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Adenine nucleotide metabolism of blood platelets
342
BIOCHIMICAET BIOPHYSICAACTA
BBA 95807
A D E N I N E N U C L E O T I D E METABOLISM OF BLOOD P L A T E L E T S . II. U P T A K E OF A D E N O S I N E AND I N H I B I T I O N OF A D P - I N D U C E D PLATELET AGGREGATION MAURICE C. ROZENBERG AND HOLM HOLMSEN Institute ]or Thrombosis Research, Rihshospitalel, Oslo (Norway)
(Received August end, 1967) (Revised manuscript received October I9th, 1967)
SUMMARY I. Inhibition of ADP-induced aggregation of human platelets in plasma b y AMP, adenosine monoacetate and adenosine monopropionate has been studied. These compounds produced inhibition only after hydrolysis to adenosine in plasma. 2. The concentration of adenosine in plasma and the amount phosphorylated in the platelets during inhibition of aggregation was estimated b y use of [14C10]adenosine and EA4C101AMP. Inhibition correlated well with the rate of phosphorylation, but poorly with the adenosine concentration. 3. Inhibition of platelet aggregation remained present after rapid clearance of the adenosine from plasma on addition of adenosine deaminase. 4- Rising concentrations of adenosine did not induce I0O % inhibition of aggregation. Both inhibition and the rate of phosphorylation were saturated at the same adenosine concentration. 5. D-2-Deoxyglucose and antimycin together strongly inhibited ADP-induced platelet aggregation, reduced adenosine phosphorylation b y 5 ° ~o and conversion of [8-1aCJadenine to nucleotides b y 80 °/o. 6. These results suggest that adenosine is transported across the membrane and perhaps phosphorylated before inducing inhibition. Inhibition might be caused by competition for energy required for both platelet aggregation and the adenosine transport-phosphorylation process.
INTRODUCTION GAARDER et al. 1 first demonstrated that ADP induced aggregation of blood platelets. The possible role of ADP in normal haemostasis was indicated b y the aggregation of platelets after infusions of ADP i n vivo ~-4 and b y the findings that both collagen a,8- and thrombinT-induced aggregation of platelets was mediated through ADP. AMP s and, more effectively, adenosine 9 inhibited ADP-induced aggregation i n vitro and the latter was also effective i n vivo 1°. Both these inhibitors, however, Biochim. Biophys. Acla, 155 (1968) 342-352
ADENOSINE UPTAKE AND PLATELET AGGREGATION
343
required preincubation with platelets or prolonged infusion in vivo, whereas the ADP action was always immediate. Adenosine and AMP were competitive inhibitors of ADP T M . BORN13 suggested that these substances, because of their similarity in structure to ADP, competed with it for receptor sites on the platelet membrane and induced inhibition b y blocking access to these sites. Prevention of dephosphorylation of AMP in plasma by cyanide also prevented the inhibition of ADP-induced platelet aggregation 14, indicating that AMP had to be converted to adenosine and perhaps be transported in the platelets before acting as an inhibitor. The present paper attempts to correlate adenine nucleotide metabolism ~5 with platelet function. The time course of adenosine inhibition of ADP-induced platelet aggregation is reported and the mode of action of AMP, adenosine monopropionate and adenosine monoacetate via adenosine is also demonstrated. Inhibition of platelet aggregation was studied simultaneously with the clearance of E~4C10~adenosine in plasma and the appearance of E14C1011abelled adenine nucleotides in platelets. Separation of platelets from plasma was not necessary in view of the strict localization of the nucleotides intracellularly and of adenosine, inosine and hypoxanthine extracellularly 15. The relation between the uptake of E14C101adenosine, of ES-~4Cladenine, the aggregation of platelets and the reduction of energy production with D-deoxyglucose and antimycin was also investigated.
MATERIALS
Adenosine 5'-monoacetate, adenosine 5'-monopropionate and D-2-deoxyglucose from Sigma Chemical, St. Louis, were made up in o.15 M NaC1, and antimycin (Sigma) was made up to I mg/ml in abs. ethanol. Other reagents have been described previously 15. Adenosine deaminase did not deaminate adenosine 5'-monoacetate, adenosine 5'-monopropionate, or AMP. E14C10]adenosine, E14C101AMPand IS-14Cladenine from Radiochemical Centre, Amersham, were diluted to desired concentrations with carriers in 68 mM glucose-o.I5 M NaC1. In experiments with D-2-deoxy-glucose and antimycin, the radiochemicals were dissolved in o.15 M NaC1 only. Human citrate platelet-rich plasma was prepared as before TM.
METHODS
Incubation experiments Basic incubation mixtures always consisted of one part of the inhibitor under investigation in 68 mM glucose-o.I 5 M NaC1 added to 19 parts of citrate-plateletrich plasma. At recorded time intervals, 1.5-ml samples were removed for measurement of the degree of inhibition of platelet aggregation (described below) and in the case of radioactive studies, 0.2- to o.5-ml samples were removed for determination of radioactivity of metabolites 15. Control mixtures containing I part of 68 mM glucose in o.15 M NaC1 to 19 parts of platelet-rich plasma were also run to enable estimation of the degree of inhibition of platelet aggregation. In some experiments, incubation mixtures containing 5 #l/ml of 1/2o adenosine deaminase 15 added prior to the inhibBiochim. Biophys. Acta, 155 (1968) 342-352
344
M. C. ROZENBERG, H. HOLMSEN
itor were also p r e p a r e d . The a m o u n t of [14C101adenosine p h o s p h o r y l a t e d after inc u b a t i o n for a given t i m e is expressed in # M a n d c a l c u l a t e d b y (#M original adenosine) × ( r a d i o a c t i v i t y of A T P + A D P + A M P ) / ( r a d i o a c t i v i t y of original adenosine) where the r a d i o a c t i v i t y is measured as counts/rain p e r 25 ~1 of basic i n c u b a t i o n m i x t u r e .
Measurement o~ inhibition o/ platelet aggregation The r a t e of A D P - i n d u c e d aggregation of platelets was m e a s u r e d at 25 ° b y t h e fall in light a b s o r p t i o n at 50 sec (A A~0~ec) in an E E L t i t r a t o r a n d recorded on a Varian g r a p h i c recorder 6-15-1. 1.5 ml samples of the basic i n c u b a t i o n m i x t u r e s were placed in the c u v e t t e a n d 1.2 ml of Tris-buffered saline 15 were added. Stirring with a p l a s t i c - c o v e r e d m a g n e t i c rod was c o m m e n c e d a n d aggregation was i n d u c e d b y 0.3 ml of A D P (final concn., 0.64 #M). The r a t e of aggregation o b t a i n e d in m i x t u r e s c o n t a i n i n g the i n h i b i t o r was c o m p a r e d to t h a t of t h e control m i x t u r e i n c u b a t e d for the same p e r i o d of time. The degree of i n h i b i t i o n was recorded as: '~A50 sec of control m i x t u r e - - a l A s 0 sec of i n h i b i t o r m i x t u r e ) AAs0 s~o of control m i x t u r e X IOO per cent inhibition
RESULTS The r a t e of aggregation (ZlAs0see) of p l a t e l e t s in platelet-rich p l a s m a i n c u b a t e d at 37 ° falls slowly with t i m e (Fig. i ) . This is n o t n e a r l y so m a r k e d a t 25 °, a n d usually, BOmin
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120 rain
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Fig. i. Variation in light absorption of platelet-rich plasma during ADP-induced platelet aggregation. Platelet-rich plasma was incubated at 37 ° (a) in the absence and (b) in the presence of adenosine (15/~M). The rate of aggregation was measured at the times indicated after incubation. The arrow indicates injection of ADP.
no difference is o b s e r v e d in t h e first 2 h of incubation. Nevertheless, i d e n t i c a l l y t r e a t e d controls are used in all e s t i m a t i o n s of r a t e of p l a t e l e t aggregation b o t h at 25 ° a n d 37 ° . The i n h i b i t i o n of A D P - i n d u e e d aggregation of p l a t e l e t s i n c u b a t e d in p l a s m a at 37 ° with adenosine increases r a p i d l y a n d reaches a m a x i m u m a p p r o x . 6 rain after a d d i t i o n of the i n h i b i t o r (Fig. I). The p l a t e l e t s then slowly recover their a b i l i t y to aggregate and, when i n c u b a t e d with 3.4 # M adenosine at 37 °, will aggregate as well as, a n d often b e t t e r t h a n , t h e control samples after 30 min (Fig. I). A t this stage, we assume t h a t the i n h i b i t i o n has been c o m p l e t e l y overcome. H i g h e r c o n c e n t r a t i o n s of
Biochim. Biophys. Acta, I55 (1968) 342-352
ADENOSINE UPTAKEAND PLATELETAGGREGATION
345
adenosine (15/~M) in platelet-rich plasma at 37 ° give greater inhibition which is also maximal at 6 min, but persisting for a longer time.
Mode o~ action o~ A MP, adenosine 5'-monoacetate and adenosine 5'-monopropionate The inhibitory effect of AMP (15/,M) is less marked than that of adenosine incubated in platelet-rich plasma but recovery of aggregation using AMP is delayed compared to adenosine. The effects of adenosine 5'-monoacetate and adenosine 5'monopropionate parallel that of AMP. KCN (o.oi M) prevents AMP-induced inhibition at an early stage ~*, but platelets exposed to cyanide for 30 rain lose their ability to aggregate. Adenosine deaminase does not itself influence aggregation and when added to the platelet-rich plasma prior to AMP, adenosine 5'-monoacetate or adenosine 5'-monopropionate, no inhibition of ADP-induced aggregation of platelets occurs (Table I). When [14C10]AMP (15 #M) was used as inhibitor in the presence of added deaminase, no [14C10]adenosine accumulated. TABLE
I
INHIBITION OF P L A T B L E T A G G R E G A T I O N W I T H CERTAIN A D E N O S I N E ESTERS Basic incubation mixtures of adenosine and its esters (15/~M) and platelet-rich plasma were incubated at 37 ° in the absence or presence of adenosine deaminase (2.5/~g/ml incubation mixture). T h e degree of inhibition expressed in % w a s determined after 5 and 45 rain. A N stands for adenosine deaminase.
Adenosine AMP Adenosine 5'-monopropionate Adenosine 5'-monoacetate
Inhibition (%) 5 rain --AD +AD
45 rain --AD
+AD
79 73 62 46
Io 24 36 33
o o o o
6 o o o
Correlation between inhibition and [14Clo]adenosine metabolism Concurrent observations on the clearance of [14C101adenosine from plasma and the inhibition of platelet aggregation show that maximum inhibition is reached while the concentration of [14Clo]adenosine is falling from 3.4 to 2.15 #M (Fig. 2). Recovery of the aggregating ability of platelet> appears to correlate with further clearance of the Ei4C10]adenosine (Fig. 2). The pattern of inhibition of platelet aggregation appears to correlate better, however, with the phosphorylation of [14C10]adenosine in the platelets TM (Fig. 2). There is inhibition of aggregation while the radioactivity of the [i4C10]adenosine phosphates in platelets rises and when the latter reaches a steady level, no further inhibition of aggregation is evident. Incubation of platelet-rich plasma with 15 #M E14C10]AMP at 22 ° shows maximal inhibition of aggregation at 25 min (Fig. 3). Comparison between the degree of inhibition of platelet aggregation and the level of [14Ci01adenosine formed shows that inhibition of aggregation and the concentration of [14C10]adenosine in plasma rise together (Fig. 3). The recovery of the aggregating ability of platelets incubated with [14C10]AMP is delayed and does not correlate well with the clearance of [14C10]adeBiochim. Biophys. Acta, 155 (1968) 3 4 2 - 3 5 2
340
M. C. ROZENBERG, H. HOLMSEN
2o0o f 80
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40 60 I n c u b a t i o n t i m e (rain)
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Fig. 2. V a r a t i o n in t h e r a d i o a c t i v i t y of a d e n o s i n e ( • - • ) , ATP+ADP+AMP ( O - O ) a n d in t h e degree of i n h i b i t i o n ( & - - - / ' , ) d u r i n g i n c u b a t i o n of p l a t e l e t - r i c h p l a s m a a t 37 ° w i t h [14C10Ia d e n o s i n e (3.4 # M a n d 18oo c o u n t s / r a i n p e r 2 5 / A i n c u b a t i o n m i x t u r e initially). 1. 3/~M a d e n o s i n e h a s b e e n p h o s p h o r y l a t e d a f t e r 6o rain of i n c u b a t i o n in t h e cells. Fig. 3. V a r i a t i o n in t h e r a d i o a c t i v i t y of e x t r a c e l l u l a r A M P ( I - I ) , a d e n o s i n e ( • - • ), intraceIlular A T P + A D P ( Q - Q ) a n d in t h e degree of i n h i b i t i o n ( A - - - & ) d u r i n g i n c u b a t i o n of p l a t e l e t - r i c h p l a s m a a t 25 ° w i t h {14C101AMP (15 # M a n d 12oo c o u n t s / m i n p e r 25 ffl i n c u b a t i o n m i x t u r e iilitially). 1.9/~M a d e n o s i n e h a s b e e n p h o s p h o r y l a t e d in t h e cells a t t h e e n d of i n c u b a t i o n .
nosine in plasma. However, there is good correlation between the duration of inhibition of platelet aggregation and the phosphorylation in the platelets of the [14C~0Iadenosine transported from the plasma (Fig. 3). The inhibition also correlates well with the rate of phosphorylation: In the first 50 min of incubation (Fig. 3) the rate of appearance of radioactive adenine nucleotides is practically constant and the degree of inhibition does not change significantly after having reached its maximum. After 50 min of incubation the rate of phosphorylation decreases to zero, at 9 ° min of incubation, as no increase in the radioactivity of the adenine nucleotides takes place after this time. The degree of inhibition decreases concomitantly with the rate of phosphorylation during the 50-90 min of incubation.
Induced clearance o[ [14Cxoladenosine [rom platelet-rich plasma and inhibition o[ aggregation After incubating platelet-rich plasma with [14C10]adenosine at 22 ° for IO min to obtain significant inhibition of aggregation, the recovery of the aggregating ability of the platelets was observed after inducing clearance of the remaining [14C10]adenosine from plasma with adenosine deaminase. Under the conditions used, all the extracellular [14Cx0]adenosine is deaminated within 20 sec while there is only a slight recovery of the aggregating ability b y this time (Fig. 4). Inhibition of platelet aggregation is evident for more than 40 sec although there is only a small concentration of [14C10]adenosine remaining in platelet-rich plasma, the same as is observed in clearBiochim. Biophys. Acta, 155 (1968) 342-352
ADENOSINE UPTAKE AND PLATELET AGGREGATION
347
ance experiments with platelet-poor plasma (Fig. 4). This small concentration of adenosine is also present in plasma after recovery of the inhibition of platelet aggregation in all the other [14C10]adenosine-platelet-rich plasma incubation mixtures (Figs. 2 and 3). lOOO 40
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Fig. 4. V a r i a t i o n in t k e r a d i o a c t i v i t y of a d e n o s i n e ([~-[]) a n d t h e degree of i n h i b i t i o n ( & - - - & ) a f t e r a d d i t i o n of a d e n o s i n e d e a m i n a s e (upper curve, 2.5/~g e n z y m e p e r m l i n c u b a t i o n m i x t u r e ) a n d saline (lower curve) to p l a t e l e t - r i c h p l a s m a w k i c h h a d b e e n p r e i n c u b a t e d a t 25 ° for io m i n w i t h E~4C10]adenosine (3.4/~M a n d 18oo c o u n t s / m i n p e r 25/,I i n c u b a t i o n m i x t u r e initially). A n a l o g o u s p l a t e l e t - p o o r p l a s m a w a s t r e a t e d ill t h e s a m e w a y a n d tile v a r i a t i o n in ?4C10]adenosine r a d i o a c t i v i t y ( O - © ) w a s d e t e r m i n e d a f t e r a d d i t i o n of a d e n o s i n e d e a m i n a s e .
The possible role o/a plasma [actor in adenosine induced inhibition o/platelet aggregation Adenosine might induce inhibition of platelet aggregation b y activation of a plasma factor which would act on platelets even after clearance of adenosine. This possibility is investigated by incubating platelet-poor plasma with adenosine for 5 min, rapidly removing the adenosine left in the plasma with adenosine deaminase and incubating the plasma with platelet-rich plasma for a further 30 sec. No inhibition of platelet aggregation is obtained in these instances.
The e[[ect o/temperature on the phosphorylation o[ ~14Cxo]adenosine and inhibition o/ platelet aggregation The correlation observed between the phosphorylation of [t4C10]adenosine and the duration of inhibition of platelet aggregation (Figs. 2 and 3) was investigated further. Adenosine-induced inhibition of platelet aggregation rises with increase in temperature (15 ° to 37°). Similarly, the rate of phosphorylation in the cells also rises and the two processes parallel each other (Fig. 5). Deamination of ~14C10]adenosine in plasma is also faster at 37 ° than at 15 °. Biochim. Biophys. Acta, 155 (1968) 342-352
348
M . C . ROZENBERG, H. HOLMSEN
The ellect o/ varying concentrations o/ E14C1o~adenosine on its phosphorylation by platelets and the inhibition o/aggregation The inhibition of platelet aggregation a~ter 5 min incubation with adenosine increases with the adenosine concentration up to 6-1o #M (Fig. 6). Further increase of adenosine concentration does not yield more inhibition and maximal inhibition varies between 60-80 %, never reaching IOO °/o (c/. ref. 12). The amount of I14C10]adenosine phosphorylated in 5 min is the same (approx. o.6#M) in all instances where maximal inhibition of aggregation is present and is then independent of the original [14C101adenosine concentration (Fig. 7). In all instances where maximal in-
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Fig. 5. Variation in the r a d i o a c t i v i t y of A T P + A D P + A M P (solid line) and in the degree of inhibition (broken line) during incubation of platelet-rich p l a s m a (5.1 × lO 6 platelets//~l) with []*Clo! adenosine (3.4/zM and 18oo c o u n t s / m i n per 25/zl incubation mixture initially) at 15 ° (E]), 22 ° ( A ) and 37 ° ( O ) . Aggregation w a s performed at 25 °. Fig. 6. Effect of v a r y i n g t h e initial concentration of [l~C10]adenosine in platelet-rich plasma (3.84 x io~ platelets/#l) on the a m o u n t of adenosine p h o s p h o r y l a t e d b y the cells (O O) and on the degree of inhibition ( A - - - A ) after 5 rain of incubation at 37 °.
hibition of aggregation is still present at IO min, the amount of E14C10]adenosine phosphorylated is also the same (approx. I #M). It is apparent that the inhibition of platelet aggregation and the rate of phosphorylation reach their m a x i m u m at the same E14C101adenosine concentration. This feature of reaching maximal inhibition at a certain adenosine concentration is not dependent on the concentration of A D P employed to produce platelet aggregation. However, the maximal degree of inhibition varies inversely with the A D P concentration. In a typical experiment, the maximal degrees of inhibition / after 5 rain of incubation (reached at 12-16 #M adenosine) were 62, 4 ° and 37 O/o when 0.48, 0.96 and 1.44/~M ADP, respectively, were used.
Dependence o/ platelet aggregation and incorporation o/ [14Clo]adenosine and ~-14C]adenine on energy metabolism As ADP-induced platelet aggregation is dependent on active energy metabolism 16 and phosphorylation of adenosine requires ATP 15, the effect of glycolytic and mitochondrial inhibitors on these processes was studied. Platelets in plasma incubated Biochim. Biophys. Acta, 155 (1968) 3 4 2 - 3 5 2
349
ADENOSINE UPTAKE AND PLATELET AGGREGATION
for 3o min with both D-2-deoxyglucose and antimycin lose their ability to aggregate. This is associated with a fall in thier A T P content of 40-5 ° % while the ADP content is little changed (Table I I L Platelets treated in this w a y show a definite reduction in
TABLE II EFFECT OF PREINCUBATION OI*"PLATELET-RICH PLASMA WITH D-2-DEOXVGLUCOSEAND/OR ANTIMYCIN ON PLATELET AGGREGATION, CONVERSION OF ?4C10~ADENOSINE AND [8-14C]ADENINE TO NUCLEOTIDES, AND ON LEVELS OF A T P AND A D P Platelet-rich p l a s m a (3.65 × lO 5 platelets/#l), w a s i n c u b a t e d at 37 ° w i t h the different inhibitors for 3 ° rain; t h e n platelet aggregation and levels of A T P a n d A D P (time to) were determined. At the s a m e time, [14C10]adenosine (3.4/zM, 18oo c o u n t s / m i n p e r 25 #1 i n c u b a t i o n m i x t u r e ) and [8A4Cladenine (3.4 #M, 9oo e o u n t s / m i n p e r 25/,1 i n c u b a t i o n m i x t u r e ) w e r e added s e p a r a t e l y to aliquots of the i n c u b a t i o n m i x t u r e s and i n c u b a t e d for a n o t h e r 20 rain. The c o n c e n t r a t i o n (time tl) and the r a d i o a c t i v i t y of the adenine nueleotides were determined. The isotopes were in glucosefree saline, D-2-deoxyglucose in saline (8.9 mM), a n t i m y c i n in absolute e t h a n o l (22.2 # g / m l ) and to the control were added saline a n d ethanol in corresponding a m o u n t s .
Addition
Saline-ethanol (control) D-2-Deoxyglucose Antimyein D-2-Deoxyglucose + antimycin
Rate o[ Radioaetivityo/ATP+ADP+AMP Amounto/nueleotides aggregation (counts/min per 251~lincubation (t~moles/Ionplatelets) (%) mixture) ATP ADP [l*Clo]Adenosine [8A4C]Adenine to
tl
to
tl
38 36 38
223 234 255
251 277 281
4 .6 4.2 4.9
3.9 3.6 4.4
3.3 2.8 3.2
3.2 2. 4 3.o
4
124
51
3.0
2.o
2.6
3.2
phosphorylation associated with a further fall in ATP. Incubation of these platelets with E8-14C]adenine shows a more marked reduction in the formation of labelled adenine nucleotides as compared to the [14C10]adenosine experiments (Table II). Treatment of platelets with D-2-deoxyglucose or antimycin separately does not show any significant deviations from the control (Table II).
DISCUSSION
In the presence of adenosine deaminase, no adenosine accumulates during dephosphorylation of AMP in platelet-rich plasma and there is no inhibition of platelet aggregation. Hence, AMP must be dephosphorylated to adenosine in plasma before inducing inhibition of platelet aggregation as was found b y SALZMAN, CHAMBERS AND NER114. Adenosine 5'-monoacetate and adenosine 5'-monopropionate are also able to induce inhibition of aggregation, but only after hydrolysis b y hitherto undescribed plasma esterases to adenosine. Adenosine thus plays a central role in the inhibition of ADP-induced platelet aggregation b y these substances. Our studies confirm the need for prior incubation with adenosine to reach maximal inhibition s, and the recovery of the ability to aggregate with time TM. Bioehim. Biophys. Acta, 155 (1968) 342-352
350
M.C. ROZENBERG, H. HOLMSEN
BOI~Nla has postulated that adenosine, because of its resemblance to ADP, m a y occupy receptor sites on the platelet membrane and prevent access of ADP to these. The need for preincubation with the inhibitor is explained by slow binding of adenosine to these receptor sites in contrast to the fast binding of ADP. Our observations of inhibition of aggregation and disappearance of [l~C101adenosine do fit in with this hypothesis. However, correlation of the increase in inhibition and the ~14Clo]adenosine formed extracellularly 15 when I14CloJAMP is added to plateletrich plasma shows that, under these circumstances, inhibition is produced rapidly. This could only be explained b y rapid binding of adenosine to receptor sites, were the above theory valid. Moreover, AMP is structurally more closely related to ADP than is adenosine and should occupy receptor sites better than adenosine, yet it does not induce inhibition per se. The theory of competition for receptor sites admits that adenosine is capable of occupying all sites used b y ADP for induction of platelet aggregation, hence producing I00 °,o inhibition. In spite of increasing concentrations of [14Cioladenosine in platelet-rich plasma, we never observe complete inhibition. We also show that, provided platelets have been incubated with adenosine, inhibition of aggregation is still present when almost all significant adenosine has been deaminated. Although adenosine induces inhibition of platelet aggregation, the compound itself does not have to be present during the whole inhibitory phase as required b y the theory for competitive inhibition. We therefore feel that our findings render this theory unlikely. The hypothesis that adenosine could induce inhibition of aggregation b y activation of a plasma factor which would then act on platelets is exlcuded b y our finding that platelet-poor plasma, incubated with adenosine, cannot induce inhibition ot aggregation if the adenosine itself is rapidly removed with deaminase prior to contact with the platelets. We have observed good correlation between the inhibition of aggregation and the duration of phosphorylation of [14C10Jadenosine b y the platelets. The appearance of labelled adenine nucleotides in the platelets in our experiments consists of two processes: firstly, the transport of adenosine across the membrane and secondly, the phosphorylation of adenosine to AMP and its subsequent conversion to ADP or ATP. These two phases are included in our term 'uptake', and the amount of phosphorylation observed is taken as a measure of uptake in view of the absence of adenosine in the platelets 15. However, which of the two processes is rate limiting is not yet clear. SALZMAN,CHAMBERS AND NER114 first suggested that uptake of adenosine might precede induction of the inhibition of aggregation. Our findings support this view. There is correlation between inhibition of aggregation and phosphorylation of adenosine. AMP does not induce inhibition and it does not penetrate the membrane if its breakdown to adenosine is prevented 15. The adenosine-induced inhibition is temperature dependent and the rate of phosphorylation varies in the same manner with temperature, which might indicate that the two phenomena are related. It is shown that, in spite of wide variation in adenosine concentrations, maximal inhibition of aggregation correlates well with the amount of adenosine phosphorylated. This maximal inhibition appears limited by adenosine uptake which has reached saturation, and which then remains uninfluenced by further increase in the concentration of adenosine. On the basis of these results, we propose that inhibition of aggregation is associated with the process of adenosine uptake b y the platelets. Biochim. Biophys. Acta, I55 (1968) 342-352
ADENOSINE UPTAKE AND PLATELET AGGREGATION
351
The mechanism of adenosine-induced inhibition during uptake requires closer examination of the latter phenomenon. Adenosine is first transported across the membrane b y an unknown process. I t is then phosphorylated to AMP b y ATP catalysed b y adenosine kinase 15. This enzyme is probably located in the membrane or close to it as all adenosine transported into the cells is phosphorylated and does not come into contact with platelet adenosine deaminase (as opposed to plasma adenosine deaminase). AMP in the platelets is then converted to ADP and A T P b y adenylate kinase. SALZMAN, CHAMBERS AND NER114A8 suggest that adenosine uptake leads to more newly formed ATP providing more energy to an ATPase which keeps the platelets in a low responsive state. Our finding that newly phosphorylated I14C10~ATP remains present in the platelets while they recover this ability to aggregate does not favour this view. Furthermore platelets, having recovered from adenosine inhibition, will often aggregate better than the controls, suggesting that more ATP leads to increased platelet aggregation. Finally, the use of inhibitors of glycolysis and oxidative phosphorylation confirm previous reports 16,2°,22 that platelets in a low energy state are less, not more responsive to ADP. The dependence of aggregation on intracellular A T P m a y mean that ATP is consumed during the process of aggregation or that ATP is consumed to maintain platelets in a responsive state. An attractive hypothesis would be that the ATP used during transport and/or phosphorylation of adenosine would then not be available for platelet aggregation. Yet adenine also requires ATP in order to be converted to nucleotides and does not produce inhibition. Platelets form adenine nucleotides from adenine b y condensation with 5-P-Rib-P-P catalysed b y adenine phosphoribosyl transferase 17. A T P is required for 5-.P-Rib-P-P synthesis but m a y originate from a pool different from that used in adenosine phosphorylation or platelet aggregation. It must be emphasized, however, that partial reduction of ATP b y the metabolic inhibitors produced complete inhibition of aggregation while some phosphorylation of adenosine was still present. This might not be compatible with the energy competition theory. An alternative theory of inhibition of aggregation would be that the transport form of adenosine induces a change in the platelet membrane independent of energy consumption. Other possibilities could be that inhibition of aggregation is somehow induced b y a change in enzyme conformation during the phosphorylation of adenosine to AMP (platelet adenosine kinase) or its subsequent conversion to ADP and ATP (adenylate kinase) irrespective of energy consumption. The inhibitory action of adenosine analogues parallels their vasodilatory effects 22. BERNE has suggested a role for adenosine in the coronary blood flow 23 and it has been shown that vasodilation occurs while adenosine is taken up and phosphorylated b y the heart 24. The mode of action of adenosine in inducing vasodilation and preventing ADP induced platelet aggregation m a y indeed be similar. The close relation between inhibition of platelet aggregation and adenosine uptake indicates that further studies on adenosine kinase and the mode of transport of adenosine across the membrane are warranted.
ACKNOWLEDGEMENT
We wish to t h a n k Miss R. JANSSEN for her technical assistance. M. C. R. was Biochim. Biophys. Acta, 155 (1968) 342-352
352
M. C. ROZENBERG, H. HOLMSEN
supported by the National Heart Foundation of Australia. Financial support was also provided by Proprietmr Christopher Overland og hustri Ingeborg Overlands legat. Det Videnskapelige Forskningsfond av 1919 and J. L. Tiedemanns Tobaksfabrik Joh. H. Andresens medisinske fond. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 I8 19 20 21 22 23 24
A. GAARDER, J. JONSEN, S. LALAND, A. HELLEM AND P. A. OWREN, Nature, 192 (1961) 531. M. G. DAVEY AND H. LANDER, Nature, 2OI (1964) 1037. A. J. HONOUR AND J. R. A. MITCHELL, Nature, 197 (1963) lO19. A. ]2~ORDOYAND A. B. CHANDLER, Scand. J. Haematol., i (1964) 16. T. HOVlG, Thromb. Diath. Haemorrhag., 9 (1963) 264. T. H. SPAET AND J. CINTRON, Thromb. Diath. Haemorrhag., Suppl. 13 (1963) 335. R. J. HASLAM, Nature, 202 (1964) 765 . R. J. O'BRIEN, J. Clin. Pathol., 15 (1962) 446. G. V. R. BORN AND M. J. CROSS, J. Physiol. London, 168 (1963) 178. G. V. R. BORN, A. J. HONOUR AND J. R. A. MITCHELL, Nature, 2o2 (1964) 761. G. V. R. BORN, Nature, 194 (1962) 927. L. SKOZA, M. ZUCKER, Z. JERUSHALMY AND R. GRANT, Thromb. Diath. Haemorrhag., in t h e press. G. V. R. BORN, Nature, 2o6 (1965) 1121. E. W . SALZMAN, D. A. CHAMBERS AND L. L. !N]'ERI, Nature, 21o (1966) 167. H. HOLMSEN AND M. C. ROZENBERG, Biochim. Biophys. Acta, 155 (1968) 326. E. MORER, A. HELLEM AND M. C. ROZENBERG, Scand. J. Clin. Lab. Invest., 19 (1967) 28o. H. HOLMSEN AND M. C. ROZENBERG, Biochim. Biophys. Acta, 155 (1968) 326. D. A. CHAMBERS, E. W. SALZMAN AND L. L. NERI, Arch. Biochem. Biophys., 119 (1967) 173. G. G. DE VREKER AND R. A. DR VREKER, Rev. Belge Pathol. Med. Exptl., 31 (1965) 79. H. HOLMSEN, Scan& J. Clin. Lab. Invest., 17 (1965) 537J. R. O'BRIEN, Nature, 212 (1966) lOl 7. G. V. R. BORN, R. J. HASLAM, M. GOLDMAN AND R. D. LOWE, Nature, 205 (1965) 678. R. N. BERNE, Physiol. Rev., 44 (1964) I. M. J. JACOB AND R. M. BERNE, Am. J. Physiol., 198 (196o) 322.
Bioehim. Biophys. Aeta, 155 (I968) 342-352
BIOCHIMICAET BIOPHYSICAACTA
BBA 95807
A D E N I N E N U C L E O T I D E METABOLISM OF BLOOD P L A T E L E T S . II. U P T A K E OF A D E N O S I N E AND I N H I B I T I O N OF A D P - I N D U C E D PLATELET AGGREGATION MAURICE C. ROZENBERG AND HOLM HOLMSEN Institute ]or Thrombosis Research, Rihshospitalel, Oslo (Norway)
(Received August end, 1967) (Revised manuscript received October I9th, 1967)
SUMMARY I. Inhibition of ADP-induced aggregation of human platelets in plasma b y AMP, adenosine monoacetate and adenosine monopropionate has been studied. These compounds produced inhibition only after hydrolysis to adenosine in plasma. 2. The concentration of adenosine in plasma and the amount phosphorylated in the platelets during inhibition of aggregation was estimated b y use of [14C10]adenosine and EA4C101AMP. Inhibition correlated well with the rate of phosphorylation, but poorly with the adenosine concentration. 3. Inhibition of platelet aggregation remained present after rapid clearance of the adenosine from plasma on addition of adenosine deaminase. 4- Rising concentrations of adenosine did not induce I0O % inhibition of aggregation. Both inhibition and the rate of phosphorylation were saturated at the same adenosine concentration. 5. D-2-Deoxyglucose and antimycin together strongly inhibited ADP-induced platelet aggregation, reduced adenosine phosphorylation b y 5 ° ~o and conversion of [8-1aCJadenine to nucleotides b y 80 °/o. 6. These results suggest that adenosine is transported across the membrane and perhaps phosphorylated before inducing inhibition. Inhibition might be caused by competition for energy required for both platelet aggregation and the adenosine transport-phosphorylation process.
INTRODUCTION GAARDER et al. 1 first demonstrated that ADP induced aggregation of blood platelets. The possible role of ADP in normal haemostasis was indicated b y the aggregation of platelets after infusions of ADP i n vivo ~-4 and b y the findings that both collagen a,8- and thrombinT-induced aggregation of platelets was mediated through ADP. AMP s and, more effectively, adenosine 9 inhibited ADP-induced aggregation i n vitro and the latter was also effective i n vivo 1°. Both these inhibitors, however, Biochim. Biophys. Acla, 155 (1968) 342-352
ADENOSINE UPTAKE AND PLATELET AGGREGATION
343
required preincubation with platelets or prolonged infusion in vivo, whereas the ADP action was always immediate. Adenosine and AMP were competitive inhibitors of ADP T M . BORN13 suggested that these substances, because of their similarity in structure to ADP, competed with it for receptor sites on the platelet membrane and induced inhibition b y blocking access to these sites. Prevention of dephosphorylation of AMP in plasma by cyanide also prevented the inhibition of ADP-induced platelet aggregation 14, indicating that AMP had to be converted to adenosine and perhaps be transported in the platelets before acting as an inhibitor. The present paper attempts to correlate adenine nucleotide metabolism ~5 with platelet function. The time course of adenosine inhibition of ADP-induced platelet aggregation is reported and the mode of action of AMP, adenosine monopropionate and adenosine monoacetate via adenosine is also demonstrated. Inhibition of platelet aggregation was studied simultaneously with the clearance of E~4C10~adenosine in plasma and the appearance of E14C1011abelled adenine nucleotides in platelets. Separation of platelets from plasma was not necessary in view of the strict localization of the nucleotides intracellularly and of adenosine, inosine and hypoxanthine extracellularly 15. The relation between the uptake of E14C101adenosine, of ES-~4Cladenine, the aggregation of platelets and the reduction of energy production with D-deoxyglucose and antimycin was also investigated.
MATERIALS
Adenosine 5'-monoacetate, adenosine 5'-monopropionate and D-2-deoxyglucose from Sigma Chemical, St. Louis, were made up in o.15 M NaC1, and antimycin (Sigma) was made up to I mg/ml in abs. ethanol. Other reagents have been described previously 15. Adenosine deaminase did not deaminate adenosine 5'-monoacetate, adenosine 5'-monopropionate, or AMP. E14C10]adenosine, E14C101AMPand IS-14Cladenine from Radiochemical Centre, Amersham, were diluted to desired concentrations with carriers in 68 mM glucose-o.I5 M NaC1. In experiments with D-2-deoxy-glucose and antimycin, the radiochemicals were dissolved in o.15 M NaC1 only. Human citrate platelet-rich plasma was prepared as before TM.
METHODS
Incubation experiments Basic incubation mixtures always consisted of one part of the inhibitor under investigation in 68 mM glucose-o.I 5 M NaC1 added to 19 parts of citrate-plateletrich plasma. At recorded time intervals, 1.5-ml samples were removed for measurement of the degree of inhibition of platelet aggregation (described below) and in the case of radioactive studies, 0.2- to o.5-ml samples were removed for determination of radioactivity of metabolites 15. Control mixtures containing I part of 68 mM glucose in o.15 M NaC1 to 19 parts of platelet-rich plasma were also run to enable estimation of the degree of inhibition of platelet aggregation. In some experiments, incubation mixtures containing 5 #l/ml of 1/2o adenosine deaminase 15 added prior to the inhibBiochim. Biophys. Acta, 155 (1968) 342-352
344
M. C. ROZENBERG, H. HOLMSEN
itor were also p r e p a r e d . The a m o u n t of [14C101adenosine p h o s p h o r y l a t e d after inc u b a t i o n for a given t i m e is expressed in # M a n d c a l c u l a t e d b y (#M original adenosine) × ( r a d i o a c t i v i t y of A T P + A D P + A M P ) / ( r a d i o a c t i v i t y of original adenosine) where the r a d i o a c t i v i t y is measured as counts/rain p e r 25 ~1 of basic i n c u b a t i o n m i x t u r e .
Measurement o~ inhibition o/ platelet aggregation The r a t e of A D P - i n d u c e d aggregation of platelets was m e a s u r e d at 25 ° b y t h e fall in light a b s o r p t i o n at 50 sec (A A~0~ec) in an E E L t i t r a t o r a n d recorded on a Varian g r a p h i c recorder 6-15-1. 1.5 ml samples of the basic i n c u b a t i o n m i x t u r e s were placed in the c u v e t t e a n d 1.2 ml of Tris-buffered saline 15 were added. Stirring with a p l a s t i c - c o v e r e d m a g n e t i c rod was c o m m e n c e d a n d aggregation was i n d u c e d b y 0.3 ml of A D P (final concn., 0.64 #M). The r a t e of aggregation o b t a i n e d in m i x t u r e s c o n t a i n i n g the i n h i b i t o r was c o m p a r e d to t h a t of t h e control m i x t u r e i n c u b a t e d for the same p e r i o d of time. The degree of i n h i b i t i o n was recorded as: '~A50 sec of control m i x t u r e - - a l A s 0 sec of i n h i b i t o r m i x t u r e ) AAs0 s~o of control m i x t u r e X IOO per cent inhibition
RESULTS The r a t e of aggregation (ZlAs0see) of p l a t e l e t s in platelet-rich p l a s m a i n c u b a t e d at 37 ° falls slowly with t i m e (Fig. i ) . This is n o t n e a r l y so m a r k e d a t 25 °, a n d usually, BOmin
5 rnin
-8(:
_~ ~ ~c 50sec
ZZ 6 0 rain
5o se~
120 rain
~s0. ~
Fig. i. Variation in light absorption of platelet-rich plasma during ADP-induced platelet aggregation. Platelet-rich plasma was incubated at 37 ° (a) in the absence and (b) in the presence of adenosine (15/~M). The rate of aggregation was measured at the times indicated after incubation. The arrow indicates injection of ADP.
no difference is o b s e r v e d in t h e first 2 h of incubation. Nevertheless, i d e n t i c a l l y t r e a t e d controls are used in all e s t i m a t i o n s of r a t e of p l a t e l e t aggregation b o t h at 25 ° a n d 37 ° . The i n h i b i t i o n of A D P - i n d u e e d aggregation of p l a t e l e t s i n c u b a t e d in p l a s m a at 37 ° with adenosine increases r a p i d l y a n d reaches a m a x i m u m a p p r o x . 6 rain after a d d i t i o n of the i n h i b i t o r (Fig. I). The p l a t e l e t s then slowly recover their a b i l i t y to aggregate and, when i n c u b a t e d with 3.4 # M adenosine at 37 °, will aggregate as well as, a n d often b e t t e r t h a n , t h e control samples after 30 min (Fig. I). A t this stage, we assume t h a t the i n h i b i t i o n has been c o m p l e t e l y overcome. H i g h e r c o n c e n t r a t i o n s of
Biochim. Biophys. Acta, I55 (1968) 342-352
ADENOSINE UPTAKEAND PLATELETAGGREGATION
345
adenosine (15/~M) in platelet-rich plasma at 37 ° give greater inhibition which is also maximal at 6 min, but persisting for a longer time.
Mode o~ action o~ A MP, adenosine 5'-monoacetate and adenosine 5'-monopropionate The inhibitory effect of AMP (15/,M) is less marked than that of adenosine incubated in platelet-rich plasma but recovery of aggregation using AMP is delayed compared to adenosine. The effects of adenosine 5'-monoacetate and adenosine 5'monopropionate parallel that of AMP. KCN (o.oi M) prevents AMP-induced inhibition at an early stage ~*, but platelets exposed to cyanide for 30 rain lose their ability to aggregate. Adenosine deaminase does not itself influence aggregation and when added to the platelet-rich plasma prior to AMP, adenosine 5'-monoacetate or adenosine 5'-monopropionate, no inhibition of ADP-induced aggregation of platelets occurs (Table I). When [14C10]AMP (15 #M) was used as inhibitor in the presence of added deaminase, no [14C10]adenosine accumulated. TABLE
I
INHIBITION OF P L A T B L E T A G G R E G A T I O N W I T H CERTAIN A D E N O S I N E ESTERS Basic incubation mixtures of adenosine and its esters (15/~M) and platelet-rich plasma were incubated at 37 ° in the absence or presence of adenosine deaminase (2.5/~g/ml incubation mixture). T h e degree of inhibition expressed in % w a s determined after 5 and 45 rain. A N stands for adenosine deaminase.
Adenosine AMP Adenosine 5'-monopropionate Adenosine 5'-monoacetate
Inhibition (%) 5 rain --AD +AD
45 rain --AD
+AD
79 73 62 46
Io 24 36 33
o o o o
6 o o o
Correlation between inhibition and [14Clo]adenosine metabolism Concurrent observations on the clearance of [14C101adenosine from plasma and the inhibition of platelet aggregation show that maximum inhibition is reached while the concentration of [14Clo]adenosine is falling from 3.4 to 2.15 #M (Fig. 2). Recovery of the aggregating ability of platelet> appears to correlate with further clearance of the Ei4C10]adenosine (Fig. 2). The pattern of inhibition of platelet aggregation appears to correlate better, however, with the phosphorylation of [14C10]adenosine in the platelets TM (Fig. 2). There is inhibition of aggregation while the radioactivity of the [i4C10]adenosine phosphates in platelets rises and when the latter reaches a steady level, no further inhibition of aggregation is evident. Incubation of platelet-rich plasma with 15 #M E14C10]AMP at 22 ° shows maximal inhibition of aggregation at 25 min (Fig. 3). Comparison between the degree of inhibition of platelet aggregation and the level of [14Ci01adenosine formed shows that inhibition of aggregation and the concentration of [14C10]adenosine in plasma rise together (Fig. 3). The recovery of the aggregating ability of platelets incubated with [14C10]AMP is delayed and does not correlate well with the clearance of [14C10]adeBiochim. Biophys. Acta, 155 (1968) 3 4 2 - 3 5 2
340
M. C. ROZENBERG, H. HOLMSEN
2o0o f 80
o
¢ c
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0
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20 40 Incubation time (min)
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---4
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20
40 60 I n c u b a t i o n t i m e (rain)
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Fig. 2. V a r a t i o n in t h e r a d i o a c t i v i t y of a d e n o s i n e ( • - • ) , ATP+ADP+AMP ( O - O ) a n d in t h e degree of i n h i b i t i o n ( & - - - / ' , ) d u r i n g i n c u b a t i o n of p l a t e l e t - r i c h p l a s m a a t 37 ° w i t h [14C10Ia d e n o s i n e (3.4 # M a n d 18oo c o u n t s / r a i n p e r 2 5 / A i n c u b a t i o n m i x t u r e initially). 1. 3/~M a d e n o s i n e h a s b e e n p h o s p h o r y l a t e d a f t e r 6o rain of i n c u b a t i o n in t h e cells. Fig. 3. V a r i a t i o n in t h e r a d i o a c t i v i t y of e x t r a c e l l u l a r A M P ( I - I ) , a d e n o s i n e ( • - • ), intraceIlular A T P + A D P ( Q - Q ) a n d in t h e degree of i n h i b i t i o n ( A - - - & ) d u r i n g i n c u b a t i o n of p l a t e l e t - r i c h p l a s m a a t 25 ° w i t h {14C101AMP (15 # M a n d 12oo c o u n t s / m i n p e r 25 ffl i n c u b a t i o n m i x t u r e iilitially). 1.9/~M a d e n o s i n e h a s b e e n p h o s p h o r y l a t e d in t h e cells a t t h e e n d of i n c u b a t i o n .
nosine in plasma. However, there is good correlation between the duration of inhibition of platelet aggregation and the phosphorylation in the platelets of the [14C~0Iadenosine transported from the plasma (Fig. 3). The inhibition also correlates well with the rate of phosphorylation: In the first 50 min of incubation (Fig. 3) the rate of appearance of radioactive adenine nucleotides is practically constant and the degree of inhibition does not change significantly after having reached its maximum. After 50 min of incubation the rate of phosphorylation decreases to zero, at 9 ° min of incubation, as no increase in the radioactivity of the adenine nucleotides takes place after this time. The degree of inhibition decreases concomitantly with the rate of phosphorylation during the 50-90 min of incubation.
Induced clearance o[ [14Cxoladenosine [rom platelet-rich plasma and inhibition o[ aggregation After incubating platelet-rich plasma with [14C10]adenosine at 22 ° for IO min to obtain significant inhibition of aggregation, the recovery of the aggregating ability of the platelets was observed after inducing clearance of the remaining [14C10]adenosine from plasma with adenosine deaminase. Under the conditions used, all the extracellular [14Cx0]adenosine is deaminated within 20 sec while there is only a slight recovery of the aggregating ability b y this time (Fig. 4). Inhibition of platelet aggregation is evident for more than 40 sec although there is only a small concentration of [14C10]adenosine remaining in platelet-rich plasma, the same as is observed in clearBiochim. Biophys. Acta, 155 (1968) 342-352
ADENOSINE UPTAKE AND PLATELET AGGREGATION
347
ance experiments with platelet-poor plasma (Fig. 4). This small concentration of adenosine is also present in plasma after recovery of the inhibition of platelet aggregation in all the other [14C10]adenosine-platelet-rich plasma incubation mixtures (Figs. 2 and 3). lOOO 40
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Fig. 4. V a r i a t i o n in t k e r a d i o a c t i v i t y of a d e n o s i n e ([~-[]) a n d t h e degree of i n h i b i t i o n ( & - - - & ) a f t e r a d d i t i o n of a d e n o s i n e d e a m i n a s e (upper curve, 2.5/~g e n z y m e p e r m l i n c u b a t i o n m i x t u r e ) a n d saline (lower curve) to p l a t e l e t - r i c h p l a s m a w k i c h h a d b e e n p r e i n c u b a t e d a t 25 ° for io m i n w i t h E~4C10]adenosine (3.4/~M a n d 18oo c o u n t s / m i n p e r 25/,I i n c u b a t i o n m i x t u r e initially). A n a l o g o u s p l a t e l e t - p o o r p l a s m a w a s t r e a t e d ill t h e s a m e w a y a n d tile v a r i a t i o n in ?4C10]adenosine r a d i o a c t i v i t y ( O - © ) w a s d e t e r m i n e d a f t e r a d d i t i o n of a d e n o s i n e d e a m i n a s e .
The possible role o/a plasma [actor in adenosine induced inhibition o/platelet aggregation Adenosine might induce inhibition of platelet aggregation b y activation of a plasma factor which would act on platelets even after clearance of adenosine. This possibility is investigated by incubating platelet-poor plasma with adenosine for 5 min, rapidly removing the adenosine left in the plasma with adenosine deaminase and incubating the plasma with platelet-rich plasma for a further 30 sec. No inhibition of platelet aggregation is obtained in these instances.
The e[[ect o/temperature on the phosphorylation o[ ~14Cxo]adenosine and inhibition o/ platelet aggregation The correlation observed between the phosphorylation of [t4C10]adenosine and the duration of inhibition of platelet aggregation (Figs. 2 and 3) was investigated further. Adenosine-induced inhibition of platelet aggregation rises with increase in temperature (15 ° to 37°). Similarly, the rate of phosphorylation in the cells also rises and the two processes parallel each other (Fig. 5). Deamination of ~14C10]adenosine in plasma is also faster at 37 ° than at 15 °. Biochim. Biophys. Acta, 155 (1968) 342-352
348
M . C . ROZENBERG, H. HOLMSEN
The ellect o/ varying concentrations o/ E14C1o~adenosine on its phosphorylation by platelets and the inhibition o/aggregation The inhibition of platelet aggregation a~ter 5 min incubation with adenosine increases with the adenosine concentration up to 6-1o #M (Fig. 6). Further increase of adenosine concentration does not yield more inhibition and maximal inhibition varies between 60-80 %, never reaching IOO °/o (c/. ref. 12). The amount of I14C10]adenosine phosphorylated in 5 min is the same (approx. o.6#M) in all instances where maximal inhibition of aggregation is present and is then independent of the original [14C101adenosine concentration (Fig. 7). In all instances where maximal in-
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Fig. 5. Variation in the r a d i o a c t i v i t y of A T P + A D P + A M P (solid line) and in the degree of inhibition (broken line) during incubation of platelet-rich p l a s m a (5.1 × lO 6 platelets//~l) with []*Clo! adenosine (3.4/zM and 18oo c o u n t s / m i n per 25/zl incubation mixture initially) at 15 ° (E]), 22 ° ( A ) and 37 ° ( O ) . Aggregation w a s performed at 25 °. Fig. 6. Effect of v a r y i n g t h e initial concentration of [l~C10]adenosine in platelet-rich plasma (3.84 x io~ platelets/#l) on the a m o u n t of adenosine p h o s p h o r y l a t e d b y the cells (O O) and on the degree of inhibition ( A - - - A ) after 5 rain of incubation at 37 °.
hibition of aggregation is still present at IO min, the amount of E14C10]adenosine phosphorylated is also the same (approx. I #M). It is apparent that the inhibition of platelet aggregation and the rate of phosphorylation reach their m a x i m u m at the same E14C101adenosine concentration. This feature of reaching maximal inhibition at a certain adenosine concentration is not dependent on the concentration of A D P employed to produce platelet aggregation. However, the maximal degree of inhibition varies inversely with the A D P concentration. In a typical experiment, the maximal degrees of inhibition / after 5 rain of incubation (reached at 12-16 #M adenosine) were 62, 4 ° and 37 O/o when 0.48, 0.96 and 1.44/~M ADP, respectively, were used.
Dependence o/ platelet aggregation and incorporation o/ [14Clo]adenosine and ~-14C]adenine on energy metabolism As ADP-induced platelet aggregation is dependent on active energy metabolism 16 and phosphorylation of adenosine requires ATP 15, the effect of glycolytic and mitochondrial inhibitors on these processes was studied. Platelets in plasma incubated Biochim. Biophys. Acta, 155 (1968) 3 4 2 - 3 5 2
349
ADENOSINE UPTAKE AND PLATELET AGGREGATION
for 3o min with both D-2-deoxyglucose and antimycin lose their ability to aggregate. This is associated with a fall in thier A T P content of 40-5 ° % while the ADP content is little changed (Table I I L Platelets treated in this w a y show a definite reduction in
TABLE II EFFECT OF PREINCUBATION OI*"PLATELET-RICH PLASMA WITH D-2-DEOXVGLUCOSEAND/OR ANTIMYCIN ON PLATELET AGGREGATION, CONVERSION OF ?4C10~ADENOSINE AND [8-14C]ADENINE TO NUCLEOTIDES, AND ON LEVELS OF A T P AND A D P Platelet-rich p l a s m a (3.65 × lO 5 platelets/#l), w a s i n c u b a t e d at 37 ° w i t h the different inhibitors for 3 ° rain; t h e n platelet aggregation and levels of A T P a n d A D P (time to) were determined. At the s a m e time, [14C10]adenosine (3.4/zM, 18oo c o u n t s / m i n p e r 25 #1 i n c u b a t i o n m i x t u r e ) and [8A4Cladenine (3.4 #M, 9oo e o u n t s / m i n p e r 25/,1 i n c u b a t i o n m i x t u r e ) w e r e added s e p a r a t e l y to aliquots of the i n c u b a t i o n m i x t u r e s and i n c u b a t e d for a n o t h e r 20 rain. The c o n c e n t r a t i o n (time tl) and the r a d i o a c t i v i t y of the adenine nueleotides were determined. The isotopes were in glucosefree saline, D-2-deoxyglucose in saline (8.9 mM), a n t i m y c i n in absolute e t h a n o l (22.2 # g / m l ) and to the control were added saline a n d ethanol in corresponding a m o u n t s .
Addition
Saline-ethanol (control) D-2-Deoxyglucose Antimyein D-2-Deoxyglucose + antimycin
Rate o[ Radioaetivityo/ATP+ADP+AMP Amounto/nueleotides aggregation (counts/min per 251~lincubation (t~moles/Ionplatelets) (%) mixture) ATP ADP [l*Clo]Adenosine [8A4C]Adenine to
tl
to
tl
38 36 38
223 234 255
251 277 281
4 .6 4.2 4.9
3.9 3.6 4.4
3.3 2.8 3.2
3.2 2. 4 3.o
4
124
51
3.0
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2.6
3.2
phosphorylation associated with a further fall in ATP. Incubation of these platelets with E8-14C]adenine shows a more marked reduction in the formation of labelled adenine nucleotides as compared to the [14C10]adenosine experiments (Table II). Treatment of platelets with D-2-deoxyglucose or antimycin separately does not show any significant deviations from the control (Table II).
DISCUSSION
In the presence of adenosine deaminase, no adenosine accumulates during dephosphorylation of AMP in platelet-rich plasma and there is no inhibition of platelet aggregation. Hence, AMP must be dephosphorylated to adenosine in plasma before inducing inhibition of platelet aggregation as was found b y SALZMAN, CHAMBERS AND NER114. Adenosine 5'-monoacetate and adenosine 5'-monopropionate are also able to induce inhibition of aggregation, but only after hydrolysis b y hitherto undescribed plasma esterases to adenosine. Adenosine thus plays a central role in the inhibition of ADP-induced platelet aggregation b y these substances. Our studies confirm the need for prior incubation with adenosine to reach maximal inhibition s, and the recovery of the ability to aggregate with time TM. Bioehim. Biophys. Acta, 155 (1968) 342-352
350
M.C. ROZENBERG, H. HOLMSEN
BOI~Nla has postulated that adenosine, because of its resemblance to ADP, m a y occupy receptor sites on the platelet membrane and prevent access of ADP to these. The need for preincubation with the inhibitor is explained by slow binding of adenosine to these receptor sites in contrast to the fast binding of ADP. Our observations of inhibition of aggregation and disappearance of [l~C101adenosine do fit in with this hypothesis. However, correlation of the increase in inhibition and the ~14Clo]adenosine formed extracellularly 15 when I14CloJAMP is added to plateletrich plasma shows that, under these circumstances, inhibition is produced rapidly. This could only be explained b y rapid binding of adenosine to receptor sites, were the above theory valid. Moreover, AMP is structurally more closely related to ADP than is adenosine and should occupy receptor sites better than adenosine, yet it does not induce inhibition per se. The theory of competition for receptor sites admits that adenosine is capable of occupying all sites used b y ADP for induction of platelet aggregation, hence producing I00 °,o inhibition. In spite of increasing concentrations of [14Cioladenosine in platelet-rich plasma, we never observe complete inhibition. We also show that, provided platelets have been incubated with adenosine, inhibition of aggregation is still present when almost all significant adenosine has been deaminated. Although adenosine induces inhibition of platelet aggregation, the compound itself does not have to be present during the whole inhibitory phase as required b y the theory for competitive inhibition. We therefore feel that our findings render this theory unlikely. The hypothesis that adenosine could induce inhibition of aggregation b y activation of a plasma factor which would then act on platelets is exlcuded b y our finding that platelet-poor plasma, incubated with adenosine, cannot induce inhibition ot aggregation if the adenosine itself is rapidly removed with deaminase prior to contact with the platelets. We have observed good correlation between the inhibition of aggregation and the duration of phosphorylation of [14C10Jadenosine b y the platelets. The appearance of labelled adenine nucleotides in the platelets in our experiments consists of two processes: firstly, the transport of adenosine across the membrane and secondly, the phosphorylation of adenosine to AMP and its subsequent conversion to ADP or ATP. These two phases are included in our term 'uptake', and the amount of phosphorylation observed is taken as a measure of uptake in view of the absence of adenosine in the platelets 15. However, which of the two processes is rate limiting is not yet clear. SALZMAN,CHAMBERS AND NER114 first suggested that uptake of adenosine might precede induction of the inhibition of aggregation. Our findings support this view. There is correlation between inhibition of aggregation and phosphorylation of adenosine. AMP does not induce inhibition and it does not penetrate the membrane if its breakdown to adenosine is prevented 15. The adenosine-induced inhibition is temperature dependent and the rate of phosphorylation varies in the same manner with temperature, which might indicate that the two phenomena are related. It is shown that, in spite of wide variation in adenosine concentrations, maximal inhibition of aggregation correlates well with the amount of adenosine phosphorylated. This maximal inhibition appears limited by adenosine uptake which has reached saturation, and which then remains uninfluenced by further increase in the concentration of adenosine. On the basis of these results, we propose that inhibition of aggregation is associated with the process of adenosine uptake b y the platelets. Biochim. Biophys. Acta, I55 (1968) 342-352
ADENOSINE UPTAKE AND PLATELET AGGREGATION
351
The mechanism of adenosine-induced inhibition during uptake requires closer examination of the latter phenomenon. Adenosine is first transported across the membrane b y an unknown process. I t is then phosphorylated to AMP b y ATP catalysed b y adenosine kinase 15. This enzyme is probably located in the membrane or close to it as all adenosine transported into the cells is phosphorylated and does not come into contact with platelet adenosine deaminase (as opposed to plasma adenosine deaminase). AMP in the platelets is then converted to ADP and A T P b y adenylate kinase. SALZMAN, CHAMBERS AND NER114A8 suggest that adenosine uptake leads to more newly formed ATP providing more energy to an ATPase which keeps the platelets in a low responsive state. Our finding that newly phosphorylated I14C10~ATP remains present in the platelets while they recover this ability to aggregate does not favour this view. Furthermore platelets, having recovered from adenosine inhibition, will often aggregate better than the controls, suggesting that more ATP leads to increased platelet aggregation. Finally, the use of inhibitors of glycolysis and oxidative phosphorylation confirm previous reports 16,2°,22 that platelets in a low energy state are less, not more responsive to ADP. The dependence of aggregation on intracellular A T P m a y mean that ATP is consumed during the process of aggregation or that ATP is consumed to maintain platelets in a responsive state. An attractive hypothesis would be that the ATP used during transport and/or phosphorylation of adenosine would then not be available for platelet aggregation. Yet adenine also requires ATP in order to be converted to nucleotides and does not produce inhibition. Platelets form adenine nucleotides from adenine b y condensation with 5-P-Rib-P-P catalysed b y adenine phosphoribosyl transferase 17. A T P is required for 5-.P-Rib-P-P synthesis but m a y originate from a pool different from that used in adenosine phosphorylation or platelet aggregation. It must be emphasized, however, that partial reduction of ATP b y the metabolic inhibitors produced complete inhibition of aggregation while some phosphorylation of adenosine was still present. This might not be compatible with the energy competition theory. An alternative theory of inhibition of aggregation would be that the transport form of adenosine induces a change in the platelet membrane independent of energy consumption. Other possibilities could be that inhibition of aggregation is somehow induced b y a change in enzyme conformation during the phosphorylation of adenosine to AMP (platelet adenosine kinase) or its subsequent conversion to ADP and ATP (adenylate kinase) irrespective of energy consumption. The inhibitory action of adenosine analogues parallels their vasodilatory effects 22. BERNE has suggested a role for adenosine in the coronary blood flow 23 and it has been shown that vasodilation occurs while adenosine is taken up and phosphorylated b y the heart 24. The mode of action of adenosine in inducing vasodilation and preventing ADP induced platelet aggregation m a y indeed be similar. The close relation between inhibition of platelet aggregation and adenosine uptake indicates that further studies on adenosine kinase and the mode of transport of adenosine across the membrane are warranted.
ACKNOWLEDGEMENT
We wish to t h a n k Miss R. JANSSEN for her technical assistance. M. C. R. was Biochim. Biophys. Acta, 155 (1968) 342-352
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supported by the National Heart Foundation of Australia. Financial support was also provided by Proprietmr Christopher Overland og hustri Ingeborg Overlands legat. Det Videnskapelige Forskningsfond av 1919 and J. L. Tiedemanns Tobaksfabrik Joh. H. Andresens medisinske fond. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 I8 19 20 21 22 23 24
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