45

Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 4 5 - - 5 5 © Elsevier Scientific 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 27839

INHIBITION OF PLATELET AGGREGATION BY ACYL-CoA THIOESTERS

C H U Y U A N LIN *, B E R T R A M L U B I N a n d S T U A R T S M I T H **

Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center of Northern California, Fifty-First and Grove Streets, Oakland, Calif. 94609 (U.S.A.) ( R e c e i v e d N o v e m b e r 5th, 1 9 7 5 )

Summary The aggregation of platelets induced by ADP, collagen, or epinephrine in human platelet-rich plasma is inhibited by long chain acyl-CoA thioesters. Palmityl-CoA exerts a concentration dependent inhibition of collagen-induced aggregation and of the primary and secondary waves of ADP-induced aggregation. Palmityl-CoA also inhibits the secondary wave of epinephrine-induced aggregation but has no effect on the primary wave. The inhibitory effect of palmitylCoA can be reversed by addition of excess ADP and cannot be attributed to a detergent action.

Introduction The involvement of ADP in platelet aggregation has been acknowledged for over a decade [1], although the exact mechanism is incompletely understood. Boullin et al. [2] were the first to demonstrate that ADP is actually bound to the platelets during the aggregation process, and that the aggregation response is correlated with the platelet-bound ADP. We have been intrigued by the possible analogy between the ADP-receptor system of platelets and the ADP translocase system of mitochondria. The mitochondrial ADP translocase system is known to be inhibited by acyl-CoA thioesters [3--10] ; we have therefore investigated the effect of these compounds on the aggregation of human platelets.

Experimental Procedure Materials Acyl-CoA derivatives (10 : 0, 12 : 0, 14 : 0, 16 : 0 and 18 : 0) were prepared * F e l l o w o f t h e Bay Area Heart A s s o c i a t i o n . * * Established Investigator o f t h e A m e r i c a n Heart A s s o c i a t i o n .

46 from both the N-hydroxysuccinimide esters [11,12] and the acyl chlorides [13]. Acyl-CoA derivatives 18 : 1, 18 : 2 were prepared from their appropriate acyl chlorides. Acyl-CoA concentrations were assayed by measuring with 5,5-dithiobis (2-nitrobenzoate), the sulfhydryl groups [14] released following mild alkaline hydrolysis, or by measuring the extinction at 232 and 260 nm. Acyl-CoA derivatives were stored in water at pH 6. ADP, obtained from Calbiochem, was dissolved in veronal buffer pH 7.35. ADP concentration was determined by extinction at 260 nm. Veronal buffer was prepared by dissolving 11.75 g sodium diethyl barbiturate and 14.6 g sodium chloride in water. The pH was adjusted to 7.35 with HCI and the volume was made up to 2 1. Epinephrine solution was freshly prepared for each experiment by diluting ampoules of adrenalin chloride solution (Parke-Davis) with 0.9% sodium chloride solution to the desired concentration. Bovine achilles tendon collagen was obtained from Sigma. Acid soluble collagen was prepared by adding 1 g of crude collagen to 8.35 mM acetic acid (100 ml) and homogenizing in a blender at 0°C [15]. Cold water (100 ml) was added to the blender and the mixture was rehomogenized. Finally 16.7 mM acetic acid (800 ml) was added and the mixture rehomogenized. The resulting sol was stored at --20°C in small aliquots. Stock solution is 1 mg/ml. Immediately before use the collagen was rehomogenized with a teflon pestle-glass homogenizer and diluted to the desired concentration with 16.7 mM acetic acid. Palmityl-CoA was purchased from P-L Biochemicals and the potassium salt of atractyloside from Sigma. All other reagents were of the highest grade commercially available.

Methods (i) Isolation of platelet rich plasma and platelet poor plasma. Platelet rich plasma and platelet poor plasma were isolated from healthy laboratory volunteers. One part of 3.8% trisodium citrate was mixed with 9 parts of fresh human venous blood. Platelet rich plasma was obtained by centrifugation at 200 X g for 10 min at room temperature. After removal of platelet rich plasma, the lower phase was centrifuged at 1200 X g for 15 min at room temperature to obtain the platelet poor plasma. Platelets were counted under a phase contrast microscope. (ii) Platelet aggregation studies. Platelet aggregation studies were performed using a Bio/Data platelet aggregation profiler model PAP-2A. In operation, 0.05 ml of effectors (or 0.05 ml water for controls) and 0.1 ml of 0.9% NaC1 were added to 0.3 ml of platelet rich plasma or platelet poor plasma in a small flatb o t t o m e d test tube. A small magnetic bar was added to the platelet rich plasma tube. The platelet rich plasma and platelet poor plasma tubes were inserted into their respective channels. The turning speed of the magnetic bar in platelet rich plasma channel was about 1 000 rev./min. The temperature was maintained at 37°C in both platelet rich plasma and platelet poor plasma channels. After incubation for 2 or 3 min. 0.05 ml of ADP, collagen or epinephrine solution was added to the platelet rich plasma tube. The final volume was 0.5 ml and the platelet rich plasma tube contained 1.2--1.5 X 108 platelets. The differ-

47 ential amplification and automatic recorder calibration of the aggregometer result in the presentation of the transmittance of the platelet rich plasma relative to the platelet poor plasma in such a manner that the initial transmittance corresponds to a value of 9 arbitrary units on the recorder. The change in relative optical density over the first 24 s was taken to represent the initial rate of aggregation. The instrument can be adjusted to provide either a filtered or unfiltered trace of the oscillations observed in aggregating patterns. Results

The effect of palmityl-CoA on platelet aggregation. The aggregation response induced by 1 pM ADP in the absence of palmityl-CoA is shown in Fig. 1A. At this concentration of ADP the initial aggregation wave is so fast that the second wave is barely perceptible. In the presence of 10 pM and 20 pM palmityl-CoA, the magnitude of the first wave is reduced considerably and the second wave is clearly visible (Fig. 1B and C). In the presence of 50 ttM palmityl-CoA, the rate and extent of platelet aggregation is decreased, no second wave is observed, and most of the platelets disaggregate within 2 min (Fig. 1D}. Higher concentrations of the acyl-CoA inhibit the aggregation response more severely; almost no aggregation is seen in the presence of 150 pM palmityl-CoA. The effect of palmityl-CoA on the initial aggregation rate was measured from the charts shown in Fig. 1, and the results are shown in Fig. 2. The initial aggregation rate was inhibited 50% by 60 pM palmityl-CoA. The inhibitory effect appears to be attributable specifically to acyl-CoA and not to any impurities in the acyl-CoA preparation. This fact was ascertained by synthesizing palmityl-CoA via two different intermediates (palmitylchloride and palmityl N-hydroxysuccinimide) and purifying the product; the palmityl-CoA prepared by both procedures, in addition to palmityl-CoA obtained from a commercial source inhibited platelet aggregation to equal extents. We next determined whether palmityl-CoA also inhibits collagen induced platelet aggregation. The effect of palmityl-CoA on platelet aggregation induced by collagen is shown in Fig. 3. The rate and extent of platelet aggregation were both decreased as the palmityl-CoA concentration was increased. The effect of palmityl-CoA on epinephrine induced aggregation was quite distinct from the effect on aggregation induced by ADP and collagen (Fig. 4). As the palmityl-CoA concentration was increased from 10 to 90 pM, there was an increased lag period before the second wave of aggregation reached maxim u m rates. When the palmityl-CoA concentration was increased to 150 pM, the second wave of aggregation was completely inhibited: addition of more epinephrine (0.27 ttmol) to this reaction mixture had no effect, however, the addition of ADP (10 nmol) elicited immediate aggregation. This observation suggested that the action of palmityl-CoA on the platelets was reversible as the inhibitive effect could be overcome by excess ADP. Comparison of the effects of various acyl-CoA 's on platelet aggregation To determine whether there were any specific structural requirements for acyl-CoA's to function as inhibitors of platelet aggregation, we compared the effect of a number of CoA derivatives on ADP-induced aggregation (Table I).

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49 0.6

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For the homologous series of even carbon number acyl-CoA derivatives C~0 : 0 to C~ s : 0, the effectiveness as an inhibitor increased with increasing chain length. An increase in the degree of unsaturation decreased the inhibitive potency.

The effect of various compounds

on

ADP-induced platelet aggregation

To ascertain whether the inhibitory effect of acyl-CoA's on platelet aggregation might result from hydrolysis of its thiolesters to CoA and subsequent breakdown of CoA to AMP and adenosine, we compared the effect of acylCoA with that of CoA (Table II). CoA inhibited the rate of aggregation by only

TABLE

I

THE EFFECT

OF VARIOUS

COENZYME-A

DERIVATIVES

* ON ADP-INDUCED

** PLATELET

GREGATION

C o n d i t i o n s as described in E x p e r i m e n t a l Procedure. Acyl-CoA

None CI0:0 C12:0 Cl4:0 C16:0 Cl8:0 C18:1 C18:2

Initial rate of aggregation

E x t e n t o f aggregation

Relative A A/MIN

% Control

Total relative* * * change

% Control

0.552 0.436 0.407 0.317 0.285 0.135 0.236 0.394

100 79 74 57 52 25 43 71

0.298 0.208 0.176 0.138 0.114 0.032 0,094 0.182

100 70 59 46 38 11 32 61

* A c y I - C o A concentration, 1 0 0 ~tM. ** A D P concentration~ 2/~M. *** The maximum relative Absorbance change from 0 to 3 rain after the addition of A D P .

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51

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10%, considerably less than palmityl-CoA. It seemed highly unlikely, therefore, that the inhibitory effect of palmityl-CoA could result from conversion to CoA. A number of other structurally related compounds, acetyl-CoA, N A D and N A D P had no effect on the rate of ADP-induced aggregation. As has been reported by other investigators, adenosine was found to be a potent inhibitor. In view of the fact that palmityl-CoA is an anionic detergent, we considered the possibility that the effect on platelet function might result from a disruption of the platelet structure. Deoxycholate, another anionic detergent, had no effect on platelet aggregation, however, rendering this possibility highly unlike-

52 T A B L E II THE EFFECT ADP *

OF V A R I O U S A G E N T S ON R A T E OF P L A T E L E T A G G R E G A T I O N

I N D U C E D BY

C o n d i t i o n s as d e s c r i b e d in E x p e r i m e n t a l P r o c e d u r e . Addition

% Control rate

None CoA, 50/~M A c e t y l - C o A , 50 pM P a l m i t y l - C o A . 50 pM A d e n o s i n e , 50 pM N A D , 50 ~M NADP. 130/~M D e o x y c h o l a t e , 2 0 0 pM * * A t r a c t y l o s i d e , 3 0 0 pM

i00 90 I00 58 11 100 100 96 1 O0

* A D P c o n c e n t r a t i o n , 1.0 pM. ** A D P c o n c e n t r a t i o n , 2.0 ~M.

ly. (Table II). The observation that the structural requirements of acyl-CoA for inhibition of platelet aggregation are the same as those required for inhibition of the mitochondrial adenine nucleotide translocation system, prompted us to investigate this analogy further. We tested the effect of atractyloside, an inhibitor of the mitochondrial translocase [8] on ADP induced aggregation of platelets. No inhibition was observed (Table II). Similar results were obtained when aggregation was induced with collagen and epinephrine. It is conceivable, however, that the spatial arrangement of the ADP receptor system in platelets is such that it is inaccessible to atractyloside. Discussion

The experiments described in this paper demonstrate that the aggregation of human platelets, induced by ADP, collagen and epinephrine is inhibited by acyl-CoA thioesters. Long chain acyl-CoA esters have been shown to inhibit numerous enzyme reactions because of the detergent nature of these compounds [16,17]. For this reason we have carefully considered the possibility that the inhibitory effect on platelet aggregation might be attributable to a detergent effect of the acyl-CoA's. Evidence which suggests that this is not the case, is summarized below: (i) The inhibitory effect of palmityl-CoA on platelet aggregation is completely reversed by the addition of excess ADP (Fig. 4). (ii) The action of acyl-CoA on platelet aggregation is selective in nature. Whereas the aggregation induced by ADP and collagen can be completely inhibited by palmityl-CoA, only the second wave of epinephrine induced aggregation is inhibited by acyl-CoA; the first wave of epinephrine induced aggregation is completely unaffected. (iii) Whereas lauryl-CoA (200/~M) inhibited 50% of the aggregation induced by 2/~M ADP, sodium deoxycholate (200/~M), a powerful anionic detergent, had no effect. (iv) High concentrations of proteins such as albumin usually afford a protective

53 effect against the disruptive action of detergents [5]. Therefore, the high concentration of albumin in the platelet rich plasma (3 g%} used in our experiments might be expected to minimize any detergent effects. Thus the evidence points overwhelmingly to a specific effect of acyl-CoA rather than to a detergent-like action. The differential effect of acyl-CoA's on aggregation induced by ADP and collagen on the one hand, and epinephrine on the other is intriguing and warrants some discussion. When platelet aggregation is induced by ADP, two distinct waves of aggregation can be seen; the first is due to the ADP added, and the second is due to the release of endogenous ADP by the aggregated platelets. If the ADP added is sufficient to aggregate most of the platelets, then a second wave may not be observed, although ADP is still released by the aggregated platelets. Thus in Fig. 1, in the absence of acyl-CoA, the ADP concentration is apparently high enough to aggregate most of the platelets, the second wave is only just perceptible. As the concentration of palmityl-CoA is increased, however, the second wave, induced by the ADP released from aggregated platelets now becomes more prominent. As the acyl-CoA concentration is further increased, the magnitude of the second wave is also reduced, since fewer platelets aggregate in the first wave and thus less ADP is released. In our experiments we have consistently observed a biphasic response to collagen. Immediately on addition of collagen, there is a small decrease in optical density, then a lag period of about 30 s before the main wave of aggregation. Day and Holmsen also made the same observation [15]. However, the small initial decrease of optical density can be reduced considerably if the soluble collagen was diluted with 139 mM sodium chloride in 16.7 mM acetic acid and kept at room temperature before the addition to the platelet rich plasma. This small initial wave was not inhibited by acyl-CoA thioesters. That the pattern of aggregation induced by epinephrine is classically biphasic, was confirmed in our own studies (Fig. 4}. However, only the second wave of aggregation is inhibited by acyl-CoA, the first wave is not altered. This would seem to indicate then that ADP is not involved in the primary aggregation induced by epinephrine. However, the situation is not quite so clear-cut. Izrael et al. [18] have used two systems to destroy ADP (creatine phosphate plus creatine phosphokinase, and apyrase) in an attempt to establish the role of ADP in platelet aggregation. They found that both waves of aggregation induced by epinephrine were abolished in the presence of the creatine phosphokinase system. In the presence of apyrase, however, only the second wave was inhibited. We have confirmed both of these observations in our laboratory. Thus the inhibitor studies with acyl-CoA and the experiments with apyrase would support a non-involvement of ADP in the primary epinephrine induced aggregation, whereas, the experiments with creatine phosphokinase suggest that ADP is involved. It has been demonstrated that epinephrine can potentiate the aggregation of platelets induced by ADP [19,20]. It is conceivable, therefore, that epinephrine might render the platelets more sensitive to trace amounts of ADP in the platelet-rich plasma: according to McPherson et al. [21], the concentration of ADP in plasma of normal heparinized blood is 0.08 pM. However, if this were the case, it is difficult to imagine why this small quantity of available ADP could be removed by creatine phosphokinase but n o t by apyrase,

54

since the Km of the former enzyme for ADP is an order of magnitude higher than that of the latter [22,23]. Thus, whether the primary wave in epinephrine induced aggregation is due to epinephrine directly, or whether ADP is involved in some way, cannot be judged with certainty at present. The inhibitory action of acyl-CoA on platelet aggregation apparently does not involve hydrolysis to free fatty acid and CoASH, since we found CoASH itself does not inhibit platelet aggregation significantly. The sodium salts of a number of fatty acids actually have been found to induce platelet aggregation [24,25]. However, we cannot rule out the possibility that the inhibitory effect is mediated by transfer of the acyl group to some other acceptor molecule. Inhibition of aggregation by acyl-CoA's depends on certain structural characteristics of the thioesters. Thus, long chain acyl-CoA's are more effective than short chain, and saturated CoA thioesters are more effective than unsaturated. It is significant perhaps that these are the same structural requirements which are associated with the inhibition of adenine nucleotide translocation in liver mitochondria [8,10]. The inhibition of adenine translocation in liver mitochondria by acyl-CoA's has been attributed to a competition by acyl-CoA for the ADPbinding site [8]. We have indeed performed experiments with human platelets which showed that the uptake of radioactivity following incubation of platelets with [U-14C] ADP is inhibited by palmityl-CoA. However, in view of the controversy surrounding the question of whether such experiments reflect the binding of ADP or its metabolites (contrast ref. 2 with refs. 26 and 27), we cannot say with certainty whether the mechanism of inhibition by acyl-CoA involves direct competition for the ADP binding site. The inhibition of adenine nucleotide translocation in mitochondria by acylCoA's has been implicated in a regulatory mechanism for mitochondrial metabolite transport. Whether these compounds play a role in platelet function has yet to be determined. Acknowledgements We would like to thank several of our colleagues for their generous donations of blood and Ms. Sue Fujimura and Ms. Klara Kleman for their services in drawing the blood samples. We would also like to thank Drs. S. Abraham and Byron Smith for their encouragement during the course of this investigation. The Merck Institute provided some financial support. References 1 Gaarder, A., Jonsen, J., Laland, S., Hellem, A. and Owren, P.A. (1961) Nature ( L o n d o n ) 192, 531-532 2 Boullin, D.J., Green, A.R. and Price, K.S. (1972) J. Physiol. 221,415---426 3 Shug, A.L., Lerner, E.0 Elson, C. and Shrago, E. (1971) Biochem. Biophys. Res. C o m m u n . 4 3 , 5 5 7 - 563 4 Lerner, E., Shug, A.L., Elson, C. and Shrago, E. (1972) J. Biol. Chem. 247, 1513--1519 5 Pande, S.V. and Blanchaer, M.C. (1971) J. Biol. Chem. 246, 402---411 6 Vaartjes, W.J., Kemp, A., Jr., Souverijn, J.H.M. and Van Den Bergh, S.G. (1972) Fed. Eur. Biochem. Soc. Lett. 23, 3 03--308 7 Harris, R.A., Farmer, B. and Ozawa, T. (1972) Arch. Biochem. Biophys. 150, 199--209 8 Shrago, E., Shug, A., Elson, C., Spennetta, T. and Crosby, C. (1974) J. Biol. Chem. 249, 5269--5274

55

9 Devaux, P.F., Bienveniie, A., Lauquin, G., Brisson, A.D., Vignais, P.M. and Vignais, P.V. (1972) Biochemistry 14, 1272--1280 10 Ho, C.H. and Pande, S.V. (1974) Biochim. Biophys. Acta 369, 86--94 11 LaPidot, Y., Rappoport, S. and Wioman, Y. (1967) J. Lipid Res. 8, 142--145 12 AI-Arif, A. and Blecher, M. (1969) J. Lipid Res. 10, 344--345 13 Seubert, W. (1960) Biochem, Prep. 7, 80--83 14 Ellman, G. (1959) Arch. Biochem. Biophys. 82, 70--77 15 Day, H.J. and Holmsen, H. (1972) Annu. Clin. Lab. Sci. 2, 63--74 16 Taketa, K. and Pogell, B.M. (1966) J. Biol. Chem. 2 4 1 , 7 2 0 - - 7 2 6 17 Pande, S.V. and Mead, J. (1968) J. Biol. Chem. 243, 6 1 8 0 - - 6 1 8 5 18 Izrael, V., Zawilska, K., Jaisson, F., Levy-Toledano, S. and Caen, J. (1974) in Platelets (Baldini, M.G. and Ebbe, S., eds.), pp. 187--195, Grune and Stratton, New York 19 Mills, D.C.B. and Roberts, G,C.K. (1967) J. Physiol. 193, 443--453 20 Ardlie, N.G., Glew, G. and Schwartz, C.J. (1966) Nature (London) 212, 415--417 21 McPherson, V.J., Zucker, M.B., Fricdberg, N.M. and Rifkin, P.L. (1974) Blood 44, 411--425 22 Molnar, J. and Lorand, L. (1961) Arch. Biochem. Biophys. 93, 353--363 23 Eppenberger, H.M., Dawson, D.M. and Kaplan, N.O. (1967) J. Biol. Chem. 242, 204--209 24 Haslam, R.J. (1964) Nature (London) 202, 765--768 25 Kerr, J.W., Piffle, R. and Tanos, B. (1965) Lancet 1, 1296--1299 26 Salzman, E.W., Chambers, D.A. and Neri, L.L. (1966) Thromb. Diath. Heamorrh. 15, 52--68 27 Ireland, D.M. and Mills, D.C.B. (1966) Biochem. J. 99, 283--296