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Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 3 6 9 - - 3 7 8 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27874

METABOLIC ASPECTS OF THE SECRETION OF STORED COMPOUNDS FROM BLOOD PLATELETS IV. EFFECTS OF IONOPHORE X537A ON WASHED PLATELETS *

E.H. M U R E R , K. D A V E N P O R T a n d H.J. D A Y

SCOR Thrombosis Center, Temple UHSC, Philadelphia, Pa. 19140 (U.S.A.) (Received October 13th, 1975)

Summary 1. X537A at concentrations below 10 pM can liberate platelet serotonin from washed h u m a n platelets without inducing the platelet release reaction. Up to 100% of serotonin preabsorbed by the platelets can be liberated before initiation of the release reaction. 2. Concentrations of X537A above 10 pM initiate the platelet release reaction, with a m a x i m u m release of adenine nucleotides and platelet factor 4 antigen comparable to that obtained with 1.25 units thrombin/ml. 3. The changes in ATP metabolism at the concentration necessary for X537A-induced release are more p r o f o u n d than those in platelets exposed to concentrations of t h r o m b i n or A23187 giving the same degree of release, and approach those seen with high concentrations of A23187. At concentrations where serotonin is liberated but no adenine nucleotide or platelet factor 4 antigen is released, short time incubation causes no change in the level of metabolic ATP. 4. In Na ÷ medium the release increases with prolonged incubation to a level which seems to be characteristic for a particular ionophore concentration. Replacing Na ÷ with K ÷ in the incubation medium produces the m a x i m u m release for a particular concentration after short-time incubation, and no further increase is seen. 5. When platelets are incubated with antimycin and glucose so t h a t anaerobic metabolism is predominant and then exposed to X537A, a marked sparing effect on ATP utilization can be observed in K ÷ medium, and an increased ATP

* Preliminary ~'eports of this study were presented at the 59th A n n u a l Meeting of the Federation of A m e r i c a n Societies for Experimental Biology, Atlantic City, N.J., April 18--18, 1975 (Abstract Fed. Proc. (1975) 34, 289) and the 5th Congress o f the International S o c i e t y o n T h r o m b o s i s and Haemostasis0 Paris° France, July 21--26, 1975 (Abstract No. 169).

370 breakdown in Na* medium. The shift in monovalent cation does n o t affect the a m o u n t liberated and released. 6. We conclude that the increased speed of release induction and of liberation of serotonin reflects a direct effect of the ion change on the ionophore action, possibly b y increasing the speed by which X 5 3 7 A is accumulated in the platelet membrane.

Introduction The ionophores A 2 3 1 8 7 and X 5 3 7 A have both been shown to bind divalent cations in a non-ionic medium although the binding affinities for different cations vary markedly for the two ionophores, decreasing from Ca 2÷ via Mg 2÷ and Sr 2÷ to Ba 2÷ for A 2 3 1 8 7 and from Ba 2÷ via Sr 2÷ and Ca 2÷ to Mg 2÷ for X537A. H o w this difference in binding affinity in a basic organic solvent [1] reflects itself in the ability of the ionophores to transport divalent cations across biological membranes is less well-known. A23187 is the more specific ionophore in that it seems not to be accessible to a number of monovalent cations including K ÷ [2] while in contrast X 5 3 7 A can bind organic monoamines as well as various inorganic cations [1]. X537A, in contrast to A23187, causes release of histamine from mast cells in the absence of Ca 2÷ [3]. Massini and Ltischer [4] and WSrner and Brossmer [5] have shown that X537A causes a liberation of serotonin from platelets at a much higher ratio to released nucleotides than do other release inducers. An intracellular accumulation of IMP is a good indicator for a disturbance in the breakdown sequence from ATP to hypoxanthine [6]. It occurs frequently under conditions where leakage of cytoplasmic material takes place, so that the extracellular IMP concentration can be useful as an indicator for cell leakage [7]. Human platelets secrete adenine nucleotides, serotonin, calcium and other constituents from a storage c o m p a r t m e n t directly into the extracellular medium [ 8 ] , [9] by a process termed the platelet release reaction, or simply release. The only c o m p o u n d in the storage c o m p a r t m e n t which seems exchangeable b y short-time incubation is serotonin [ 1 0 ] . When serotonin is translocated from the storage c o m p a r t m e n t to the extracellular medium without the triggering of the platelet release reaction, we reserve the term serotonin liberation for this process. The liberation of serotonin from platelets may represent a specific transport of this c o m p o u n d through the granule and plasma membrane to the extracellular medium. This assumption will be tested here, along with the effect of incubation with X537A on platelet nucleotide metabolism. The decline in metabolic ATP, liberation of serotonin, release of adenine nucleotides and platelet factor 4 antigen and accumulation intra- and extracellularly of IMP will be used as markers for the effect of X 5 3 7 A on platelet structure, function and metabolism. Materials and Methods Radiochemicals were [3H]serotonin-binoxalate (4.3 Ci per mmole) from New England Nuclear, Boston, Mass. and [U-14C]adenine (281 Ci per mol) from Amersham/Searle Corporation, Chicago, Ill. 2,5-Diphenyloxazole (PPO),

371 bis (2(5-phenyloxazolyl)) benzene (POPOP), antimycin A, Type III and 2-deoxyD-glucose from Sigma Chemical Co., St. Louis, Mo. X537A was a gift from Drs. Zane Gaut and W.E. Scott, Hoffmann-LaRoche, Inc., Nutley, N.J., A23187 from Dr. Robert L. Hamill, Eli Lilly et Co., Indianapolis, Ind. and imipramine from the Ciba-Geigy Corporation, London, U.K. Thrombin was provided by Parke-Davis, Detroit, Mich. Blood from healthy donors who had not used anti-inflammatory drugs during the last 7 days was collected in 11 mM sodium citrate of pH 7.9 (final concr.). The plate-rich plasma prepared by 15 min centrifugation at 250 × g and room temperature was incubated with 3H-labeled serotonin-binoxalate (1 pCi/ml) and 14C-labeled adenine (250 nCi/ml) by shaking 20 min at 37°C in a Dubnoff metabolic incubator (75 strokes/min) after which the pH was adjusted to 6.0 with 0.1 M citric acid to facilitate resuspension of the platelets in the wash medium. The platelets were washed twice in 0.15 M NaC1 containing 11 mM sodium citrate of pH 6.0, and finally resuspended in 0.15 M NaC1 or KC1. The experiments were performed by shaking in a 25 ml Erlenmeyer flask at 37°C a mixture of the platelet suspension (2--4 • l 0 s platelets per ml) with 10 mM sodium citrate of pH 7.9, 25 mM Tris, HC1 of pH 7.4, 2.5 pM imipramine added to prevent reabsorption of serotonin [11] and additions to be tested (total volume 4 ml and NaC1 or KC1 concentration 0.13 M). The flask was then cooled in an icebath and aliquots removed for different tests (the "total incubate" referred to in the tables) after which the remainder of the incubation mixture was centrifuged and new aliquots taken from the supernatant material. To measure 14C-labeled metabolites of ATP, aliquots from the incubation mixture and from the supernatant were mixed with an equal amount of 90% ethanol with 7.7 mM EDTA of pH 7.4. The 14C-labeled metabolites were separated by paper strip electrophoresis [12] and counted in a Beckman LS330 scintillation counter in toluene with PPO and POPOP. 50-pl aliquots for the determination of 3H-serotonin were taken from the supernatant or incubation mixture and added directly to a scintillation fluid which had been mixed 1 : 1 with Triton X-100. The counts in the 3H channel were corrected for spillover of z4C. Adenine nucleotides were determined as 260 nm-absorbing material in the HC104-treated supernatant from platelet incubate by scanning in an Acta III Beckman spectrophotometer, after subtraction of "unspecific" absorption as previously described [6]. Platelet factor 4 was determined by a radial immunodiffusion technique by the courtesy of Dr. S. Niewiarwoski [13]. 100 mg ionophore X537A was stored in 1 ml ethanol/acetone (1 : 1, v/v) at --20°C and the stock solution diluted with ethanol to the desired concentration (0.8--24 mM) which was likewise stored at --20°C. The ionophore was added to the incubation medium in 5 pl volume. Stock solution of A23187 was 8 mM, which was likewise diluted to the desired concentration for 5-pl additions. Results

When washed, [3H]serotonin-labeled human platelets were incubated with the ionophore X537A, radioactive serotonin was recovered in the extracellular medium. The liberation of serotonin was slower than the release induced by thrombin or by the ionophore A23187 (Table I). A23187 released serotonin

372 TABLE I Comparison between extracellular nucleotides and serotonin after exposure of platelets to X537A, A23187 a n d t h r o m b i n . E x t r a c e n u l a r s e r o t o n i n w a s d e t e r m i n e d as t r i t i u m l a b e l in s u p e r n a t a n t as p e r c e n t o f t h a t o f w h o l e i n c u b a t e , A T P a n d ADP by t h e f i r e f l y m e t h o d [ 1 2 ] . (a) A 2 3 1 8 7 a d d e d in t h e c o l d , t h e n i n c u b a t e d f o r 1 0 r a i n a t 3 7 ° C . (b) A 2 3 1 8 7 a d d e d a f t e r 9 r a i n p r e i n c u b a t i o n a t 3 7 ° C , t i m e w i t h i o n o p h o r e 1 rain. W h e n A 2 3 1 8 7 w a s a d d e d w i t h o u t p r e i n c u b a t i o n , t h e release i n d u c t i o n w a s less d e v e l o p e d [ 7 ] . T h e o t h e r a d d i t i o n s w e r e in t h e c o l d , a n d t i m e g i v e n is i n c u b a t i o n t i m e w i t h release i n d u c e r . Addition: Incubation (min): W i t h i n d u c e r (rain): [ 3 H ] S e r o t o n i n (%) ATP (pM) ADP (pM)

None 10 0 4.5 1.0 0.7

X537A 1 1

X537A 3 3

X537A 10 10

A23187 10 lO(a)

A23187 10 l(b)

Thrombin 1 1

4.6 1.0 0.5

22.9 1.0 0.5

56.0 1.0 0.8

47.6 5.1 7.2

81.4 10.0 11.2

83.0 9.2 11.7

and adenine nucleotides in tandem, as did thrombin. 1 gM X537A induced serotonin liberation only (Table I}, b u t increasing the amount of ionophore p r o d u c e d release of nucleotides and of platelet factor 4 antigen (PF4) with a m a x i m u m equal to that observed with high thrombin concentrations at between 20 and 30 pM concentration of X537A (Fig. la). Prolonged incubation increased the efficiency of release induced by 10 pM X 5 3 7 A from insignificant to 70% of maximal. At 3 pM X 5 3 7 A concentration, however, there seemed to be little or no release-induction even with prolonged incubation, although maximum serotonin liberation was obtained. Even with maximal release of nucleotides and PF4 the leakage of cytoplasmic material did not exceed 10% (measured as IMP) when total incubation time was kept to one min. Release was, however, accompanied by a sharp drop in metabolic ATP and irreversible accumulation of IMP (Fig. 1). The changes were also considerably greater than those accompanying one minute incubation with concentrations of A 2 3 1 8 7 below 0.4 pM [7] or with thrombin (a 10% drop in metabolic ATP was found with the same platelet preparation) even though the amounts released were comparable. With longer incubation a drop in metabolic ATP was observed even at concentrations below those necessary for release induction. In contrast the increase in metabolic IMP seemed strictly to parallel the release induction (Fig. 1). Leakage of cytoplasmic material expressed as IMP in the extracellular medium occasionally exceeded 20% with 10 pM or higher concentration of X537A, but only with prolonged incubation (10 min). The degree of leakage varied considerably from preparation to preparation. When K ÷ replaced Na ÷ as the dominant ion in the incubation medium (Table II), shorter incubation time was needed for the same a m o u n t of serotonin liberation or nucleotide release. However, the release or liberation obtained with one specific ionophore concentration seemed to reach the same level, indicating that the ion change influenced the time needed for induction, but not the strength of induction or the efficiency of the induced reaction. The drop in metabolic ATP coincided with the induction of release (Table II). When oxidative phosphorylation was blocked with antimycin and glycolysis stimulated by the addition of glucose, the change from Na ÷ to K ÷ medium did n o t cause a great alteration in metabolic ATP level or adenylate energy charge.

°/o

cl

100-

80

/I / 60

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....

/

20

l

I

3

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20 X 5 3 7 A (~M)

30

b

100-

80

60

40-

20-

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I

10 X 537A

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20 (,.uM)

30

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I00-

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Fig. I . E f f e c t o f d i f f e r e n t X 5 3 7 A c o n c e n t r a t i o n s a n d t i m e s o f i n c u b a t i o n o n p l a t e l e t m e t a b o l i s m , l i b e r a t i o n a n d release. C o n d i t i o n s a r e e x p l a i n 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 . • m, 2 6 0 r u n - a b s o r b i n g m a t e r i a l in t h e p l a t e l e t s u p e m a t a n t as % o f 2 6 0 n m - a b s o r b i n g m a t e r i a l r e l e a s e d w i t h 1 . 2 5 N I H u n i t s t h r o m b i n p e r m l , a n d is a n e x p r e s s i o n f o r t h e a d e n i n e n u c l e o t i d e c o n c e n t r a t i o n i n t h e s u p e r n a t a n t [ 6 ] . • a is p l a t e l e t f a c t o r 4 a n t i g e n as % o f t h a t r e l e a s e d b y t h r o m b i n [ 1 3 ] . o o, [ 1 4 C ] A T P a n d • ...... 0, [ 1 4 C ] I M P a r e g i v e n in p e r c e n t o f 14C l a b e l i n t h e t o t a l i n c u b a t e . ~ . . . . . . A, [ 3 H ] s e r o t o n i n i n t h e p l a t e l e t s u p e r n a t a n t as p e r c e n t o f [ 3 H ] s e r o t o n i n i n t h e w h o l e i n c u b a t e . (a) 1 m i n i n c u b a t i o n w i t h X 5 3 7 A . M e a n o f 3 e x p e r i m e n t s . ( b ) 3 r a i n i n c u b a t i o n w i t h X 5 3 7 A . M e a n o f 2 e x p e r i m e n t s . (c) 1 0 m i n incubation with X537A. Mean of 3 experiments.

374 T A B L E II C o m p a r i s o n b e t w e e n X 5 3 7 A - i n d u c e d s e r o t o n i n liberation and n u c l e o t i d e release in N a + a n d K + m i l i e u . Ino c u b a t i o n m i x t u r e c o n s i s t e d o f either 0 . 1 3 M N a C I ( u p p e r c o l u m n s ) o r 0 . 1 3 M KCI ( l o w e r c o l u m n s ) , the o t h e r a d d i t i o n s w e r e c o m m o n for all e x p e r i m e n t s as e x p l a i n e d i n M a t e r i a l s a n d M e t h o d s a n d legend for Fig. I . M e t a b o l i c [ 1 4 C ] A T P in % o f t o t a l 14C; A N is 2 6 0 n m - a b s o r b i n g m a t e r i a l ( e q u i v a l e n t s o f p.M a d e n i n e n u c l e o t i d e [ 6 ] ) in s u p e r n a t a n t , [ 3 H ] s e r o t o n i n i n s u p e r n a t a n t as percent of [3HI s e r o t o n i n c o n c e n t r a t i o n in t o t a l i n c u b a t e . M e a n o f 2 e x p e r i m e n t s . X537A (pM): Incubation (rain): Dominant

10 1

3 3

10 3

3 1O

10 10

ion Na +

14C in A T P (%) In s u p e r n a t a n t [ 3 H ] S e r o t o n i n (%) A N (juM) Dominant

3 1

79.5

71.8

68.4

25.1

47.8

16.5

16.6 0.3

58.9 1.7

47.4 0.9

94.2 16.7

99.3 3.6

99.8 15.9

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41.8

70,7

28.7

52.1

12.7

66.3 1,4

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93.9 1.3

94.9 14.4

99.9 2.7

102.8 14.2

ion K +

14C in A T P (%) In s u p e r n a t a n t [ 3HI S e r o t o n i n (%) A N (~M)

In the presence of 10 pM X 5 3 7 A the change from Na ÷ to K ÷ medium produced only a slight effect. But when the conditions were combined, the level of metabolic ATP d r o p p e d further in the presence of Na ÷ and increased in the K ÷ medium; the latter indicating a conservation and/or an increased regeneration o f ATP. It also suggested that the ionophore increased the movement of K ÷ to the interior of the platelet, activating glycolysis via the pyruvate kinase system. However, there were no significant changes in the a m o u n t of liberated serotonin or released nucleotides under these circumstances (Table III). Discussion These studies indicate that X 5 3 7 A induces m o v e m e n t of stored c o m p o u n d s from platelets to the exterior medium by two different mechanisms, dependent u p o n the concentration of added ionophore and the monovalent extracellular ion. Lower concentrations of X 5 3 7 A liberate serotonin from human blood platelets w i t h o u t concomitant release of stored adenine nucleotides and PF4. The liberation is not accompanied b y the metabolic changes which in other systems a c c o m p a n y the platelet release reaction. This suggests that the liberation of serotonin occurs through an energy-independent translocation to the extracellular medium. A similar, b u t much slower, liberation of serotonin can be seen when platelets are exposed to reserpine [ 1 5 ] , tyramine [16] or methylene blue [ 1 7 ] . All are thought to affect the uptake or storage of 8erotonin b y the platelet storage granules. Monoamines have been shown to bind very effectively to X 5 3 7 A in a nonpolar medium [1]. That X 5 3 7 A most likely acts as a m o n o a m i n o p h o r e for serotonin is supported b y the latter's very fast liberation -- more than 50% in 1 min with 10 pM X537A. Foreman et al. [3] showed that "release" of hista-

OF CHANGE

III

FROM

Na + TO K + MEDIUM

ON IONOPHORE-TREATED

PLATELETS

WITH BLOCKED

NON-GLYCOLYTIC

METABOLISM

Serotonin A N (/~M)

In s u p e r n a t a n t

(% 3H)

ATP IMP Inosine + hypoxanthine Adenylate energy charge

% 14C i n

In total incubate

Added X537A: Added antimycin and glucose: Dominant ion:

2.3 1.1

2.2 1.7

74.0 2.5 4.7 0.882

0 K+

0 Na ÷

79.4 2.0 3.3 0.908

0

0

2.2 1.3

72.8 2.4 8.2 0.897

+ Na +

0

2.0 2.0

70.9 2.5 11.0 0.896

+ K+

0

100.0 12.0

36.5 29.6 13.8 0.754

0 Na +

+

99.4 13.3

41.7 23.1 11.4 0.750

0 K+

+

99.8 12.9

23.9 37.3 12.1 0.625

+ Na ÷

+

101.0 10.4

53.6 21.0 11.7 0.868

+ K+

+

125 ng antimycin per ml and 10 mM glucose were added to the incubation medium, other conditions are explained in the legend of Fig. 1 and Table II. Adenylate energy charge was calculated by Atkinson's [14] formula: [ATP] + lh[ADP]/[ATP] + [ADP] + [AMP], using the total radioactivity of the adenylates as measure for concentration. AN is 260 nm-absorbing material [6]. Incubation time 3 rain, [X537A] = 10 #M. Mean of 3 experiments.

EFFECT

TABLE

..,,] 01

376 mine from mast cells was induced by X537A in the absence of Ca 2÷ and independent of energy, which suggests an aminophore mechanism. Higher concentrations of X537A induce the platelet release reaction [8] accompanied by a drop in metabolic ATP and build-up of IMP. The metabolic changes are much greater than those observed with concentrations of A23187 sufficient to cause almost maximal release [7]. In some cases there is considerable leakage even when the release reaction is only partially activated. This suggests that X537A is considerably less specific than A23187, and induces a number of intracellular changes in addition to triggering the release reaction -perhaps because of an ability to facilitate movement of other compounds in addition to and partly in preference to Ca 2÷, among them serotonin. Ca 2÷ has been shown to move into platelets exposed to X537A [4], but at high concentrations of the ionophore. We have thus no direct evidence as yet for the participation of calcium in the triggering of the X537A-induced release reaction. Lingjaerde [18] has shown that the uptake of serotonin into platelets is dependent on the presence of Na ÷. It is thus possible that in contrast K ÷ will increase the efficiency of serotonin movement out of the platelets. This should not affect the induction of the platelet release reaction. An involvement of (Na ÷ + K ÷)-ATPase in the uptake mechanism for serotonin is indicated by the fact that this uptake is inhibited by ouabain [19] and accompanied by K ÷ influx [20]. The uptake mechanism is not likely to influence the apparent rate of efflux in our experiments, as they are performed in the presence of imipramine. ADP-induced platelet functions are accompanied by Na ÷ influx supposedly resulting from an inhibitory action on the platelet membrane (Na ÷ + K÷)-ATPase [21]. Moake et al. [22] suggest that such an effect is caused by ADP specifically rather than by a general initiation of platelet function. But it is more likely that the substitution of K ÷ for Na ÷ increases the efficiency of X537A uptake by the cell membrane or its specificity for the parts of the membrane which control ion movements, if ion liberation is indeed the mechanism for the release induction. The main arguments for this are: (1) K ÷ has a similar "speeding u p " effect on both serotonin liberation and nucleotide release indicating that this effect is not specific for the movement of serotonin. But if the movement of serotonin competes with the release-triggering effect of the ionophore, then faster movement of serotonin by K ÷ might free the ionophore for the suggested Ca 2÷ movement at an earlier stage. This could thus support either hypothesis, i.e. that the K ÷ affects either the movement of serotonin out of, or the movement of ionophore into, the platelet. (2) There seems to be a maximal a m o u n t released or liberated for any given ionophore concentration, indicating a saturation point for the induction; thus an exclusive effect on serotonin movement seems unlikely. A slow uptake of the ionophore and a competition between the monoamine movement and the suggested movement of Ca 2÷ may explain the increased induction with time which contrasts strongly the fast release curve seen with most other release inducers (A23187 [7], thrombin, latex particles [23]) whose a m o u n t affects the total a m o u n t released, but not the time course [23]. In this respect the X537A-induced release resembles the release induction by fluoride [6], which also seems to owe its slowness to an impaired membrane penetration (Miirer, unpublished observation).

377 Incubation with X 5 3 7 A in K ÷ medium results after 3 min in considerably higher metabolic ATP concentration and adenylate energy charge than external Na ÷ indicating an effect of monovalent cation on the internal metabolic apparatus. The release is not influenced; hence there is no indication that the K * affects X537A-induced release via glycolysis. The ionophore may cause alterations in the ionic constitution of the membrane itself, with resultant changes in membrane potential or structure (see also ref. 22). Altogether X 5 3 7 A seems to have a more complex m o d e of action than A23187. One metabolic pattern found with other release inducers [6,7] is conspicuously absent: the conversion of initially accumulated IMP to inosine and hypoxanthine. The increase in releasing effect with time may hide it: at the stage where release is being induced, a number of metabolic and ultrastructural changes are already in progress, giving an effect similar to that of high concentrations of A23187 -- namely, a poisoning of the platelet metabolic apparatus, one aspect of which is the irreversible accumulation of metabolic IMP [7]. With X537A we can obtain almost total depletion of platelet serotonin witho u t inducing the platelet release reaction. This may be a useful tool for studying the release of different elements and possibly for determining whether Ca 2÷, ADP, ATP and serotonin are stored in the same or different storage compartments. It is d o u b t f u l that the ionophore can substitute for reserpine in vivo because of the relatively steep concentration curve from effect on serotonin storage to induction of the general platelet release reaction, as well as its lack of specificity which might lead to unfortunate side effects. Still, the obvious advantage of X537A -- namely its fast action compared to other serotonin liberators -- may make it a very useful agent for studies connected with the storage of serotonin in platelets. Acknowledgment This study was supported b y grant No. HL 14217 from the Heart and Lung Institute of the National Institutes of Health, Bethesda, Md. References 1 Pressman, B.C. (1973) Fed. Proc. 32, 1698--1703 2 Reed, P.W. and Lazdy, H.A. (1972) In The Role o f Membranes in Metabolic Regulation (Mehlman, M.A. and Hanson, R.W., eds.), pp. 111--131, A c a d e m i c Press, New York - London 3 Foreman, J.C., Mongsx, J.L. and Gomperts, B.D. (1973) Nature 245, 249--251 4 Massini, P. and Liischez0 E.F. (1974) Biochim. Biophys. Acta 372, 109--121 5 W~rner, P. and Brossmer, R. (1975) Thrombosis Res. 6 , 2 9 5 - - 3 0 5 6 Miirer, E.H., Day, H.J. and Lieberman, J.E. (1974) Biochim. Biophys. A c t a 362, 266--275 7 Miirer, E.H., Stewart, G.J., Rausch, M.A. and Day, H.J. (1975) Thromb. Diathes. Haemorrh. 34, 72--82 8 Holmsen, H., Day, H.J. and Stormorken, H. (1969) Scand. J. Haemat., Suppl. 8 9 Milrer, E.H. and Holme, R. (1970) Biochim. Biophy$. A c t a 261,435--443 10 Minter, B. and Crawford, N. (1967) Biochem. J. 105, 22P--23P 11 Walsh, P.N. and Gagnateni, G. (1974) Blood 44, 157--168 12 Holmsen, H. and Weiss, H.J. (1970) Br. J. Haemat. 19,643--649 13 Niewiarowski, S., Lowery, C.T., Hawiger, J., Minman, M. and Timmons, S. J. Lab. Clin. Med., in the press 14 Atkinson, D.E. (1969) Annu. Rev. Microbiol. 23, 47--68 15 Carlsson, A., Shore, P.A. and Brodie, B,B. (1957) J. Pharmacol. Exp. Therap. 120, 334--339

378 16 Pletscher, A., Bartholini, G. and DaPrada, M. (1966) In Mechanisms of Release of Biogenic Amines (von Euler, U.S., Rosell, S. and Uvn~s, B., eds.), Pergamon Press, Oxford 17 Schick, P. and Yu, B.P. (1973) J. Lab. Clin. Med. 82, 5 4 6 - - 5 5 3 18 Lingjaerde, O. (1969) FEBS Lett. 3 , 1 0 3 - - 1 0 6 19 Barthel0 W, a n d Markwardt, F. (1971) Biochem, Pharmacol. 20, 2597--2601 20 Born, G.V.R. (1967) J. Physiol. 190, 273--280 21 Feinberg, H., Scorer, M., LeBreton, G., Grossman, B. and Born, G. (1974) Circulation 49 and 50, III--277 (ahs.) 22 Moake, J.L., A h m e d , K., Bachur, N.R. and Gutfreund, D.E. (1970) Biochim. Biophys. Acta 211, 337--344 23 Miirer, E.H. (1968) Biochim. Biophys. Acta 162, 320--326

Metabolic aspects of the secretion of stored compounds from blood platelets. IV. Effects of ionophore X537A on washed platelets.

1. X537A at concentrations below 10 muM can liberate platelet serotonin from washed human platelets without inducing the platelet release reaction. Up...
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