Adenine nucleotide metabolism of blood platelets

46

Biochimica et Biophysica Acta 497 (1977) 46--61

© Elsevier/North-Holland Biomedical Press

BBA 28177 ADENINE NUCLEOTIDE METABOLISM OF BLOOD PLATELETS X. FORMALDEHYDE STOPS CENTRIFUGATION-INDUCED SECRETION AFTER A23187-STIMULATION AND CAUSES BREAKDOWN OF METABOLIC ATP

HOLM HOLMSEN and CAROL A. SETKOWSKYDANGELMAIER Specialized Center for Thrombosis Research, Temple University, Philadelphia, Pa. 19140 (U.S.A.)

(Received July 21st, 1976)

Summary A23187 induced shape change, aggregation and secretion of platelets in plasma. When rapid cooling was used to stop secretion and centrifugation to separate the cells from the medium, maximal amounts of storage ATP plus ADP and preadsorbed [~4C]serotonin were found in the supernatant immediately (<5 s) after A23187 addition. These results suggested that A23187 could cause shape change and aggregation through secreted ADP and not directly. When secretion was stopped with chilling and formaldehyde treatment before centrifugation, the secreted substances appeared after a lag of 60--120 s, i.e. after shape change was terminated and aggregation was well on its way. These two platelet responses thus seemed to be independent of secretion and induced directly by A23187. The absence of a lag period when secretion was stopped by chilling alone was t h o u g h t to be due to centrifugation-induced secretion of platelets conditioned by A23187. Formaldehyde completely inhibited centrifugation-induced secretion. At 37°C, formaldehyde caused rapid breakdown of metabolic ATP in platelets with a pattern dependent on the formaldehyde concentration: Below 50 mM, ATP was converted to inosine plus hypoxanthine via ADP, AMP and IMP and the adenylate energy charge was preserved. Above 100 mM, AMP was the end product with a drastic reduction in the adenylate energy charge. These changes were not due to lysis of the platelets, but were apparently caused by an formaldehyde-induced increase in cellular ATP consumption. Platelet secretion is usually associated with a conversion of metabolic ATP to hypoxanthine. Formaldehyde had to be used to stop secretion and since it caused breakdown of ATP, additional samples were taken out for nucleotide

47 determination during stirring of platelet-rich plasma with A23187. It was found that metabolic ATP was converted to inosine plus hypoxanthine only during the secretion step.

Introduction The ionophore A23187 causes immediate shape change and aggregation of platelets when added to stirred platelet-rich plasma [1--3]. Consecutive sampling of the aggregation mixture and rapid cooling and centrifugation of the samples showed that serotonin had already been secreted from the cells in maximal amounts during the shape change phase, i.e. less than 5 s after addition of the ionophore [1]. Since ADP which is secreted concomitantly with serotonin [4] is a p o t e n t inducer of shape change and aggregation [5], it is unclear from these studies [1--3] whether A23187 induces shape change and aggregation directly or acts via the secreted ADP. When secretion of Ca 2÷ and ATP {also secreted together with serotonin [4]) was monitored by a continuous recording technique which did n o t require centrifugation, a lag period of 5--10 s was observed between the time of addition of A23187 and the onset of secretion [6,7]. This lag period is longer than the time normally required for the initial morphological changes that are observed by direct optical methods [5] and suggests that the ionophore can itself initiate p l a t e l e t shape change and aggregation before ADP secretion occurs. A similar lag period was observed in platelets treated with thrombin when secretion was measured by the direct continuous recording techniques [8,9], b u t n o t when measured after centrifugation [4]. Recently, another m e t h o d has been described for measuring the exact onset of platelet secretion which reproduces the pre-secretion lag period observed with the continuous recording method; secretion of serotonin induced by both thrombin and A23187 was efficiently stopped by treatment with formaldehyde prior to centrifugation [10]. In the present study which was undertaken to delineate the time relationship between shape change, aggregation and secretion in platelet-rich plasma as induced by A23187, formaldehyde was used to stop secretion at various stages during aggregation. The platelets were removed by centrifugation and the amounts of ATP and ADP in the supernatants were determined. This procedure necessitated an investigation of the effects of formaldehyde on metabolic and storage adenine nucleotides [11] in platelets, and the results of these studies are also reported here. Materials and Methods Chemicals. A23187 was kindly given to us by Dr. R.L. Hamill (Lilly Laboratories, Indianapolis, Ind.) and kept dissolved in acetone in a 40 mM concentration at --20°C. The concentration of A23187 increased by storage (loss of solvent), and was accurately determined spectrophotometrically [12] prior to each experiment as follows: 5--20 pl of the stock solution mixed with 100 pl acetone in a r o u n d - b o t t o m e d glass tube (0.6 mm diameter) and the mixture

48 evaporated to dryness. The ionophore film was dissolved in a known volume of ethanol and the absorbancy at 378 nm was determined. Freshly prepared solutions of A23187 in 95% ethanol had an E)TcSm = 5950, which is lower than the value of 8600 reported by Pfeiffer et al. [12]. The exact concentration of the stock solution was c o m p u t e d and dilutions to desired concentrations in acetone were prepared. Formaldehyde was obtained as a 37.4% formaldehyde solution containing 12--15% methanol (Formaldehyde Solution, Fisher Scientific Co., Fair Lawn, N.J., Code F-79) and stored at room temperature. Dilutions in 0.15 M NaC1 were made prior to each experiment. Other chemicals were of reagent grade. Radiochemicals. [14C]Adenine was obtained from Amersham/Searle Corp., Arlington Heights, Ill. ([U-14C]adenine, Code CFA 4 3 6 , 2 8 2 Ci/mol) and stored as a 160 ~M solution in 0.15 M NaC1 at --20°C. [~4C]Serotonin was obtained from Amersham/Searle Corp., Arlington Heights, Ill. ([side-chain-2-~4C]serotonin, Code CFA 170, 59 Ci/mol) and stored as a 50 uM solution in 0.15 M NaC1 at --20 ° C. Biological materials. Platelet-rich plasma was obtained from blood drawn from normal donors into sodium citrate as described previously [11]. [~4C]Adenine-labeled platelet-rich plasma was used in experiments on the effect of formaldehyde on adenine nucleotide metabolism in order to differentiate between metabolic and non-metabolic adenine nucleotide pools [ 11]. [~4C]Serotonin-labeled platelet-rich plasma. The serotonin in the storage granules was labeled [13] by incubation of platelet-rich plasma with 1 pM [~4C]serotonin for 30 min at room temperature prior to secretion experiments.

Methods General procedures. All centrifugations in the secretion experiments were done at 12 000 × g for 2 min at room temperature in an Eppendorp Centrifuge 3200 (Brinkman Instruments, Westbury, N.Y.). EDTA/ethanol extracts were prepared by mixing equal volumes (200--500 pl) of sample and freshly prepared, ice-cold 10 mM EDTA/87.4% ethanol. The mixtures were kept at 0 ° to --20°C until precipitates were removed by centrifugation and the extracts analyzed. The radioactivity of the [14C]adenine metabolites in the EDTA/ethanol extracts was determined in a Beckman LS 100C scintillation counter after their separation on a Savant Flat Plate high voltage electrophoresis apparatus as described elsewhere [14]. The concentration of ATP and ADP in the EDTA/ethanol extracts was determined by a luciferin-luceriferase method [15]. Formaldehyde in the concentration used in the experiments did n o t interfere with the determination. Platelets were counted in a Coulter Counter, Model ZBI (Coulter Electronics, Hialeah, Fla.). Special procedures. Effects of formaldehyde on adenine nucleotide metabolism in platelet-rich plasma: One volume of [14C]adenine-labeled platelet-rich plasma was incubated with 1/20 volume of formaldehyde in various concentrations or of 0.15 M NaC1 (control) at 37°C. EDTA/ethanol extracts were made of aliquots of the incubation mixtures at noted times. Occasionally aliquots

49 (0.5 ml) of the incubation mixture was centrifuged after addition of 50 pl 77 mM EDTA, pH 7.4, and EDTA/ethanol extracts were made from the supernatant and from the sedimented cells after resuspension in 0.55 ml of i c e , o l d 6.7 mM EDTA/137 mM NaC1. The sum of the radioactivities of the metabolites investigated (ATP, ADP, AMP, IMP and inosine plus hypoxanthine) did not change during 1 h of incubation with [ ~4C]adenine-labeled platelet-rich plasma. The radioactivity of each individual metabolite in a sample is expressed in percent of the sum of all metabolite radioactivity in the sample. Adenylate energy charge was calculated according to the formula [16], [ATP] + 1/2[ADP]/[ATP] + [ADP] + [AMP], using the percent radioactivity of the individual adenylates instead of the concentrations [ 17 ]. Similarly, these radioactivities were substituted for concentrations in the expression for the equilibrium constant of adenylate kinase, [ATP] × [AMP]/[ADP] 2. Platelet aggregation was performed in a Payton dual channel aggregometer module connected to a Payton two-channel recorder (Payton Assoc., Inc., Buffalo, N.Y.). Portions (0.5--7 ml) of platelet-rich plasma, labaled with either [14C]adenine or [14C]serotonin were stirred at 37°C (900 rev./min) and 2 ut A23187 (test) or acetone (control) was added per ml. Secretion in response to A23187 was measured as follows: Aliquots taken from the stirred mixtures in the aggregometer (see above) were mixed with 0.075--0.1 ml of either 0.050 M NaC1/0.051 M EDTA (pH 7.4) or 0.933 M formaldehyde/0.051 M EDTA that were cooled in 1.35 ml Eppendorf tubes immersed in ice. The aliquots were then centrifuged, and EDTA/ethanol extracts were made from both supernatants and from non-centrifuged samples of platelet-rich plasma (total extracts) for estimation of ATP and ADP; [14C]serotonin radioactivity in the supernatants or non-centrifuged platelet-rich plasma was determined by counting 200o~1 aliquots in 10 ml Triton X-100/toluene (1 : 1, v/v) in a Beckman LS-100C scintillation counter. Percent secretion was computed after the formula,

Stest Scontro 1 X I00 Tcontrol--Scontrol -

-

where S designates the amount of ATP plus ADP or [~4C]serotonin radioactivity in the supernatants and T the amounts in total platelet-rich plasma. Results

Effects o f formaldehyde on platelet adenine nucleotides Incubation of [~4C]adenine-labeled platelet-rich plasma with formaldehyde in the 5--50 mM range caused a conversion of [14C]ATP to [14C]IMP, [14C]inosine and [14C]hypoxanthine. With 10 mM formaldehyde (Fig. 1A) the level of [~4C]ATP decreased after a 2 min delay and this decrease was completely balanced by accumulation of [14C]IMP, [14C]inosine and [~4C]hypoxanthine with no accumulation of [14C]AMP. The level of [14C]ADP and [~4C]AMP (not shown in Fig. 1A) fell almost in parallel to that of [~4C]ATP. Consequently, the adenylate energy charge was nearly constant throughout the 20 min incuba-

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Time (min) Fig. 1. Effect o f l o w c o n c e n t r a t i o n s o f f o r m a l d e h y d e o n [ 14C] adenine n u c l e o t i d e s . F o r m a l d e h y d e was added to [ 1 4 C ] adenine-labeled platelet-rich plasma (4.1 • 108 cells and 78 0 0 0 c p m per ml) at 37°C and the radioactivity (percent o f total) o f A T P ( e ~), AMP (A A) and IMP + inosine + h y p o x a n t h i n e (c -') as well as a d e n y l a t e energy charge (o . . . . . . o) w e r e d e t e r m i n e d as the times indicated. A, 10 mM f o r m a l d e h y d e ; B, 30 mM f o r m a l d e h y d e . In the c o n t r o l platelet-rich plasma (not s h o w n ) , w h i c h was incubated w i t h 0 . 1 5 M NaCI instead o f f o r m a l d e h y d e , the A T P radioactivity decreased f r o m 8 0 . 4 to 78.3%, that o f IMP plus h y p o x a n t h i n e plus inosine increased from 2.2 to 5.6% whereas the adenylate energy charge was unaltered ( 0 . 9 2 4 ) over the 20 rain i n c u b a t i o n period.

tion period despite the fall in the [14C]ATP level (Fig. 1A). When 30 mM formaldehyde was used, the level of [14C]ATP decreased more rapidly than at 10 mM and the greater part of the radioactivity was recovered as IMP, inosine and hypoxanthine. However, although the level of [14C]ADP decreased in parallel to that of [14C]ATP, small amounts of [14C]AMP accumulated which resulted in a decrease in adenylate energy charge (Fig. 1B). Incubation of [~4C]adenine-labeled platelet-rich plasma at 37°C with 100-500 mM formaldehyde gave changes in the 14C distribution that were distinctly different from those observed with the lower concentrations. Fig. 2 shows the results from a typical experiment with 135 mM formaldehyde. The decrease in [14C]ATP was balanced by formation of [14C]ADP and [14C]AMP, the former showing a transient increase while the latter accumulated during the 30 min incubation period. At the end of the incubation 4/5 of the ATP radioactivity was recovered as [14C]AMP. Only small amounts of radioactive IMP and inosine plus hypoxanthine accumulated during the conversion of [~4C]ATP to [14C]AMP. Concomitant with these conversions the adenylate energy charge decreased rapidly from 0.932 to below 0.3 after 5 min, and below 0.2 after 30 min of incubation. With higher concentrations of formaldehyde, i.e. 250 and 500 mM, the decrease in the level of [~4C]ATP occurred more slowly and the reduction in adenylate energy charge and in particular the accumulation of AMP was smaller than at 135 mM formaldehyde. The adenylate kinase equilibrium was not much altered by the changes in the adenylates caused by formal-

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Fig. 2. E f f e c t o f 1 3 5 m M of f o r m a l d e h y d e o n [ 14C] a d e n i n e n u c l e o t i d e s . T h e e x p e r i m e n t was p e r f o r m e d a n d t h e s a m e s y m b o l s used as in Fig. 1. I n a d d i t i o n , t h e s e s y m b o l s axe used: A D P ( v v ) a n d AMP (a 4). T h e p l a t e l e t - r i c h p l a s m a c o n t a i n e d 5.6 • 108 c e l l / m l a n d 84 0 0 0 c p m / m l . I n s i g n i f i c a n t c h a n g e s t o o k p l a c e in t h e c o n t r o l .

dehyde (Table I). The formation of radioactive IMP, inosine and hypoxanthine was the same at all formaldehyde concentrations, but greater than that in control platelet-rich plasma. Formaldehyde lowered the pH of platelet-rich plasma (500 mM formaldehyde decreased the pH from 7.69 to 6.67); the formaldehyde preparation used contained methanol (1 : 3 on a mol base). However, neither lowering of pH in platelet-rich plasma by HC1 nor addition of methanol alone in concentrations corresponding to those introduced by formaldehyde caused TABLE I C H A N G E S IN [ 1 4 C ] A D E N I N E M E T A B O L I T E S IN P L A T E L E T ° R I C H PLASMA D U R I N G INCUBATION WITH DIFFERENT CONCENTRATIONS OF FORMALDEHYDE [ 1 4 C ] A d e n i n e . l a b a l e d plateletorich p l a s m a ( 3 . 9 6 • 108 cells/ml; 6 0 2 0 0 c p m / m l w a s i n c u b a t e d w i t h various c o n c e n t r a t i o n s o f f o r m a l d e h y d e at 3 7 ° C a n d t h e level o f r a d i o a c t i v i t y in v a r i o u s m e t a b o l i t e s was d e t e r m i n e d at t h e t i m e s i n d i c a t e d

Formaldehyde (raM)

0 135 250 500

Incubation Radioactivity(%) time (rain) ATP ADP

AMP

0 5 5 5 5

0.6 0.9 50.5 27.7 12.7

87.2 82.6 11.4 31.5 49.5

9.6 10.6 27.0 33.4 27.8

IMP + Ino + hypoxanthine

Adenylate kinase equilibrium constant

Adenylate energy charge

3.0 5.0 10.9 7.1 9.8

0.83 0.75 0.75 0.86 0.83

0.947 0.943 0.280 0.520 0.704

OF ADENINE

METABOLITES

AND THE CONCENTRATION

10.5 0.0

5.6 0.1

5.4 1.1

PRP PPP

PRP PPP

PRP PPP

5

2O

3O

9.2 1.0

11.7 0.2

26.4 0.0

9.0 0.0

ADP

66.3 5.0

66.5 2.1

55.2 0.0

0.9 0.I

AMP

* Supernatant obtained by centrifugation of platelet-rich plasma.

87.6 0;0

PRP PPP

ATP

R a d i o a c t i v i t y (%)

0

(rain)

Time

1.1 0.2

0.8 0.0

2.2 0.0

0.5 0.0

IMP

17.8 16.9

15.2 12.9

5.7 3.9

1.8 1.7

Ino + hypoxanthine

1.59 0.02

1.99 0.01

2.41 0.01

6.03 0.01

2.50 0.05

2.68 0.01

4.12 0.00

2.75 0.02

(mmol/1011 cells) ATP ADP

Amounts

and the metabolite

--

--

--

313

321

523

2000 --

(cpm/nmol) ATP

345 --

625 --

772 --

364 --

ADP

radioactiv-

OF ADP AND ATP IN PLATE-

Specific radioactivity

[ 14C]Adenine_labele d platelet-rich plasma (PRP) (3.8 • 108 cells; 80 000 cpm/ml) was incubated at 37°C with 135 mM formaldehyde ity and nucleotide concentrations determined in platelet-rich plasma and platelet-poor plasma (PPP) * at the times shown.

THE EFFECT OF FORMALDEHYDE ON THE RADIOACTIVITY LET-RICH AND PLATELET-POOR PLASMA

T A B L E II

cjl bo

53 changes in the radioactive adenine nucleotides. Table II shows that insignificant amounts of [14C]ATP, [~4C]ADP or [14C]IMP were present in the supernatant plasma when aliquots of the platelet-rich plasma/formaldehyde mixture were centrifuged at different stages during incubation. No [~4C]AMP was present in plasma early during incubation, whereas significant amounts (up to 8% of those in the total incubation mixture) were present at the later stages of incubation. The level of radioactive inosine plus hypoxanthine in plasma was practically the same as in the non-centrifuged incubation mixtures. There was no measurable (non-radioactive) ATP or ADP in plasma during incubation of formaldehyde with platelet-rich plasma (Table II). Although the level of ATP radioactivity decreased to less than 1/10 of its original level by incubation of platelet-rich plasma with formaldehyde (Fig. 2, Table II), the chemical amounts of ATP decreased only to about one-fourth of the initial amounts (Table II). Hence, at the end of the incubation, the specific radioactivity of ATP had decreased to 15% of its original value. Both the radioactivity and the amounts of ADP showed a transient increase during the incubation period, but the radioactivity increased more than the amounts. Consequently, the specific radioactivity rose to twice the initial value during the first part of incubation, and thereafter decreased to the initial value at the end of incubation. The amounts of ATP plus ADP decreased only by 50% during incubation (Table II). The above findings show that formaldehyde cannot be used at 37°C to stop secretion rapidly in experiments that involve determination of metabolic adenine nucleotides in platelets. The degree of changes in nucleotide levels induced by formaldehyde could be markedly reduced, but not completely abolished by combining formaldehyde treatment with rapid chilling. After equilibration of '4C-labeled platelet-rich plasma at 37°C aliquots were transferred to tubes kept at various temperatures and containing a small volume of either 0.15 M NaC1 or formaldehyde. After 2 min the mixtures were extracted and the levels of metabolite radioactivity and adenylate energy charge were determined. Results from one experiment are presented in Table III and show that when the platelet-rich plasma was added to formaldehyde i n tubes chilled in ice, the nucleotide radioactivity and adenylate energy charge were practically identical to the control prepared at 37 ° C. The control at 4 ° C, however, had distinctly higher levels of ['4C]ATP and adenylate energy charge and a lower level of [~4C]ADP than the control at 37°C. Table IV shows that platelets isolated by centrifugation of ice-cooled mixtures of [~4C]adenine-labeled platelet-rich plasma and EDTA/formaldehyde followed by resuspension and extraction, had distinctly lower levels of [~4C]ATP and adenylate energy charge than platelet-rich plasma extracted directly or platelets isolated from mixtures of platelet-rich plasma and EDTA/NaC1. When the platelet-rich plasma was briefly pretreated with A23187, a far greater reduction in [~4C]ATP and adenylate energy charge was observed in platelets isolated from mixtures of platelet-rich plasma and EDTA/formaldehyde as well as in platelets isolated from platelet-rich plasma/NaC1 mixtures. The decrease in [14C]ATP was accompanied with increases in [14C]ADP, [14C]AMP and [ ~4C]IMP (formaldehyde) or only [~4C]ADP (NaC1).

54 TABLE III E F F E C T OF T E M P E R A T U R E ADENINE NUCLEOTIDES

ON

THE

FORMALDEHYDE-INDUCED

C H A N G E S IN P L A T E L E T

A l i q u o t s ( 5 0 0 pl) o f [ 1 4 C ] a d e n i n e - l a b e l e d p l a t e l e t - r i c h p l a s m a (3.8 • 108 cells/ml; 80 0 0 0 c p m / m l ) t h a t was w a r m e d t o 3 7 ° C w e r e t r a n s f e r r e d t o t u b e s t h a t w e r e k e p t a t d i f f e r e n t t e m p e r a t u r e s a n d c o n t a i n e d 0.1 m l o f e i t h e r 0 . 1 5 M NaC1 or 8 2 0 m M f o r m a l d e h y d e . T h e m i x t u r e s w e r e e x t r a c t e d b y E D T A / e t h a n o l 2 rain afte~ t h e t r a n s f e r .

Temperature (°C)

Medium

NaC1 Formaldehyde NaC1 Formaldehyde NaC1 Formaldehyde

37 23 4

Radioactivity (%) ATP

ADP

AMP

I M P + Ino + hypoxanthine

83.1 28.5 85.0 42.5 90.5 82.4

11.2 34.6 8.9 34.5 5.9 12.2

0.3 31.4 0.3 16.4 0.0 0.6

5.2 5.6 5.6 5.6 3.6 4.2

Adenylate energy charge

0.937 0.484 0.940 0.639 0.969 0.920

Secretion of ATP, ADP and [14C]serotonin in platelet-rich plasma by A23187 Insignificant amounts of ATP or ADP were present in the supernatants after centrifugation of cold platelet-rich plasma/formaldehyde or plateletrich plasma/NaC1 mixtures (Table V). Considerable amounts of these nucleotides were present in supernatants after centrifugation of platelet-rich plasma that had been exposed to A23187 for a few seconds before dilution in ice-cold NaC1 (Tables V and VI, Fig. 3). The specific radioactivity of these extracellular nucleotides were 15 (ATP) to 25 (ADP) times lower than that of these nucleotides in the control platelet-rich plasma (Table V). When aliquots of the same platelet-rich plasma that had been exposed to A23187 for less than 5 s were diluted with ice-cold formaldehyde before centrifugation, no ATP or ADP was present in the supernatant plasma. This difference between NaC1

T A B L E IV C E N T R I F U G A T I O N O F P L A T E L E T S . E F F E C T O F F O R M A L D E H Y D E A N D A 2 3 1 8 7 ON P L A T E L E T BOUND METABOLIC NUCLEOTIDES P o r t i o n s o f [ 14C] a d e n i n e - l a b e l e d Platelet-rich p l a s m a (3.3 • 108 cells/nil; 78 0 0 0 c p m / m l ) t h a t h a d b e e n or h a d n o t b e e n stirred w i t h 20 #M A 2 3 1 8 7 f o r less t h a n 2 s at 3 7 ° C w e r e e x t r a c t e d d i r e c t l y w i t h E D T A / e t h a n o l . O t h e r p o r t i o n s (0.5 m l ) w e r e m i x e d w i t h 75/~1 of ice-cold 52 m M E D T A / 5 0 m M NaCI or 52 m M E D T A / 1 . 0 3 M f o r m a l d e h y d e a n d c e n t r i f u g e d . T h e cells w e r e s u s p e n d e d in ice-cold 6.7 m M E D T A / 1 3 6 m M NaC1 a n d e x t r a c t e d . Treatment of plateletrich p l a s m a

Extraction

None

A23187

R a d i o a c t i v i t y (%) *

Adenylate energy change

ATP

ADP

AMP

IMP

Direct Cells, NaCI Cells, f o r m a l d e h y d e

81.5 85.9 56.3

11.3 6.2 23.9

1.1 0.3 8.8

0.9 1.3 6.0

0.928 0.962 0.766

Direct Cells, NaC1 Cells, f o r m a l d e h y d e

79.2 52.9 29.1

14.3 13.1 30.1

0.9 1.8 18.3

0.9 20.2 11.2

0.914 0.876 0.569

* I n P e r c e n t o f t o t a l , i.e. cells a n d p l a s m a .

55 TABLE V PREVENTION

BY FORMALDEHYDE

OF THE

ATP AND ADP FROM A23187-TREATED

APPEARANCE

PLATELETS

DURING

IN PLASMA

Portions (0.5 ml) of [14C] adenine-labeled platelet-rich plasma (4.1 s t i r r e d i n t h e a g g r e g o m e t e r . 2 ~zl a c e t o n e ( c o n t r o l ) o r 2 . 5 m M A 2 3 1 8 7 content was immediately transferred to ice-cooled tubes containing NaC1 o r 51 m M E D T A / 9 3 3 m M f o r m a l d e h y d e . A f t e r c e n t r i f u g a t i o n for amounts and radioactivity of ATP and ADP. Sample

Formaldehyde

Total * * *

Amounts

*

ATP

ADP

3.60

1.89

OF NON-METABOLIC

CENTRIFUGATION • 108 cells/ml; 75 000 cpm/ml)were w a s a d d e d , a n d 2 5 0 ~1 o f t h e c u v e t t e 75 pl 51 m M E D T A / 5 0 m M 0 . 1 5 M the supernatant plasma was assayed

Percent extracellular ATP + ADP

Specific radioactivity * * ATP

ADP

--

4485

845

---

---

312 --

32 --

Supernatants Control

-+

0.03 0.02

0.03 0.03

1.1 0,7

A23187

-+

0.39 0.02

0.59 0.02

18.0 0,6

* pmol/1011 platelets. ** I n c p m / n m o l . N o r a d i o a c t i v e A T P , A D P , A M P o r I M P w e r e p r e s e n t i n t h e p l a s m a s u p e r n a t a n t s o f the control. *** Extract of non-centrifuged control platelet-rich plasma.

chilling and formaldehyde chilling was the same whether the samples were centrifuged immediately after chilling or they were kept on ice for 20 min before centrifugation. When [14C]serotonin-labeled platelet-rich plasma was treated briefly with A23187, large amounts of platelet-bound serotonin radioactivity were found TABLE

VI

PREVENTION BY [14C] S E R O T O N I N

FORMALDEHYDE OF BY CENTRIFUGATION

APPEARANCE IN P L A S M A OF ATP OF A23187-TREATED PLATELET-RICH

pLUS ADP PLASMA

AND

T h e procedure was as described in Table IV, except that [14C]serotonin-labeled platelet-rich plasma (3.56 • 108 cells/ml) was used and samples transferred from the aggregometer at the times indicated. Sample

Formaldehyde

ATP + ADP Amounts

Total

*

[ 14C]Serotonin Extrac e l l u l a r (%) **

cpm/ml

--

6782

Extrac e l l u l a r (%)

--

7.64

--

Control

--

0.06

0.0

1470

0.0

A23187, lOs

-+

1.73 0.05

24.5 0.0

3810 1480

44.0 0.5

A23187, 30 s

-+

1.02 0.19

12.6 1.9

2540 1635

20.9 2.3

A23187,

--

100

+

1.79 1.70

25.7 24.0

3890 3800

46.1 43.8

Supernatants

s

* n m o l / 1 0 1 1 platelets. ** See M e t h o d s for calculation.

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Fig. 3. A p p e a r a n c e o f e x t r a c e l l u l a r A T P p l u s A D P a n d s e r o t o n i n r a d i o a c t i v i t y d u r i n g p l a t e l e t a g g r e g a t i o n with A23187. 5 ml of [14C]serotonin-labeled platelet-rich plasma (4.3 • 108 cells/ml; 10.35 #tool ATP plus A D P / 1 0 1 1 cells; 1 4 0 7 8 0 c p m / m l ) w a s s t i r r e d w i t h 1 0 # M A 2 3 1 8 7 in t h e a g g r e g o m e t e r . A l i q u o t s ( 4 5 0 #1) w e r e t a k e n a t t h e t i m e s i n d i c a t e d and a d d e d t o i c e - c o o l e d t u b e s c o n t a i n i n g 7 5 #1 o f 0 . 0 5 NaC1/ 0 . 0 5 M E D T A ( A ) o r 0 . 9 3 M f o r m a l d e h y d e / 0 . 0 5 M E D T A (B). T h e m i x t u r e s w e r e c e n t r i f u g e d and the a m o u n t s o f A T P plus A D P (~) a n d p l a t e l e t - b o u n d s e r o t o n i n r a d i o a c t i v i t y (o) in the s u p e r n a t a n t s w e r e d e t e r m i n e d . T h e s e values axe s u p e r i m p o s e d o n the a g g r e g o m e t e r t r a c i n g .

together with ATP and ADP in the supernatants obtained after centrifugation of chilled NaC1/platelet-rich plasma mixtures, while no radioactivity above control levels were found in supernatants after centrifugation of the chilled formaldehyde/platelet-rich mixtures (Table VI, Fig. 3). When platelet-rich plasma was stirred for 30 s with A23187 at 37°C before chilling with NaC1 or formaldehyde, less ATP plus ADP and [14C]serotonin were present in the supernatants of NaC1/platelet-rich plasma mixtures than when platelet-rich plasma was chilled immediately after exposure to A23187 (Table VI). After the 30 s exposure, small amounts of ATP plus ADP and platelet-bound serotonin radioactivity were present in the supernatants from chilled formaldehyde/platelet-rich plasma mixtures. When platelet-rich plasma was exposed to A23187 for increasing periods of time before chilling, the amounts of ATP plus ADP and [14C]serotonin in the supernatants of the NaCl/plateletrich plasma mixtures eventually became the same or higher than they were by brief exposure of platelet-rich plasma to A23187. By this prolonged exposure of platelet-rich plasma to A23187 the amounts of ATP plus ADP and ['4C]serotonin in the supernatants from the formaldehyde/platelet-rich plasma mixtures also increased and eventually became as large as in those supernatants of NaC1/platelet-rich plasma mixtures. The extent of these changes and the time for them to occur varied with platelet-rich plasma from different donors, b u t the qualitative patterns of the changes were the same (compare Table VI and Fig. 3).

57 40-

-

100

80

3C

f-, < +

50

9 "5

< 2C

~,

,

o 10

2o ~

o

~

' Time (min)

~,

6

Fig. 4. S e c r e t i o n o f A T P plus A D P and changes in [ 1 4 C ] adenine n u c l e o t i d e s during platelet aggregation w i t h A2 3187. 7 ml [14C] adenine-labeled platelet-rich plasma (3.5 • 108 cells/ml, 9.3 #tool A T P plus A D P / 1 0 I 1 cells, 74 000 c p m / m l ) w a s stirred w i t h 10 pM A 23187 in the aggregometer. A t n o t e d t i m e s samples (200 pl) were t a k e n for d e t e r m i n a t i o n o f adenine n u c l e o t i d e radioactivity and samples 4500 #1) were m i x e d w i t h 75 pl 0.93 M f o r m a l d e h y d e / 0 . 0 5 M EDTA in ice, centrifuged and the a m o u n t s o f A T P plus A D P d e t e r m i n e d in the supernatant. The levels o f secreted A T P plus A D P CA), [ ' 4 C ] ATP (e) and [14] IMP plus inosine plus h y p o x a n t h i n e (m) have b e e n s u p e r i m p o s e d on the aggregometer tracing. No changes in the [ 14C] adenine n u c l e o t i d e s occurred w h e n the same platelet-rich plasma was stirred w i t h a c e t o n e (solvent for A23187).

In Fig. 3 the amounts of ATP plus ADP and [14C]serotonin in the two types of supernatants have been plotted against exposure time together with the optical changes in the stirred platelet-rich plasma/A23187 mixture (aggregation trace). When compared to the levels in the NaC1/platelet-rich plasma supernarants, shape change (the transient increase in absorbance immediately following addition of A23187) and aggregation (decrease in absorbance) appeared to be subsequent to the appearance of ATP plus ADP and serotonin extracellularly. In contrast, when compared to the ATP plus ADP and ['4C]serotonin levels in the formaldehyde/platelet-rich plasma supernatants, it appears as if shape change and most of the aggregation takes place before these substances appear extracellularly. The level of ['4C]ATP started to fall 1 min after addition of A23187 to platelet-rich plasma and leveled off when aggregation and secretion were terminated. Secretion was associated with the second phase of aggregation (Fig. 4). ['4C]ATP was recovered as ['4C]IMP and ['4C]inosine plus hypoxanthine (Fig. 4) and variations in the levels of ['4C]ADP, ['4CLAMP and ['4C]IMP took place (not shown in Fig. 4) that were typical for an ATP -~ ADP -> AMP -+ IMP conversion.

58 Discussion At 37°C formaldehyde caused rapid breakdown of [~4C]ATP in platelets previously labeled with [ ~4C]adenine. IMP and inosine plus hypoxanthine were the products at low (10--30 mM) formaldehyde concentrations, while at high concentrations (100--500 mM) AMP was the final product. The formaldehydeinduced conversion of ATP to hypoxanthine was not caused by the corresponding fall in pH or presence of methanol, but was similar to that seen in platelets incubated with metabolic inhibitors [18--20], glyoxylate [21] or H202 [22], and during platelet secretion [2,11,23]. This conversion has been thought to occur by the following pathway: ATP-~ ADP-~ AMP-* IMP-* inosine hypoxanthine + Rib-l-P, involving ATPase, adenylate kinase, adenylate deaminase, 5'-nucleotidase and purine riboside phosphorylase [24]. Since high concentrations of formaldehyde caused ATP -~ ADP -~ AMP conversion only, the initial conversion probably also occurred with low formaldehyde concentrations and the further breakdown of AMP followed the route outlined above. The accumulation of AMP at high formaldehyde concentrations indicated that AMP deaminase was inhibited. When the formaldehyde concentration was increased above 135 mM the rate of ATP breakdown decreased, while ATP, ADP and AMP coexisted in adenylate kinase equilibrium, which indicated that the ATP-consuming process, and not adenylate kinase, became progressively inhibited. In human platelets the major part of the metabolic adenine nucleotide pool is ATP, as exemplified by the [14C]ATP/[~4C]ADP ratio close to 9 in the present study; the storage pool constitutes 50--60% of the total ATP plus ADP with an ATP/ADP ratio of 0.6--0.8 [11,25]. The far greater reduction in the metabolic than in the non-metabolic pool strongly indicates that formaldehyde causes changes primarily in the metabolic pool, and probably leaves the storage pool unaltered. The great fall in the specific radioactivity of ATP and transient increase in that of ADP support this view. Since formaldehyde hardly affected the storage pool or caused extracellular accumulation of [~4C]ATP or ['4C]ADP, its effect on the metabolic adenine nucleotide pool was not due to intracellular damage or general cell lysis. The appearance of [ 14C]inosine plus hypoxanthine extracellularly occurred with and without formaldehyde, and represents accumulation of adenine nucleotide breakdown products [26]. At low formaldehyde concentrations metabolic ATP disappeared at constant adenylate energy charge. This indicates that formaldehyde lowers ATP by increased usage rather than inhibition of regeneration of ATP, which is associated with decrease in both the level of ATP and of adenylate energy charge [18]. The decrease of both the level of ATP and adenylate energy charge with high formaldehyde concentrations was probably due to the complete inhibition of AMP deaminase. There is no change in the level of inorganic phosphate during formaldehyde-induced ATP depletion (Holmsen, H., unpublished), indicating that formaldehyde does not induce simple ATP hydrolysis (ATPase activation) but rather activates ATP-dependent phosphorylation processes. Other experiments (unpublished) have shown that glyoxal and methylglyoxal lower ATP levels similar to formaldehyde, while glycoaldehyde, acetaldehyde and propionaldehyde are without effect. This substrate spe-

59 cificity suggests involvement of formaldehyde dehydrogenase [27,28] in the formaldehyde-induced ATP depletion in platelets. No changes in the [14C]adenine nucleotides were observed when plateletrich plasma was added to chilled (135 mM) formaldehyde, and neither ATP nor ADP were present in the supernatants after centrifugation of such formaldehyde/platelet-rich plasma mixtures. However, marked alterations occurred, however, in the centrifuged cells and this effect was greater with A23187-treated platelets. Treatment of platelet-rich plasma with cold formaldehyde before centrifugation was used in order to prevent centrifugation-induced secretion (see below). Therefore, studies on the relation of A23187-induced secretion to adenine nucleotide metabolism were performed by sampling for the determination of [~4C]adenine metabolites directly from platelet-rich plasma/A23187 mixtures before formaldehyde treatment and centrifugation. Formaldehyde rapidly stops uptake of serotonin by platelets [10]. Electron microprobe examination indicated that other secretable substances [4] of the dense granules, such as Ca 2÷ and phosphate (most probably present in ATP, ADP and pyrophosphate [4]), remained in the cells after the formaldehyde treatment [ 10]. Secretion of serotonin induced by either thrombin or A23187 was efficiently stopped by formaldehyde, which suggests that the fixative does n o t specifically inactivate thrombin or interfere with thrombin receptors, b u t seems to block the transfer of granule-stored constituents to the extracellular space independently of the type of inducer. In the present paper, when platelet-rich plasma stirred with A23187 for a few seconds was added to chilled EDTA/saline and then centrifuged, large amounts of ATP and ADP were present in the supernatant. That these nucleotides originated from the storage pool is indicated by their ATP/ADP ratio of 0.7 and a specific radioactivity 15--25 times lower than that of the ATP and ADP in extracts of control platelet-rich plasma. The appearance in the supernatants of storage pool ATP and ADP was completely prevented by treating the platelet-rich plasma that had been briefly exposed to A23187 with cold formaldehyde before centrifugation. When platelet-rich plasma was stirred with A 2 3 1 8 7 for 30--60 s before chilling in EDTA/ NaCl and subsequent centrifugation, less ATP plus ADP was present in the supernatants; in some platelet-rich plasma there was no difference between such supernatants and those from controls. Since extracellular ATP and ADP do n o t penetrate the platelet membrane [29], the smaller amounts found in the supernatants after longer than after brief exposure of platelet-rich plasma to A23187 could n o t have been due to reuptake by the cells of secreted nucleotides. In addition the absence of ATP or ADP in supernatants from platelet-rich plasma exposed briefly to A23187 and pretreated with formaldehyde show that ATP and ADP could not have been extracellular in the A23187/platelet-rich plasma mixtures before centrifugation. Therefore, centrifugation itself caused secretion of ATP and ADP from platelets exposed briefly to A23187. The behavior of [14C]serotonin radioactivity in the two types of supernatants from A23187treated ['4C]serotonin-labeled platelet-rich plasma was the same as for the storage ATP and ADP. However, secreted serotonin is easily reabsorbed [30]. The difference between the ['4C]serotonin and ATP plus ADP levels in supernatants from EDTA/NaC1 and EDTA/formaldehyde-treated A23187/platelet-rich plasma mixtures decreased as the incubation time of platelet-rich plas-

60 ma with the ionophore increased. At a certain time (1--3 min) which varied among platelet-rich plasma from different donors, the levels became practically identical. A plausible explanation for this behavior is as follows: When plateletrich plasma is stirred with A23187, the platelets are immediately preconditioned for secretion, b u t do not secrete until another stimulus is applied, in our case, centrifugation. The preconditioned state was preserved during cold storage since the same degree of centrifugation-induced secretion was observed with platelet-rich plasma centrifuged immediately as after 20 min of chilling. The secretory response due to preconditioning is completely abolished by formaldehyde and is reversible, since most platelet-rich plasma showed decreased centrifugation-induced secretion after 1 min stirring with A23187. The platelets aggregated during stirring for 1 min, suggesting a connection between aggregation and loss of preconditioning and/or the secretory response to centrifugation. Upon further stirring with A23187 the platelets underwent secretion in platelet-rich plasma and when no difference was observed between supernatants with and without formaldehyde, maximal secretion had taken place before centrifugation. Secretion during centrifugation of platelets has been reported previously [31--35] and was thought to be induced by close cell contact in the presence of extracellular fibrinogen, Ca 2÷ and ADP. The lack of secretion during centrifugation of non-stimulated platelets in the present study was possibly due to the presence of EDTA. The secretion of A23187-conditioned platelets during centrifugation t o o k place in the presence of EDTA, which indicated that a mechanism different from that occurring during centrifugation of non-stimulated platelets was involved. Centrifugation-induced secretion obscures the temporal relation between secretion and aggregation. In Fig. 3A, where centrifugation-induced secretion was not prevented by formaldehyde, shape change and aggregation appear to be a result of secretion. (In fact, the sigmoidal shape of the aggregation curves that frequently were obtained with A23187 (see Fig. 4) strongly resembled the usual biphasic aggregation response to ADP, with secretion occurring during the second phase [11]). In Fig. 3B, where centrifugation-induced secretion is prevented by formaldehyde, it is clear that both shape change and the greater part of aggregation t o o k place before secretion. Thus, A23187 does not cause shape change and aggregation as a result of secreted ADP, b u t induces these platelet responses directly. This is in accordance with the findings [37] that A23187 causes a normal shape change-aggregation pattern in platelets previously depleted for their secretable ADP by thrombin. Metabolic ATP was converted to inosine plus hypoxanthine concurrently with secretion in the A23187-induced shape change -- aggregation --secretion secretion sequence in platelet-rich plasma. The ATP-hypoxanthine conversion is specific for the secretory step in the sequence induced by other agents, i.e. thrombin, collagen, adrenaline, ADP and bovine factor VIII [38]. The lowering of the metabolic ATP level apparently reflects greater usage than production of ATP in the responding platelet [4]. In platelet-rich plasma this lowering was 20% [4], in citrate-washed platelets [39] as much as 50% of the ATP content disappeared, while in gel-filtered platelets [4] only 8% reduction was seen. It appears, therefore, as if the coupling of metabolism to function induced by A23187 is highly dependent on the experimental conditions.

61

Acknowledgements This work was supported by U.S.D.H.E.W. grant No. 5 PI 7HL 14217-05 and was done during the tenure of an Established Investigatorship of the American Heart Association (H. H.). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

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