Release-induced changes in adenine nucleotide metabolism of human blood platelets

XiROMBOSIS RESEARCH 15; 309-318 Pergamon Press Ltd.1979. Printed in Great Britain

RULEAS%INDUCED CHANGES IUADENIUENUCIiSCTIDE METABCLISM OF HUHAH BLCCD PLATELETS I. VOW Pusch and W. Wesemann Institute of Physiological Chemistry

Philipps University MarburR. Germany (Received 30.12.1978; in revised form 9.2.1979 Accepted by Editor N. Goossens) ABSTRACT Human platelets incybated in platelet-rich lasma (PRP) accumulated [ 4C]5-~dmxytryptamine P S-ET) yi;h a K of 2.3juM and [ Huenine with a Kt of

3reincubationwith 1.8/W adenine decreased 5:P &ake by 2096.The incorporatedadenine was mainly converted to ATP and ADP. Tyramine, tryptamine, imipramine,reserpine, 1-aminoadamantane(D 1) snd its 3.5.dimethylderivative (D 145) feleased more than 6596of previously accumulated [ 4C]5-HT. In addition D 145, reserpine,andimipramine also reduced SndOgenOUS ATP and ADP within platelets by more tha?+4C96part of which was recovered extracellularly. In[ 4C]adenine_labeledplatelets the same 3 drugs cause a marked decrease in adenylate energy charge and [04C]ATP which was metabolised mainly to hypoxanthine + inosine. The results indicate that release of S-UT plus ATP/ADP from platelet storage organelles requires energy which is supplied by the metabolically active adenine nucleotide pool whereas 5-HT alone is liberated rather by passive transport.

According to Holmsen et al. (1,2) at least two different adenine nucleotide poola are found in human platelets. One compartment is labeled when platelets are incubated with radioactive adenine or adenosine. However, on treatment with aggregating ents like thrombin (3), collagen (l), AI@ or adrenaline (4 Y unlabeled ATP and ADP are released from the platelets. These metabolicallyinert nucleotides are stored together with S-UT and calcium in a second pool (5). Specific subcellular organelles, very eleotron-densegranules, were identified as storage compartment (6,7) whereas the non-

309

310

RELEASE

FRO31 PLATELET

ORGGELLES

i’ol

.l?, so.

3/b

releasable,metabolically active pool of labeled AT?/ADP aontaining about l/3 of totsl platelet adenine nucleotides is localized in mitochondria,membranes, and cytoplasm (1,2). Though during platelet aggregation radioactivelylabeled ATP is not found in the extracellularmedium, the amount of metabolically active ATP within the platelets decreases (1,2,3,4). Since during ag regation ATP is degraded to hypoxanthine Holrasen et al. 7 1,4,5) suggest that part of the metabolically active ATP (the release energy pool) delivers the energy required for the "release reaction", the secretion of compounds from the storage organelles. To test the assumption that the secretion of metabolically inert ATP/ADP from the storage organelles requires energy, in the present paper the adenine nucleotide metabolism of human blood platelets is studied after the addition of drugs which induce the release reaction without concomitant aggregation. Hence changes which occur in the adenine nucleotide metabolism of human blood platelets during the release reaction can be separated from possible shifts in the nucleotide pattern during aggregation. The effect of substances which liberate only 5-HT is compared to that of compounds releasing 5-HT as well as ATP/ADP from platelets. I'IATERIA3.S AND METHODS 5-Hydroxy [side chain-2-14C tryptamine creatinine [14C]5-RT),54mCi/mmol; [8-_$ H] adenine, 25 Ci/mmol; 61 mCi/mmol: Amersham Buchler, Braunschweig. -Aminoadamantane*HCl(D 1); I-amino-3.5-dimethyladamanfiane. HCl (D 145): Merz, Frankfurt/Main.Imiprsmine (Tofranil ); reserpine: Geigy, Basel. Firefly desiccated tails (lanterns): Sigma, St. Louis, MO. Pyruvate kinase (PK) from rabbit muscle (EC 2.7.1.40): Boehringer, Mannheim. Platelet-richPlasma (PBPl was prepared from venous blood of althy donors as ciescribedby von Pusch et al. (8). Platelet c&nt was obtained after fixation in 0.35% formaldehyde/3.8% trisodium citrate (8). Uptake of 114C15-ET and [3dadenine was vsayed after incubation of 3 ml aliquots of PW (2-5.5 x 10 platelets/ml)with of the labeled compound at 37OC. The reaction was termi30 naped by cooling and centrifugationat 1500 g 10 min, 4OC. After washing and recentrifugationthe platels'were lysed with 0.01 E HCl and the radioactivity was measured by liquid scintillationspectrometry as described previously (9). In some experiments the uptake of labeled compounds into platelets was calculated from the difference of the radioactivity found in PRP and platelet-poor plasma PPP obtained after ) these cases 0.5ml centrifugationat 1500 g 10 min, 46C. In of PRP or PPP were absorm'by glass fibre paper, Whatqn GF/A, prior to radioactivitymeasurement. nine metabolites. After enine m/crCifI), 2 h,

vol.l3,xo.3/~

RELEASE

FROM PLATELET

ORGANELLES

311

37'C, platelet adenylate kinase was inactivated by 0.01 M Nap HDTA (10) prior to centrifugationand ethanol extraction of the platelets (9). Separation was achieved by high-voltage electrophoresison Whatman 3 MM paper with 0.05 M citrate buffer, pH 3.8, at 50 V/cm, 75 min, using 5O/ul of the ethanolic extract mixed with 5 ul of buffer containing 90 nmoles each of ATP, ADP, IMP, fnosine, hypoxanthine,and adenine as carriers (11). tabolites)_ was studied 1 '+CJ5-HT.12.5 /uCi/l, stop the reaction'3 ml or 3.0-$?4 [jH]adenine,0.15 mCi/l. samples were centrifuged and the platelets washed and reauspended in 2 ml of Gaintner medium, pH 7.4, (12) prior to incubation with the release-inducingagents dissolved in 0.15 M NaCl or 96% ethanol (reserpine).After the release reaction platelets were lysed for radioactivitymeasurement as described above. In order to study changes in adenine nucleotide metabolism due to e release reaction PHP was incubated for 2 h with 50.5p 11%C]adenine,278pCi/l, prior to the drug-induced relea8e. Ethanolic extracts for electrophoreticseparation were prepared as already described. Release of endogenous ATP and ADP was studied in ethanolic extracts of platelets which had seen incubated in PRP or Gaintner medium, pH 7.4, with the release-inducingdrugs for 30 or 60 min at 37OC and also in the supernatantsobtained by centrifugation.ATP and ADP were determined with the luciferinluciferase assay according to-Holmsen et al. (10) modified by Wesemann et al. (9). ADP was converted to ATP by pyruvate kinaae and phosphoenolpyruvate(10). The activity of LDH (L-Lactate:NAD oxidoreductase,EC 1 1 27) was assayed according to Bergmeyer and Ber& (13) iA Ehd supernatantsobtained after centrifuging treated PHP. UltraSOniCatedPHP Served as control. RESULTS

used t Calculate the transport constants and uptake rates per 108 platelets; 5-HT: Kt= 2.3/u~, v = 2.5 nmoles/h; V max= 3.6 nmoles'pp(Fig.2). adenine: Kt= l.lp, Simultaneousincubation of PPP with adenine and 5-HT did not affect the uptake of either compound. However, when PRP was preincubatedfor 45 min in the presence and absence of adenine a 2096decrease of 5-HT incorporationwas observed in the adenine-treatedsamples (Fig. 3). After 2 h incubation of PRP with[14Cjadeninethe metabolism of adenine was followed in platelets and PPP by high-voltage

312

RELEASE FRO?4PLATELET ORGXNELLES

Vol.lj,No.3/i

FIG. 1 Time dependence of 5-HT and agenine uptake uman blood plateizs. PRP was incubated gt 37 C with 2.1 /uM CJ5-HT ----a) and 1.8 [ H ladenine ( ?? respectively. Uptake was calculat pd from the difference of radioactivity found in PRP and PPP and corrected for uptake at 0 min incubation time. The mean of 4 - 5 experiments is given.

iim0

FIG. 2 Fig.

(mlnl

FIG. 3

2. Lineweaver-Burk plot of 5-HT (6----d ) and adenine (a---e) uptake by human blood platelets. Uptake was calculated as described in the legend of Fig. 1 after 60 min incubation at 37OC. Mean of 5 experimenta.

Fig. 3. Influence

of adenine on 5-HT incorpration into hflan 1 C] 5-HT platelets. PRP was incubated at 37 with 2.1 or after 4 f” min preeither simultaneously (0-O) [ 3HJadenine. Adenine) with 1.8 incubation (e-0 free controls were obtained wit Y (&-----a) and with45 min preincubation.Uptake was Calout (b -----A) culated as indicated on Fig. 1. The mean of 5 experiments is given.

RELEASEFROMPLATELET ORGAXELLES

Vo1.15,No.3/4

313

TABLB1 [14C]Adenine

Platelets

Metabolites in HumanBlood and Platelet Poor Plasma (PPP), E % 1

ATP

HYP+I

Ade

PPP

0.10

0.06

0.08 LO.01

0.09

2.84

fJ.03

~0.25

96.83 217.55

;;;;e-

73.40 21.74

12.19 21.30

3.32 20.33

3.38 20.54

5.46 ~1.25

2.25 20.63

LO.04

20.03

[ 14CJadenine. PPP was incubated for 2 h at 37’C with 50.5 The labeled compounds in platelets and PPP (?ere separated by high-voltage electrophoresis at pH 3.8. The total radioactivity recovered from the electropherogram was taken 100%. The mean of 6 experiments 2 SEMis given. paper electrophoresis. The incorporated adenine was mainly converted to ATP, 7396, and to a lesser degree to ADP, 12% (Table l . Per log platelets a total of 3.8 + 0.525 (SBM) nmoles [ 14C]ATT4 0.641 2 0.015 nmoles [ ‘4C]Abp, and 0.204 2 0.033 nmoles [ CJAHPwere found. The adenylate energy charge (14) was calculated as 0.887 + 0.009 (SBM). In the PPP besides unmetabolized adenine (96.8fl about 3% hypoxanthine + inosine were found, which were not separated under the electrophoretic conditions employed.

genie amines tyramine and tryptamine, the I-aminoadamantanes D 1 and D 145, and the psychotropic d#gs imipramine and reserpine (Table 2). In addition to [ C]5-HT release, D 145, imipramlne, and reaerpine decreased the concentration of endogenous ATP and ADP within the platelets by more than 40% as determined by the lueiferin-luciferase assay. However, only part of the ATP and ADP which disappeared from the cells could be deteoted in the extracellular medium (Table 3), suggesting intracellular catabolism of adenine nucleotides. A time-dependent release of radioact vity was observed when platelets preincubated with 3/W [3Hf adenine were incubated at 37’C with D 145, imipramine, or reserpine. After 60 min incubation 5 mMD 145 liberated 8896, 0.5 mMimipramine 48%. and 50 reserpine 35% of the incorporated radioactivity. Sample$Mincubated without drugs served as control (100%). No significant release of radioactivity was observed after treatment wfth tyrsmfne, tryptamlne, or D 1. Bigh-voltage electrophoresis of extracts from platelets treated with D 145, reserpine, and imipramine shows that the observed decrease of radioactivity can be attributed mainly

TABizl2 _ Drug-Induced Decrease in ['4CJ5_RT, ATT and ADP Content of Human Blood Platelets Cone.

mM

[14C]5-HT

ATP

Decrease ( % 1 LDH ADP

Tyramine

5.0

71 2 2.2

5 2 2.3

5 + 2.7

1.8 2 0.8

Tryptamine

5.0

88 2 3.8

11 2 4.4

8 + 4.5

1.0 2 0.5

Dl

5.0

66 z 5.7

1.6

2 _c 1.5 3.8 + 1.0

D 145

5.0

88 2 1.0 87 2 1.3

82 2 1.1 4.5 2 0.3

82 2 3.1

57 + 4.3 4.1 + 1.0

Imipratuine 0.5 Reserpine

3 2

57 2 4.2

0.05 69 2 3.0 43 2 9.4

40 + 4.4

1.5 2 0.5

PRP was incubated for 60 min at 37'C with the release-inducing drugs prior to ATP/ADP assay with luciferin-luciferasein washed platelets. In untreated platelets a total of 58 + 1.5 nmoles ATP and 33 + 1.5 nmoles ADP was found per 109 blzod platebets. - Br 5=HT analysis PRP was incubated for 60 min at 37 C with 2.1 [14Cj5-HT prior to drug treatment. - LDH was assayed in tYe supernatant;ultrasonicatedPRP served as control (100%). - The mean of 6 - 8 experiments2 SEX is given.

to a decrease in ATP and, to a lower degree, in ADP concentration within the platelets (Fig. 4). Only small changes were found regarding the levels of the monophosphatesand hypoxanthine + inosine. In the extracellularmedium, however, high concentrationsof hypoxanthine + inosine were detected reflecting the loss of intracellularadenine (metabolites). Substances,~hiohrelease only 5-HT but no ATP and ADP do not liberate [ Cjadenine metabolites significantlyand induce at most small changes in he adenine nucleotide pattern of platelets prelabeled with [t4CJadenine. Thus the concentrationof labeled ATP is diminished by 2.3% if platelets are incubated e.g. for 45 min with 5 mM tyramine. Contrasting with the rather high decrease of the energy charge after concomitant 5-HT and ATP release (D 145: 0.31, imipramine: 0.13 reduction) tyrsmine diminished the control value of 0.887 only by 0.04. To test the integrity of the plateletaLDH activity was assayed in the plasma of drug-treatedPRP. As compared with the control obtained after 20 set ultrasonicationof PRP only up to 4.5% of platelet LDH activity was released after 60 min incubation.

RELEASE

Vol.lg,xo.3/t

FROM

PLATELET

ORCAlriLLES

313

Drug-InducedRelease of AT? and ADP from Human Platelets Recovery in extracellyular medium Cone. mM

D 145

5.0

Imipramine 0.5 Reserpine

0.05

ATP nmoles/lOg platelets

46 ’

ADP nmoles/109 platelets

54

12

24

12

43

10

30

9

50

7

28

6

43

The total amount of ATP and ADP, respectively,which disappears in the platelets, is taken 100 %.

DISCUSSION According to Holmsen et al. (1,2) l/3 of whole platelet ATP belongs to the metabolic pool in the cytoplasm. The studies descr'bed in this paper show that from 58 nmoles ATP present in 104 human blood platelets 3.8 ninolesare labeled after 120 min incubationwith [ Cjadenine. This corresponds to about 20% of the metabolicallyactive ATP assuming that exogenous adenine is incorporatedand metabolized only in the cytoplasm of the platelets (1,2,8). According to this hypothesis the metabolicallyinert adenine nucleotide compartt of the storage organelles which can be labeled with ?a C I5-HT (7,8) cannot incorporate exogenous adenine. In accordancewith this view the present study shows that substances like imipramine, reserpine, and D 145, which in high concentrationsinduce the release reaction without ag regation, liberate radioactivelylabele 5-HT and unlabeled ATP$ADP from platelets preincubatedwith I4Cj5-HT and [3H]adenine.This finding indicates that these substances deplete the inert ATP storage compartment.The inhibitory action of D 145 on platelet aggregation,which has been shown previously (15), supports this conclusion.However, low concentrationsof reserpine (16), imipramine (16), and D 145 (15) and substances like tryptamine, tyramine, and 1-aminoadamantaneliberate only 5-HT from blood platelets without appreciable effect on adenine nucleotide metabolism and release. On the contrary, substanceswhich release 5-HT and ATP/ADP also decrease labeled adenine nucleotides in the cytoplasm considerably, though no significantamounts of labeled nucleotides can be detected extracellularly.Labeled adenine nucleotides are not released but metabolized intracellularlymainly to hypoxanthine which passes the platelet membrane and appears extracellularly. To give one example: 5 mM D 145 decreases total platelet ATP

RELEASE

FRO?1 PLATELET

IM~PRAM~NE

ORGASELLES

RESERPINE

Vol.lj,?io.3/~

NRAMINE

_m%

I

-00

40

-20

ADPINP ANPnypyP

*DT

INP AMP Npp ATPAOP

INPANPN@

FIG. 4 Adenine nucleotide pattern of human blood platelets after 45 min incubationwith 5 mM D 145, 0.5 mM imipramine, 50 reserpine or 5 mM t ramine. PRP was preincubated for 2 h ac"M 37'C with 50.5/W 1s 4C]adenine, 278 Ci/l, followed by drug treatment ( ) and high-voltage p%per electrophoresisof r4c EDTA-ethanol xtracts of washed platelets. The total radioactivity recovered from the electropherogramof the untreated control (0) was taken to be 100%. The values represent the mean of 2 - 3 experiments. by 87% after 60 min incubation (Table 2) and degrades 95% of labeled ATP correspondingto 31% of total ATP. Thus, it can be calculated that 56% of whole platelet ATP is released though only 21% can be found extracellularly.Using other release-inducingsubstances like imipramine or reserpine, a difference was always observed between the amount of ATP and ADP lost from the platelets and the amount found in the supernatant. Besides spontaneoushydrolysis (10 ensymatic breakdown, e.g. by ATPases present in plasma (21 and platelet membrane (17), may account for diminished extracellularadenine nucleotide concentrations. The experiment8 described in this paper show that intracellular breakdown of metabolically active ATP occurs concomitantly with the release reaction even if no platelet ag regation is observed. This finding confirms the suggestion of Holmaen

vo1.15,No.3/4

RELEASE FROM PLATELET ORGANELLES

317

(5) that energy is required mainly for the extrusion process. Apparently less energy is necessary for 5-HT liberation which is achieved rather by passive transport and displacement than by an active extrusion of granule contents. The small changes of the adenylate energy charge observed after 5-m liberation as compared with the rather drastic decrease of the energy charge after ATP/ADP plus 5-HT release support this view.

Further work has to elucidate the mode of inhibition of 5-HT uptake by adenine. Drummond and Gordon (18) explained the observed inhibitory action of ADP on 5-HT uptake into rat blood platelets by inhibition of platelet membrane ATPase and resuiting energy deficiency. ACKNOWLEDG~TS The authors wish to thank Miss Ina Buschmeyer for skillful technical assistance. - This work was supported by the Deutache Forschungsgemeinschaft,Sonderforschungsbereich103 "Zellenergetik und Zelldifferenzierung".

1.

HOLMSEN, H., DAY, H. J., and STORM, E. Adenine nucleotide metabolism of blood platelets. VI. Subcellular localization of nucleotide pools with different functions in the platelet release reaction. Biochim. Biophys. Acts 186, 254, 1969.

2. HOLMSEN, H.,and DAY, H. J. Adenine nucleotides and platelet function. Ser. Haemat. 3, 28, 1971. 3. IRELAND, D. M. Effect of thrombin on the radioactive nucleotides of human washed platelets. Biochem. J. 105, 857, 1967. 4. HOLMSEN, H., DAY, H. J., and SETKOWSKY, C. A. Secretory mechanisms: Behaviour of adenine nucleotidea during the platelet release reaction induced by adenosine diphosphate and adrenaline. Biochem. J. 129, 67, 1972. 5. HOLMSEN, H. Biochemistryof the platelet release reaction. In: Biochemistryand pharmacology of platelets. Ciba Found. Sympos. 35. Amsterdam-Oxford-NewYork: Elsevier/Excerpta Medics/North-Holland,1975, p. 175. 6. DA PRADA, M., TRANZER, J. P., and PLETSCHER, A. Storage of 5-hydroxytryptaminein human blood platelets. Experientia (Baeel) 28, 1328, 1972. 7. FUKAMI, M. H., BAUER, J. S., STEWART, G. J., and SALGANICOFF,L. An improved method for the isolation of dense storage granules from human platelets. J. Cell. Biol. 77, 389, 1978.

315

a.

RELEASE FRO>1PWTELET

ORGANELLES

Vol.lj,Xc.3/t

VON

PUSCH, I., WSSSMANN, w., and ?AUL, N. Biochemical the intracellulardistribUtiOn Of 5-hydroxytryptsmineand adenine in human blood platelets. Cytobiologie (accepted for publication). and. autoradiographic studies on _ _-_ _

9.

WBSBMANN, W., VON PUSCH, I., and PAUL, N. 5-Hydroxytryptamine- and ATP-storing organelles in nucleated thrombocytes.Morphologicaland biochemical studies on spindle cells of the duck. Cytobiologie (submittedfor publication).

IO.

HOLMSEN, H., STORM, E., and DAY, H. J. Determinationof ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma. Anal. Biochem. 46, 489, 1972.

11.

HOLMSEN, H. and WEISS, H. J. Hereditary defect in the platelet release reaction caused by a deficiency in the storage pool of platelet adenine nuoleotides. Brit. J. Haemat. s, 643, 1970.

12.

GAINTNBR, J. R., JACKSON, D. P., and MAYNERT, E. W. The action of thrombin on platelet 5-hydroxytryptamine.Bull. Johns Hopkins Hosp.

13.

BERGMEYER, H. U. and BERNT, E. W-Test mit Pyruvat und NADH. In: Methoden der enzymatiechenAnalyse, Band I. H. U. Bergmeyer (Ed.) Weinheim: Verlag Chemie, 1970, p. 533.

14.

ATKINSON, D. E. and WALTON, G. M. Adenosine triphoaphate conservationin metabolic regulation. J. Biol. Chem. 242, 3239, 1967.

15. HAACKE,

U. and WRSEMANN, W. Induction of the platelet release reaction by 1.3.dimethyl-5_aminoadamantane, a new $mannne derivative. Thrombos. Haemostas. (Stuttg.)g7, ,

16.

.

PAASONEN, M. K. Release of 5-hydroxytryptaminefrom blood platelets. J. Pharm. Pharmac. l7, 681, 1965.

17. MOAKJZ,

J2 I,.,AHMED, K., BACHUR, N. R., and GUTFREUND, D. E. Mg +-dependent, (Na++ K+)-stimulatedATPase of human platelets: properties and inhibition by ADP. Biochim. Biophys. Acta 211, 337, 1970.

18.

DRUMMOND, A. H. and GORDON, J. L. Uptake of 5-hydrowtryptamine by rat blood platelets end its inhibition by adenosine 5*-diphosphate.Brit. J. Pharmac. 56, 417, 1976.