Metabolic aspects of the secretion of stored compounds from blood platelets III Effect of NaF on washed platelets

266

Biochimica et Biophysica Acta, 3 6 2 ( 1 9 7 4 ) 2 6 6 - - 2 7 5 © Elsevier Scientific Publsihing Company, Amsterdam -- Printed in The Netherlands

BBA 27486

METABOLIC ASPECTS OF T H E SE C R E T I ON OF ST O RE D COMPOUNDS FROM BLOOD P L A T E L E T S III E F F E C T OF NaF ON WASHED P L A T E L E T S * , **

E. tt. M I ] R E R , H.J. D A Y a n d J.E. L I E B E R M A N

Specialized Center for Thrombosis Research, Temple University Health Sciences Center, Philadelphia, Pa. 19140 (U.S.A.)L {Received March 18th, 1974)

Summary Two metabolic effects are described as results of incubation of washed blood platelets with NaF: (1) Before release an early transfer of the terminal Pi ~rom ATP to the glycolytic intermediates fructose 1,6-diphosphate and 3-phosphoglycerate, resulting in a small decrease in ATP and a similar increase in ADP and AMP and (2) c o n c o m i t a n t l y with the release of stored c o m p o u n d s a drop in the ATP and ADP which participate in metabolic processes, a transient accumulation of IMP and a final accumulation of inosine and h y p o x a n t h i n e which occasionally exceeded 60% of total radioactivity in platelets prelabeled with radioactive adenine. When antimycin was added together with fluoride, there was a massive accumulation of inosine and h y p o x a n t h i n e even in the absence o f the platelet release reaction, but in some cases m a x i m u m accumulation was n o t reached unless release t o o k place. Addition of antimycin during fluoride-induced release resulted in an instantaneous block of this release. It is suggested that the conversion of IMP to inosine and h y p o x a n t h i n e is partly discharged by the platelet release reaction and possibly caused by structural alterations accompanying the reaction.

Introduction Fantl [1] has shown that while most inorganic salts below 300 mosM c o n cen tr atio n cause a transient swelling of blood platelets and decrease in light

* Articles I and II were published in Biochim. Biophys. Acta, 162 (1968) 320--326 and Biochim. Biophys. Acta, 192 (1969) 138--140. * ~ Part of the data have been presented to the 9th International Congress of Biochemistry, S t oc khol m 1973, abstract 8b 18, p. 358.

267 absorption of a platelet suspension (where N-ethylmaleimide blocked the reversal b u t did n o t affect the swelling stage), similar changes induced by NaF were irreversible and could be prevented by N-ethylmaleimide [2]. Skfilhegg et al. [3] found that an initial inhibition of ADP-induced platelet adhesiveness induced b y NaF was followed b y an activation a few minutes later; their suggestion that this latter effect might be caused b y a fluoride-induced release of ADP contradicts Fantl's suggestion [2] that the NaF effect is independent of ADP. Mtirer [4] demonstrated that, in addition to inhibiting platelet functions, fluoride also caused a release of adenine nucleotides which reached the same level as that induced b y thrombin. The release had a slow time course and at 37°C the release rate reached a maximum between 3 and 10 min after fluoride addition, whereas the major part of thrombin-induced release was completed within 30 s. The released adenine nucleotides were not radioactive when platelets were prelabeled with 32 Pi [5]. This indicates that these c o m p o u n d s arise not from metabolically active adenine nucleotides (those participating actively in the cell's metabolic processes and available extracellularly only through lysis of the whole cell) b u t from stored or metabolically inactive adenine nucleotides (those released by the platelet release reaction) [6]. The extruded material also included calcium, which was released with the same time course as adenine nucleotides [7]. Studies of clot retraction revealed that fluoride inhibited platelet functions and anaerobic glycolysis simultaneously [8]; this explains why the effect described by Sk~lhegg et al. [3] was not influenced by another glycolytic inhibitor (monoiodoacetic acid), since the action of the t w o inhibitors would overlap. In contrast, an inhibitor of mitochondrial respiration blocked the release induced by fluoride [4]. The release induced by thrombin was little affected by the inhibition of only one pathway for energy metabolism [4], as thrombin does n o t directly affect either pathway of energy metabolism. The present study investigates the link between alterations in platelet metabolism induced b y NaF and the fluoride-promoted platelet release reaction. Materials and Methods 150-ml portions of blood from normal human donors were collected in 0.07 volume 0.077 M EDTA, Tris, pH 7.4. Platelet-rich plasma was obtained as the supernatant after centrifugation at 250 X g (max) at room temperature for 15 min, and platelets isolated by centrifugation at 900 X g (max) for 20 min at 4--10°C. The platelets were washed twice with 5 0 m l 0.154 M NaC1 with 100 pl 0.077 M EDTA, Tris, pH 7.4 added, and resuspended in 8--12 ml 0.154 M NaC1 with 100 pl 0.077 M EDTA, Tris, pH 7.4. These steps were performed at 0--4 ° C. Antimycin A, t y p e III, and ~3-diphosphopyridine nucleotide, disodium, reduced form, grade III, were acquired from Sigma Chemical Co., St. Louis, Mo. For radioactive experiments platelet-rich plasma was labeled by incubating with shaking at 37°C either for 2 h with 1 pCi per ml 32 Pi (carrier-free), source Norsk Institutt for Atomenergi, Kjeller, Norway, or for 20 min with 4 pCi in 1 nmole [ 3H] serotonin, creatinine sulfate per 6 ml, source Radiochemical Centre, Amersham, England, or 3.3 pCi in 0.4 nmole [14 C]- or [3 H] adenine per ml, source New England Nuclear, Boston, Mass. All other

268

incubations were done by shaking for 20 min at 37°C in a Dubnoff Metabolic Incubator. Radioactivity was counted in a Beckman Lowbeta II counter (32 p), or in a Nuclear Chicago Mark I Scintillation Counter or a Beckman LS 330 Liquid Scintillation Counter with scintillation fluid one part toluene with 0.104 g POPOP and 2.1 g PPO per 1 and one part Triton X-100, 0.25 ml liquid sample added to 10 ml scintillation fluid (when paper strips were counted, Triton was omitted). Adenine nucleotides in the supernatant after centrifugation and deproteination with 0.5 M HC104 were determined from the ultraviolet absorption spectrum (260 nm) read with a Beckman Acta Spectrophotometer. Metabolites of [ 3 H] adenine or [14 C] adenine were separated by electrophoresis as described by Hotmsen and Weiss [9] on a Savant High Voltage Paper Electrophoresis apparatus (Model FP-22A) or by strip chromatography of an HC1Oa-treated sample neutralized with Na2CO3, as described by Randerath and Struck [10]. A mixture either of 18 mM ATP, ADP, IMP, AMP, h y p o x a n t h i n e and adenine, or of the same concentration of ATP, ADP, IMP, AMP, inosine, cyclic AMP, hypoxanthine, adenosine and adenine was used as marker. Total ATP and ADP was determined in a DuPont Luminescence Biometer as described elsewhere [11]. Lactate dehydrogenase activity was measured as described by Wu and Racker [12]. Results

Incubation of 32 P-labeled platelets with NaF resulted in a drop of about 30% in [32 p] ATP within 10 s and 70% in 5 min (Table I), after which there was a slow but steady decrease in radioactive ATP. The drop was accompanied by a simultaneous increase in fructose 1,6-di-[ 32 p] phosphate and in 3-[ 32 p]. phosphoglyceric acid, and a removal of 32 Pi, while other glycolytic intermediates did not show accumulation of radioactivity [8]. Figs 1 and 2 show that when washed platelets were incubated with NaF after preincubation with [14C] - or [3 H]adenine, there was a significant decrease in radioactive ATP and a corresponding increase in radioactive hypoxan-

TABLE I CHANGES IN 32p-CONTAINING METABOLITES WITH FLUORIDE

IN W A S H E D P L A T E L E T S

DURING INCUBATION

P l a t e l e t s w e r e l a b e l e d b y i n c u b a t i o n o f p l a t e l e t - r i c h p l a s m a w i t h 32Pi, a n d t h e t w i c e - w a s h e d p l a t e l e t s i n c u b a t e d in 4 m l 0 . 1 3 M NaC1 w i t h 25 m M Tris--HC1, p H 7.4, f o r 30 rain. 10 m M N a F w a s a d d e d 5 rain o r 10 s b e f o r e t h e e n d o f i n c u b a t i o n , w h e r e a f t e r t h e p l a t e l e t s u s p e n s i o n w a s c o o l e d quickly, in an i c e - w a t e r b a t h ( t h e 10-s s a m p l e s w e r e p o u r e d i n t o p r e c o o l e d vessels). E x t r a c t i o n o f p l a t e l e t p e l l e t s a f t e r c e n t r i f u g a t i o n a n d d e t e r m i n a t i o n o f m e t a b o l i t e s b y t w o - w a y c h r o m a t o g r a p h y a n d a u t o r a d i o g r a p h y are d e s c r i b e d earlier [ 5 ] . M e a n of t w o e x p e r i m e n t s . % r a d i o a c t i v i t y in

ATP Fru-106-P 2 3-Phosphoglycerate

T i m e o f i n c u b a t i o n w i t h N a F (s) 0

10

300

19.1 2.5 2.2

13.0 9.9 9.7

4.8 22.2 23.9

269 25

20

3C

15

._~ 2C

10 Q)

.--

C

5

8

0

5 10 15 Time with NoF (rain)

20

Fig. 1. Correlation between release and accumulation o f h y p o x a n t h i n e and o f inosine. Plate|et-rich plasma was prelabeled w i t h [3 H i - or [ ] 4 C] adenine as described in Materials and Methods whereafter the platelets were isolated and washed twice w i t h 0.154 M NaC] containing 0.15 m M E D T A , pH 7.4. A 4 m i suspension o f washed platelets in 0.15 M NaCi w i t h 25 m M Tris--HCl, pH 7.4, and 0.2 m M E D T A o f same pH was incubated at 37°C f o r 9.0 rain. 10 m M NaF was added at varying times before the end o f i n c u b a t i o n w h i c h w a s s t o p p e d as d e s c r i b e d in T a b l e I. A f t e r c e n t r i f u g a t i o n a s a m p l e o f t h e s u p e r n a t a n t w a s c h r o m a t o g r a p h e d as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s , a n d t h e u l t r a v i o l e t s p e c t r u m m e a s u r e d as p r e v i o u s l y d e s c r i b e d [ 3 ] . M e a n o f 4 e x p e r h n e n t s . "o, h y p o x a n t h i n e ; ~ o adenine nucleotides; © o inosine.

thine and inosine, the latter closely paralleling the release of non-labeled adenine nucleotides. At the end of the 20 min incubation period the radioactivity in inosine and h y p o x a n t h i n e together reached about 60% of total radioactivity taken up by the platelets. During the first 30 s after fluoride addition there was a drop in adeninelabeled ATP and an increase in adenine-labeled ADP and AMP which might reflect the changes seen in Table I. The main decrease in adenine-labeled ATP preceded the appearance of the bulk of released material outside the platelet, as did the initial increase in IMP concentration. Later the IMP stabilized at a low level, while the metabolically active ATP, ADP and AMP seemed to have reached a stable level relatively early during the time course of the release reaction. The IMP accumulation reached a peak relatively early, but fell below the AMP level at a later stage (Fig. 2). While almost 90% of the accumulated inosine and h y p o x a n t h i n e was recovered extracellularly, there seemed to be no penetration of the platelet membrane by IMP. The small a m o u n t of IMP found extracellularly could be explained by platelet lysis. At no time was the lactate dehydrogenase activity

270

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I

1

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l

1C I

i

I

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20

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Fig. 2. C o r r e l a t i o n b e t w e e n release a n d c h a n g e s in i n t e r m e d i a t e s o f n u c l e o t i d e m e t a b o l i s m . P l a t e l e t - r i e h p l a s m a w a s p r e l a h e l e d w i t h [ 14 C] a d e n i n e as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e w a s h e d p l a t e l e t s u s p e n s i o n w a s t r e a t e d a n d t h e e x p e r i m e n t p e r f o r m e d as d e s c r i b e d in t h e l e g e n d t o Fig. 1. A f t e r i n c u b a tion and cooling samples were added directly to precooled mixtures of 90% ethanol and 10 mM EDTA a n d m e t a b o l i c i n t e r m e d i a t e s d e t e r m i n e d e l e c t r o p h o r e t i c a U y as d e s c r i b e d b y H o l m s e n a n d Weiss [ 9 ] . T h e u l t r a v i o l e t s p e c t r u m o f t h e s u p e r n a t a n t w a s d e t e r m i n e d as d e s c r i b e d p r e v i o u s l y [ 3 ] , M e a n o f t h r e e e x p e r i m e n t s . (a) [ ! 4 C ] A T P ~ (b) [ 1 4 C ] A D P , ( e ) [ 1 4 C ] A M P , (d) [ 1 4 C ] I M P , (e) [ 1 4 C ] i n o s i n e + [ 1 4 C ] h y p o x a n t h i n e in w h o l e i n c u b a t e , (f) /~M a d e n i n e n u c l e o t i d e s in s u p e r n a t a n t , a ~ in % o f t o t a l r a d i o a c t i v i t y in t h e p l a t e l e t s u s p e n s i o n .

271 found outside the platelets during incubation with fluoride more than 13% of the activity of lysed platelets. When the washed platelets were incubated with antimycin (an inhibitor of mitochondrial respiration) for 20 min in the absence of fluoride, there was a 50 to 70% drop in adenine-labeled ATP and 100--200% increase in IMP. The increase in the inosine + h y p o x a n t h i n e level was only 50%, however, indicating that the conversion of IMP was little affected by the presence of the inhibitor. Also characteristic was the high level of labeled ADP retained with antimycin alone, indicating that later steps of adenine nucleotide metabolism are only partially activated (Table II). The release induced by NaF was slow with about 3 min lag period, and showed the same course whether measured as adenine nucleotides or serotonin (Fig. 3). When energy metabolism was blocked by the addition of both fluoride and antimycin (producing a combined inhibition of glycolysis and mitochondrial respiration [8] ) the release was inhibited from the m o m e n t the second inhibitor was added. In most experiments there was a significant increase in inosine + h y p o x a n t h i n e when antimycin was added late enough during incubation to block the release reaction only partially (Table II). The accumulation of IMP was significantly enhanced when antimycin was added early enough during incubation to prevent release. When the inhibitor was added later, the IMP decreased to 50% of the level observed when platelets were incubated with antimycin alone, while h y p o x a n t h i n e + inosine increased to 3 times the level observed under the latter conditions (Table II). TABLE II METABOLIC CHANGES IN WHOLE INCUBATE IN NUCLEOTIDES W I T H [14C] A D E N I N E - - E F F E C T O F A N T I M Y C I N

OF PLATELETS PRELABELED

E x p e r i m e n t s p e r f o r m e d as d e s c r i b e d in t h e l e g e n d f o r Fig. 2, w i t h a n t i m y c i n as d e s c r i b e d i n t h e l e g e n d f o r Fig. 4. T o t a l i n c u b a t i o n f o r all s a m p l e s 20 rain. R e s u l t s are m e a n o f 3 e x p e r i m e n t s , a d e n i n e n u c l e o t i d e s in s u p e r n a t a n t axe m e a n o f 2 e x p e r i m e n t s . I n o n e e x p e r i m e n t t h e d i f f e r e n t c o m p o u n d s w e r e d e t e r m i n e d b y c h r o m a t o g r a p h y , in o n e b y e l e c t r o p h o r e s i s , a n d in o n e b y b o t h m e t h o d s w h e r e t h e n u m b e r s u s e d w e r e a mean of the numbers from each method. Hx = hypoxanthine. % radioactivity in

T i m e with NaF: C o n c n adenine nucleot i d e s in s u p e r n a t a n t :

ATP ADP AMP IMP Hx + inosine % radioactivity in

ATP ADP AMP IMP Hx + inosine

T i m e with NaF: T i m e with antimycin: C o n e n adenine nucleotides in supernatant:

0

3

3.5

7.3

60.8 17.9 2.8 4.2 12.8 0 20

5

10

15

20 (min)

11.8

21.2

24.3

25.0 (pM)

33.8 20.2 5.8 19.2 21.7

18.7 18.1 9.0 21.9 31.2

10.9 16.3 11.3 9.0 51.8

9.1 15.1 11.1 6.3 58.0

8.4 14.4 8.8 6.3 61.0

20 20

20 17

20 15

20 10

20 (rain) 5 (rain)

4.6

11.0

11.8

13.9

22.0

24.5 (pM)

27.3 22.8 13.7 13.4 18.9

5.2 12.6 17.8 19.5 43.6

5.4 12.3 15.3 19.2 46.9

5.2 12.7 15.1 16.0 49.6

5.4 12.6 15.8 9.9 57.5

5.7 12.5 14.7 7.3 58.9

272

E

i

15

f

Z

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U 5 Time with

10 NoF ( m i n )

15

20

Fig. 3. C o m p a r i s o n o f r e l e a s e o f a d e n i n e n u c l e o t i d e s a n d s e r o t o n i n a s a p r o d u c t o f t i m e w i t h f l u o r i d e . Platelet-rich plasma was preincubated w i t h [3 H ] s e r o t o n i n , creatine sulfate as described under Materials a n d M e t h o d s . E x p e r i m e n t s w e r e p e r f o r m e d a s d e s c r i b e d i n t h e l e g e n d o f F i g . 1. A f t e r i n c u b a t i o n , c o o l i n g and centrifugation the ultraviolet spectrum and radioactivity of the supernatant was measured. Mean of two experiments. • - • , m e a s u r e d as a d e n i n e n u c l e o t i d e s ; • • m e a s u r e d as [ 3 H ] s e r o t o n i n .

The combined addition of fluoride and antimycin from the beginning of incubation gave a considerable increase in extracellular 260 nm absorbing material (Fig. 4), but very little of this was ATP and ADP. The enhanced concentration of h y p o x a n t h i n e resulting from the combined effect of antimycin and fluoride resulted in an increase in the 2 5 0 / 2 6 0

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20

to

0

T i m e w i t h ontimycin ( m i n ) F i g . 4. C o m p a r i s o n o f r e l e a s e o f a d e n i n e n u c l e o t i d e s a n d s e r o t o n i n as a p r o d u c t o f t i m e w i t h a n t i m y c i n . Experiments w e r e p e r f o r m e d a s d e s c r i b e d i n l e g e n d s o f F i g s 1 a n d 3. T h e p l a t e l e t s u s p e n s i o n w a s i n c u bated with 10 mM NaF for 20 •in and 60 ng antimycin per ml suspension added at varying time before t h e e n d o f i n c u b a t i o n , as i n d i c a t e d . T h e t w o p o i n t s o n t h e o r d i n a t e b e l o w t h e c u r v e s i n d i c a t e a m o u n t o f extracellular material after incubation with antimyein in the absence of fluoride. Mean of two experiments. • - - • , m e a s u r e d as a d e n i n e n u c l e o t i d e s ; • o, m e a s u r e d as [ 3 H ] s e r o t o n i n .

273 nm ratio o f a b o u t 10%. There was no significant difference between the release curve f o r 260 nm absorbing material and t hat for [3 H] serotonin (Figs 3 and 4); however, the ratio of ATP + ADP to 260 nm absorbing material did increase dramatically when the addition of a nt i m yci n was post poned. When released ATP and ADP, as d e t e r m i n e d by the firefly m e t h o d (total ATP + ADP), were c o m p a r e d with 260 nm absorbing material (AN) and with carbon-labeled ATP and ADP ([14 C ] A T P + [14 C] ADP), the following results were obtained: The increase in AN when only antimycin was added, contained only 17% ATP + ADP. The f ur t her increase in AN when fluoride and antimycin were present together from the beginning of incubation contained only 18% ATP + ADP; when the addition of antimycin was delayed 3 and 5 min, the ATP + ADP c o n t e n t in the increased a m o u n t of AN was 59 and 84%, respectively. The a m o u n t of ATP + ADP in the AN released between 5 and 20 min of uninhibited exposure to NaF was 95%. The radioactivity of ATP and ADP decreased f r o m 21.4 in the material " r e l e a s e d " w i t h o u t release inducer and 21 for t h e part o f " r e l eas e " caused by the presence of ant i m yci n and fluoride from the beginning o f incubation, to 5 for the released material when antimycin addition was delayed 3 min, to 1 for the additional ATP + ADP released when the delay was e x t e n d e d 2 min. For the total ATP + ADP f o u n d extracellularly after incubation with fluoride in the absence of antimycin the specific radioactivity was reduced f r o m 21.4 to 2. The increase in h y p o x a n t h i n e + inosine (from 16 to 47% o f total radioactivity) seems to constitute a major part of the increase in 260 nm absorbing material when fluoride and ant i m yc i n were present from the beginning of incubation, b u t their influence on the spectrum in the latter part of the release reaction seems minor. The technique of drawing a base line for " u n s p e c i f i c " release [13] partly corrected for the 260 nm c o n t r i b u t i o n of the inosine + h y p o x a n t h i n e spectrum, especially in the presence of greater concentrations of adenine nucleotides. The use of 260 nm absorbance as a quantitative determination o f adenine nucleotides seems justified when release is significant, even in the presence o f considerable accumulation of inosine and h y p o x a n t h i n e .

Discussion The results indicate that the initial decrease in ATP during incubation with NaF was caused by the transfer of terminal Pi from ATP to fructose 1,6-diphosphate and 3-phosphoglycerate, suggesting t hat an increased turnover o f ATP had been initiated. The changes did n o t result in a comparable decrease in total metabolic ATP, measured as [3 H] adenine-labeled nucleotide. There was, however, a small b u t significant decrease in adenine-labeled ATP and an increase in ADP and AMP during the first 30 s, w i t h o u t a c o n c o m i t a n t effect on the IMP and hypoxanthine + inosine levels. This change was most likely caused by the initial transfer o f the terminal Pi. When release t o o k place, adenine-labeled ATP was converted to ADP, AMP, IMP, inosine and h y p o x a n t h i n e as described by Holmsen and Day [ 1 4 ] . This is clearly illustrated by the consecutive accumulation of intermediates in the course o f incubation with fluoride (Fig. 2). T he conversion from IMP to

274 inosine and h y p o x a n t h i n e did not appear until release occurred, seemed strictly to parallel the degree of completion of the platelet release reaction, and might be correlated with the energy-requiring processes connected with release, as proposed by Holmsen and Day [14] and Holmsen et al. [15]. The release of adenine nucleotides came after the initial transfer of terminal Pi from ATP of the glycolytic intermediates. Thus, resynthesis seemed to conserve the level of ATP until the drain caused by the release reaction resulted in the irreversible conversion into hypoxanthine. Antimycin's effect in immediately stopping the release reaction, even when added after fluoride, supports the suggestion that continuous production of ATP is needed for the fluoride-induced release reaction, since the inhibitor of mitochondrial respiration would have blocked only the resynthesis of ATP, but n o t the utilization of preformed ATP [8]. When energy metabolism was completely blocked, an accumulation of h y p o x a n t h i n e and inosine, as well as of IMP, was observed. Similar findings have been reported in platelet-rich citrated plasma in the presence of KCN and iodoacetate [16]. Maximum conversion to inosine and hypoxanthine was reached only in the presence of release. The addition of antimycin alone, which resulted in a dramatic decrease in metabolic ATP and increase in ADP, AMP and IMP, had a minor effect on the h y p o x a n t h i n e level, in agreement with the finding of Ball et al. [16] that the addition of cyanide + monoiodoacetate, but not of cyanide alone, resulted in a significant enhancement of h y p o x a n t h i n e production in platelet-rich plasma. The m a x i m u m accumulation of hypoxanthine + inosine appeared to be the same whether antimycin was added or not, so that the effect of inhibitors in promoting accumulation seems not to be superimposed upon the effect of release. There was an early accumulation of IMP after fluoride addition, which was also found when antimycin blocked the fluoride-induced release. The level of IMP dropped to the preinitiation level after significant release had taken place. This indicates the presence of a somewhat different mechanism for the activation of the conversion to IMP than for the ensuing conversion to inosine and hypoxanthine, with the latter more strictly linked to the release reaction. An accumulation of IMP is found when platelets in plasma are disrupted by freezing and thawing [17] or other means (Mfirer, E.H., unpublished observations) indicating that under these circumstances IMP is the end product. The results indicate that in the resting state under physiological conditions the blood platelets are in an energy-requiring metabolic equilibrium in which adenine nucleotides are preserved for resynthesis. When platelets undergo changes presumed to be irreversible (irreversible aggregation, release reaction or breakdown of the equilibrium by blockage of energy metabolism or incubation under non-physiological conditions), one of the indicators is the accumulation of inosine and hypoxanthine, depriving the platelet's metabolic pool of the essential adenine moiety which the platelets cannot resynthesize [14]. This may be used as an indicator for the quality and viability of platelets for transfusion. Chapman and Atkinson [18] have demonstrated an increase in AMP deamination with decreasing energy charge and suggested this as a means of preserving the latter. In the case of the reduction of ATP-concentration caused

275 b y the release reaction or by the addition of a combination of metabolic inhibitors, a new equilibrium between the adenine nucleotides would appear to be established at a lower level of energy charge after the initial shock of the violent changes imposed upon the cell. Other explanations should also be explored. Holmsen and Day [14] suggest that removal of AMP from the adenylate kinase equilibrium will result in increased conversion to ATP, and thus to a better utilization of available adenine nucleotides. Such a mechanism might be favorable when there is an immediate and non-repeatable demand for energy (e.g. the platelet release reaction). The regulation of the conversion from IMP to inosine and hypoxanthine, and specifically for the drop in the IMP-concentration which takes place during release b u t not when metabolic inhibitors block the reaction, may be caused b y structural changes inside the platelet. The enzymes responsible for the breakdown of IMP may be inactive in the resting cell due to compartmentalization or other reasons. By irreversible changes the activities might be released, and the chemically favorable production of inosine and hypoxanthine (which is further favored by their complete penetration through the cell membrane) would result. The greater the structural breakdown, the greater would be the accumulation of inosine and hypoxanthine, indicating that the structural changes caused by the platelet release reaction are greater even than those resulting from a block of energy metabolism. In the presence of antimycin alone there is still enough energy produced to keep the platelet structurally intact, and the extensive b r e a k d o w n of IMP will be prevented. When the platelets' plasma membrane is disrupted, the breakdown product of adenine nucleotides seems to be IMP (a fact which is difficult to explain along these lines). Acknowledgments The study was supported by grant no. HL 14217 from the H e ~ t and Lung Institute of the National Institutes of Health, Bethesda, Md. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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