay for ATP was 0.1 uM. (17). was converted to ATP for assay (17).
RESULTS Thrombin
treatment
When washed rabbit platelets were exposed to thrombf; (0.45 Cunits per ml) they released 99.7 f 2.5 percent of their After 30 minserotonin content within one minute (Table 1). of the radioactivity remained in the utes, 76.9 + 2.2 percent indicating that approximately 23 platelet suspending medium, 14C-serotonin had been taken up again by percent of the released since released adenine nucleotides are the platelets. Hovever, it seemed likely that the extent of not taken up by platelets, to deplete the the initial release reaction was sufficient Further stimulation granules of releasable adenine nucleotides.
EFFECT
Vo1.7,No.3
OF IONOPHORE
ON PLATELETS
439
with 5 units per ml of thrombin or with collagen did not result in the release of detectable ATP or ADP determined by the During the luciferin-luciferase assay (CO.1 uM ATP or ADP). first treatment with 0.45 units per ml of thrombin there was a slight increase in the amount of lactic dehydrogenase in the suspending medium (from 0.7 to 1.4 percent) (Table 1) indicating Control that a slight amount of platelet lysis had occurred. platelets exposed to the same procedures (except that thrombin treatment was omitted) did not release their granule contents, nor was there any increase in LDH in the suspending fluid. TABLE Effect of Treatment with Thrombin of Radioactivity and Loss of LDH
Platelets with
or Tyrode Solution on Release from Washed Rabbit Platelets
Time of Exposure to Treatment
Treated
1
Supernatant Radioactivity LDH (4 of Total in Suspension)
Thrombin (0.45 units/ml)
1 min. 30 min.
99.7 76.9
?: 2.5 + 2.2
0.75 1.4
f 0.1 f 0.2
Tyrode Solution
1 min. 30 min.
2.8 3.1
+ 0.4 2 0.4
0.6 0.6
2 0.1 2 0.1
Mean
? S.E.M.
Aggregation
of
10 experiments.
Responses
of Degranulated
Platelets
ADP Both control platelets and platelets pretreated with thrombin aggregated upon the addition of ADP. (Fibrinogen was added to the suspensions before ADP.) The thrombin-treated platelets aggregated more extensively than the control platelets (Figure la). Thrombin Degranulated platelets did not aggregate or change from their disc shape as Indicated by the oscillations In light transmission through a stirred suspension upon the addition of a high concentration of thrombin (0.45 units per ml) (Figure lb) and did not release any further serotonin or ATP and ADP. Collagen The addition of collagen to degranulated platelets always induced shape change and in some cases, collagen induced an increase in light transmission through the suspension, although
440
much
less
EFFECT
OF IONOPHORE
in
controls
than
the
ON PUTELETS
(Figure
lc).
LIGHT TRANSMISSION lb
la
DEGRANULATED
CONTROL
DEGRANULATED
AbP
t THROMGIN 2
4
6
6
4
2
0
MINUTES
MINUTES LIGHT TRANSMISSION
CONTROL
1C
Id
f
I
DEGRANULATED
CONTROL
Jt
DEGRANULATED
A
COLLAGEN 2
4 MINUTES
6
0
FIG.
23167 2
4 MINUTES
6
1
and control Effect of aggregating agents on thrombin-degranulated df fibrinogen, (b) platelets (a) ADP 0.83 IlH, In presence thrombin 0.45 D/ml, (c) collagen, (d) A23,187, 0.1 PM.
EFFECT
vo1.7,?l’o.3
OF IO?jOPHORE ON PLATELETS
4icl
Microscopic examination of the platelet suspensions which showed increased light transmission following the addition of collagen showed platelets adherent to the collagen fibres in the suspensions and platelets adhering to those already adherent to the Although the addition collagen but no free platelet aggregates. of CP/CPK inhibited the increase in light transmission of the control suspension it had little effect on the response of the suspension of degranulated platelets to collagen. A23,187 The addition of A23,187 at a final concentration of 0.1 u?I caused aggregation of both thrombin-degranulated and control platelets (Figure Id). The aggregation induced by this concentration of ionophore was associated with the release of serotonin from the control platelets. These platelets had retained their amine storage granule contents during the preparation procedure and their aggregation could therefore be attributed, at least in ADP. liowever, the ionophore did not cause the part, to released release of 14C, ATP or ADP from the degranulated platelets during the aggregation reaction. The extent of LDH loss from platelets upon exposure to A23,187 was 3.8 + 1.3% of the total in the suspension. The small amount of lysis indicated by LDH loss could not result in the loss of sufficient metabolic pool adenine nucleotides to cause the extent of aggregation seen with A23,187. The presence of fibrinogen in both control suspensions and suspensions of thrombin-degranulated platelets was associated with more extensive aggregation upon the addition of A23,187 than was observed when fibrinogen was not present in the platelet suspending medium (Figure 2).
LIGHT TRANSMISSION
I
NO
FIBRINOGEN
FIBRINOGEN
FIG. 2. DEGRANULATED
I
I OEGRANULATED
A
CONTROL
L
0
2 MINUTES
4
I
0
2 MINUTES
4
Effect of fibrinogen (0.01%) on aggregation induced by 0.1 uM A23,187 with thrombin-degranulated or control platelets. Fibrinogen was added 1 minute before A23,187 in the experiments shown at the right. One of 3 experiments shown.
442
EFFECT
OF IONOPHORE
Effect of calcium or magnesium degranulated platelets
ON
PL4TELET.S
on aggregation
of
Vo1.7,No.3
thrombin-
Figure 3 shows that degranulated platelets aggregated upon the addition of ADP in the presence of added fibrinogen when they were suspended in Tyrode-albumin containing 2 mM calcium and 1 mM magnesium (A), or 2 mM calcium (B). There was less exten sive aggregation when the medium contained only 1 mM magnesium (C) Irrespective of the calcium or magnesium content of the suspending medium, thrombin-degranulated platelets did not aggregate, nor did they change their shape upon exposure to units per ml of thrombin (Figure 3).
0.45
LIGHT TRANSMISSION
r-A.B.C -7 THROMBIN
-\
Lc
ti
f
COLLAGEN
6
2
4
6
0
MINUTES
4
6
MINUTES
FIG. Effect of medium on platelets Suspending B: Ca++ shown.
2
3
++ divalent cation content (Ca Mg++) of suspending aggregation of thrombin-degra:ulated or control by ADP (0.83 PM), thrombin (0.45 U/ml), or collagen. media contained: A: Ca++ (2 mkf) and Mg++ (1 mM); (2 mM) and C: One of three experiments Mg++ (1 mM).
It was possible to induce increase in light transmission
a change of platelet through suspensions
shape and of thrombin-
Vo1.7,No.3
EFFECT
OF IONOPHORE
443
ON PLATELETS
degranulated platelets with high concentrations of collagen when alone (B) were both calcium and magnesium (A), or when calcium However, when there was in the suspending fluid (Figure 3). only magnesium present in the medium (C), platelets did not change their shape updn exposure to collagen and there was no increase in light transmission. Extensive aggregation of thrombin-degranulated platelets in a occurred upon the addition of A23,187 (0.1 PM) to platelets There was medium with calcium and magnesium (A) (Figure 4). less extensive aggregation in the presence of calcium alone (B) and there was no aggregation in the presence of magnesium alone In all cases,the ionophore caused an initial (C) (Figure 4). change in platelet shape as indicated by a change in the amplitude of the oscillations of light transmission.
LIGHT TRANSMISSION
FIG.
4
Effect of divalent cation content (Ca++, Mg++) of suspending medium on aggregation of thrombindegranulated platelets induced by addition of 0.1 PM A23,187. Suspending media contained: A: Ca++ (2 mM) and Fig++ (1 mM), B: Ca++ (2 mM), and C: Mg++ (1 mM). One of 3 experiments shown. A 23187/
0
5
i MINUTES
Inhibitors of ted platelets
ionophore-induced
6
aggregation
of
thrombin-degranula-
The addition of EGTA to suspensions of degranulated platelets in media containing calcium (2 mM) and magnesium (1 mM) inhibited aggregation induced by 0.1 pM A23.187 (Table 2).
444
EFFECT
OF IONOPHORE
TABLE Effect of Inhibitors duced by A23,107
on Platelet
No.
Tyrode EGTA
EDTA
Change
Shape Change
and
Aggregation
+
100.0 97.2
+ + +
1 mM 2 mM 5 mM
3 3 3
+ + +
80.6 3.9
7
+
87.2
2
+
100.0
4
+
94.4
3
+
100.0
7 7
+ +
21.1
4 4 4
+ + +
100.0 9.9 7.4
3 .3
+ +
95.7
2 2
+ +
100.0 100.0
Units/ml
*
5 mM
Adenosine
0.01
mM
0.01 mM 0.001 mM
Theophylline
Caffeine
Aspirin
0.1 mM 1 mM 5 mM
1 mM 5 mM 0.05 mH 0.1 mM
In-
Platelet Aggregation+ (X of Control)
3 3 3
Apyrase
PGEl
Vo1.7,No.3
1 mM 2 mM 5 mM
CP/CPK 1 mM/lO
AMP
PLATELETS
2
Shape
of Experiments
Agent
ON
38.3 4.4 97.2
2.9
6.2
Platelets were exposed to inhibitors for one minute before the +The maximum change in light transaddition of 0.1 UM A23,187. mission through the platelet suspension when Tyrode solution was Maxadded 1 minute before A23,187 was assigned a value of 100%. imum change in light transmission through the samples with inhibitors was expressed as a percentage of this control value. *See Materials and Methods. The extent of inhibition depended upon the concentration of EGTA; aggregation was completely inhibited by 5 mM EGTA but a concentration of 1 mM EGTA did not modify aggregation. EDTA (5 mM) was also an effective inhibitor of A23,187-induced aggregation,
vo1.7,xo.3
EFFECT
OF IONOF'HORE
ON PLATELETS
445
The ionobut was also slightly inhibitory at 2 mM (Table 2). phore caused platelet shape change in the presence of the highest concentrations of the chelating agents (Table 2). phosphokinase Addition of the creatine phosphate :creatine enzyme system or apyrase to thrombin-degranulated, washed rabbit platelets had little effect on the aggregation induced A23,187 (Table 2) whereas it completely inhibited aggregation caused by 8 uM ADP.
by
AMP did not affect aggregation of thrombin-degranulated platelets induced by A23,187 even when it was used at a concentration of 5 mM (Table 2). PGEl at concentrations of 1 and 10 uM inhibited aggregation of thrombin-degranulated platelets induced Adenosine by A23,187 but did not modify platelet shape change. (10 uM) had no effect on aggregation caused by the ionophore. Both theophylline (1 or 5 mM) and caffeine (5 mM) caused inhibition of A23,187-induced aggregation when added one minute Theophylline was more inhibitory than before the ionophore. None of these agents inhibited the ionophorea caffeine (Table 2). induced shape change. Ionophore-induced shape change and aggregation of thrombindegranulated platelets was not modified by a one minute preincubation of the platelets with aspirin (0.1 mbl).
DISCUSSION A number of investigators have demonstrated that divalent release reaction and platecation ionophores cause the platelet None of these studies has establet aggregation (5,6,7,8,9). whether the ionophore A23,187 will induce platelished however, let shape change and aggregation independent of the release To investigate ionophore-induced aggregation in the reaction. absence of the release reaction a method was developed to deplete rabbit platelets of all of their amine storage granule contents of serotonin, ATP and ADP. This was accomplished by treating suspensions of washed platelets with thrombin in the presence of plasmin, recovering the platelets and resuspending them in fresh medium. These platelets aggregated normally upon the addition of ADP provided fibrinogen were in the medium. The necessity for fibrinogen indicates that plasmin treatment has removed the fibrinogen which usually remains associated with rabbit platelets during washing procedures (16). These degranulated platelets did not undergo shape change or aggregation upon the addition of thrombin, nor did they release ATP or ADP. Although there was an increase in light transmission upon the addition of collagen these changes appeared to be predominantly due to the adherence of platelets to the collagen fibres. These changes were not mediated through the release of ADP because CP/CPK or apyrase did not affect them. The concentrations of CP/CPK or apyrase used are sufficient to change the extent of platelet aggregation induced by thrombin or collagen with normal rabbit platelets (22). in light The collagen-induced increase transmission of degranulated platelets did not occur when the Platelets were suspended in a medium containing magnesium but no
446
EFFECT
calcium. It seems the collagen-induced to collagen. This external calcimm. of normal platelets (23).
OF IONOPHORE
ON PLATELETS
Vo1.7,No.3
likely that in a calcium-containing medium changes are caused by platelet adherence adherence, thefefore, is dependent upon It has been shown previously that adherence to collagen also is calcium dependent
The addition of the ionophore A23,187 to these thrombindegranulated rabbit platelets induced shape change and aggregation but no detectable release of serotonin, ATP or ADP. These effects of the ionophore were not mediated by ADP because the CP/CPK system or apyrase was not inhibitory nor were AMP or adenosine. At the concentrations used, A23,187 did not cause appreciable lysis because there was only slight loss of LDti. It seems reasonable to conclude that platelet shape change and aggregation induced by A23,187 are independent of the release of platelet granule ADP or loss of cytoplasmic ADP. White et al. (8) have reported that platelets from human patients with storage pool deficiency are aggregated by A23,187. Although they discuss this as evidence that release of calcium stored in dense bodies is not required for ionophore-induced aggregation it could also be interpreted to mean that the release of ADP is not required for ionophore-induced aggregation of human platelets. The observations of White et al. (8) with storage pool deficient platelets therefore are in agreement with our observations with thrombin-degranulated rabbit platelets. The effect of the ionophore in inducing shape change and aggregation is similar to that of ADP: the ionophore induces shape change independent of external calcium and magnesium in the suspending medium. For aggregation to occur when the degranulated platelets are exposed to the ionophore, calcium is required in the suspending medium. Although A23,187 could induce aggregation of degranulated platelets without the presence of fibrinogen in the suspending medium, the presence of fibrinogen potentiated ionophore-induced aggregation. It should be pointed out that these platelets have been exposed to plasmin which removes the fibrinogen that is normally adsorbed to the surface of washed rabbit platelets. The results with the ionophore indicate either that it does not require fibrinogen to induce aggregation or that it causes the release or loss of platelet fibrinogen that was not accessible to plasmin during the degranulation procedure. In these experiments ionophore-induced platelet shape change occurred in the presence of EDTA or EGTA but aggregation did not White et al. (8) made similar observations with human occur. It is unlikely that the platelets in the presence of EDTA. change of platelet shape in the presence of these strong chelating agents was mediated by the transport of external calcium across it seems more likely that the ionophore can exert the membrane: or on intracellular calcium and/or an effect either on membrane thereby influencing the reaction which is involved in magnesium, This is in agreement with the suggestion platelet shape change. the effect of calcium put forward by White et al. (8) concerning Studies with inhibitors support on platelet contractile protein.
Vol.7
,No.3
EFFECT
OF IONOPHORE
447
ON PLATELETS
the interpretation that A23,187-induced aggregation of thrombinagents which degranulated platelets is not due to released ADP: inhibited ADP-induced aggregation (CP/CPK, apyrase, adenosine, and AMP) had no detectable effect on the ionophore-induced aggreIonophore-induced aggregation cannot be attributed to gation. the formation of the endoperoxide intermediates of prostaglandin synthesis (26) because aspirin had no effect on aggregation inseveral compounds which increased In contrast, duced by A23,187. and theophylline) all platelet cyclic AMP levels (PGEl, caffeine Adenosine which causes inhibited ionophore-induced aggregation. only a slight increase in platelet cyclic AMP levels (24,25) was It may be that the action of PGEl is related to ineffective. the reputed effect of cyclic AMP on calcium availability within the platelets (8). Although White et al. (8) were studying human platelets in a system in which ionophore-induced aggregation and release could not be investigated separately, they obtained similar inhibitory effects with PGEl and theophylline and also observed that adenosine and aspirin were not inhibitory. They interpreted their results with agents that increase cyclic of AMP levels as indicating that "CAMP may block the response platelets by preventing an increase in the cytoplasmic concenIt seems likely that an effect of tration of calcium ions." cyclic AMP on platelet calcium may be the basis for its inhibition of aggregation induced either by ADP or the ionophore, both of which can cause aggregation independent of the release reaction.
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REED, P.W. and LARDY, H.A. phore. J. Biol. Chem. 247,
2.
PRESSMAN, B.C. Properties cation selectivity. Fed.
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FOREMAN, J.C., MONGAR, J.L. and GOMPERTS, B.D. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process. Nature 245, 249, 1973.
4.
COCHRANE, D.E. and DOUGLAS, W.W. Calcium-induced extrusion of secretory granules (exocytosis) in mast cells exposed to 48180 or the ionophores A23,187 and X537A. Proc. Nat. Acad. Sci. 71, 408, 1974.
5.
FEINMAN, R.D. and DETWILER, T.C. Platelet secretion induced by divalent cation ionophores. Nature 249, 1972, 1974.
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YUEN, M. and MACEY, R. Platelet aggregation induced by a calcium ionophore. Fed. Proc. 33, 269 (Abs.), 1974. .. MASSINI, P. and LUSCHER, E.F. Some effects of ionophores for divalent cations on blood platelets. Comparison with the effects of thrombin. Biochim. et Biophys. Acta 372, 109, 1974.
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A divalent A23,187: 6970, 1972.
cation
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iono-
range
WHITE, J.G., RAO, G.H.R., and GERRARD, J.M. Effects of the ionophore A23,187 on blood platelets. I. Influence on aggregation and secretion. Amer. J. Path. 77, 135, 1974.
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WHITE, J.G. by adenosine
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MOLNAR, J. and LORAND, L. Biochem. Biophys. 93, 353,
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M.A. and MUSTARD, KINLOUGH-RATHBONE, R.L., PACKHAM, The effect of prostaglandin El on platelet function vitro and in vivo. Brit. J. Haemat. 19, 559, 1970.
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PACKHAM, M.A., WARRIOR, E.S., GLYNN, M.F., SENYI, A.S. and Alteration of the response of platelets to MUSTARD, J.F. surface stimuli by pyrazole compounds. J. Exp. Med. 126, 171, 1967.
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M.A. and MUSTARD, J.F. Adenosine diARDLIE, N.G., PACKHAM, phosphate-induced platelet aggregation in suspensions of washed rabbit platelets. Brit. J. Haemat. 19, 7, 1970.
17.
JENKINS, C.S.P., PACKHAM, M.A., KINLOUGH-RATHBONE, R.L. and Interactions of polylysine with platelets. MUSTARD, J.F. Blood 37, 395, 1971.
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Fine structural alterations induced diphosphate. Blood 31, 604, 1968. and STORMORKEN, H. Stand. J. Haemat.,
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J.F., PERRY, D.W., KINLOUGH-RATHBONE, R.L. and M.A. Factors responsible for ADP-induced release of human platelets. Amer. J. Physiol. (in press).
NIEWIAKOWSKI, S., SENYI, platelet aggregation and Invest. 52, 1647, 1973.
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MUSTARD, J.F., HEGARDT, B., ROWSELL, H.C. and MacMILLAN, R.L. Effect of adenosine nucleotides on platelet aggregation and clotting time. J. Lab. Clin. Med. 64, 548, 1964.
20.
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