Vitamin E inhibits the release of calcium from a platelet membrane fraction in vitro

Prostaglandins

and

Medicine

2:

203-216,

1979

VITAMIN E INHIBITS THE RELEASE OF CALCIUM FROM A PLATELET MEMBRANE FRACTION IN VITRO A.M. Butler, J.M. Gerrard, J. Peller, S.F. Stoddard, G.H.R. Rao, J.G. White. University of Minnesota Health Sciences Center, Departments of Pediatrics and Laboratory Medicine and Pathology, Box 446 Mayo Memorial Building, Minneapolis, Minnesota 55455 USA (Reprint requests to JMG). ABSTRACT Vitamin E, an inhibitor of platelet aggregation, was evaluated for its effects on platelet intracellular calcium flux. These studies used a platelet membrane fraction containing membranes of the dense tubular system which actively After these membrane sequesters calcium in the presence of ATP and magnesium. vesicles have accumulated calcium, the cation can be released by addition of the calcium ionophore A23187. Vitamin E had no effect on uptake of calcium by the membrane vesicles, but showed a concentration dependent inhibition of the release of calcium induced by A23187. In similar or slightly higher concentrations than inhibited calcium release,vitamin E also inhibited platelet aggregation, internal contraction and secretion, but had no effect on prostaglandin and thromboxane synthesis and potentiated phospholipase A2 activity. It is suggested that vitamin E acts to inhibit platelet internal contraction and secretion by preventing efflux of calcium from the dense tubular system. The potentiation of phospholipase A2 by vitamin E could be explained by a localized increase of calcium at the site of the phospholipase A2 on the inner side of the dense tubular system membrane proximal to the vitamin E block. INTRODUCTION Vitamin E (a-tocopherol) is a required nutrient first identified by Evans and Bishop in 1922 (1) which has been a focus of interest as a result of clinical trials which suggest that it may be effective in reducing the incidence of certain thrombotic disorders including intermittent claudication (2-6), cerebral arteriosclerosis (7,8), and possibly angina and coronary heart disease (9-13). As a result, intense interest has focused on the mechanism of action of this compound which is widely believed to act as an antioxidant to protect polyunsaturated lipids from destructive oxidation (14). In support of this concept, deficiency of this vitamin in premature infants can lead to anemia with the erythrocyte showing an unusual sensitivity to in vitro auto-oxidation (15,16), and evidence has been presented that such infants also show a tendency for their platelets to synthesize increased prostaglandins (a form of oxidized fatty acid) in response to aggregating agents (17). However, vitamin E deficient animals and humans have other characteristic features including 203

degeneration of somatic muscle tissue (18) which have yet to be clearly related to lipid peroxidation as measured by such techniques as malondialdehyde formation (19). An alternate hypothesis of vitamin E action has been put forward suggesting that it plays a role in stabilizing membranes (20) and in support of this concept is evidence that vitamin E deficiency is accompanied by an impairment of intestinal and hepatic cell integrity (21-24), and by increased fragility of lysosomes (25,26). A cell which has been the focus of several recent studies into the mechanism of action of vitamin E is the platelet. These studies have shown that vitamin E can inhibit platelet aggregation stimulated by a variety of agents when the vitamin E is administered in vitro or in vivo (27-30). Evaluation of the action of vitamin E showed that the vitamin inhibits aggregation by arachidonic acid even though the arachidonic acid is converted to prostaglandins and thromboxanes (31-33), suggesting that vitamin E inhibits at a step after prostaglandin and thromboxane synthesis has occurred. Other studies suggest vitamin E suppresses prostaglandin synthesis in response to aggregating agents other than arachidonic acid (29), and when coupled with the apparent lack of effect on arachidonic acid conversion to prostaglandins and thromboxanes, these findings suggest an effect of vitamin E at a step before synthesis of prostaglandins and thromboxanes occurs. Recent studies have demonstrated that calcium regulates prostaglandin synthesis in platelets at a step preceeding formation of endoperoxides and thromboxanes - activation of the phospholipase A2 to cleave arachidonic acid from membrane phospholipids (34-37). Calcium flux in platelets also serves as the final common pathway for triggering activation of the contractile protein system resulting in internal contraction (34,38). Vitamin E could exert its inhibitory influence on platelets by suppressing calcium flux rather than actThe present investigation has examined this concept ing as an anti-oxidant. in detail. METHODS Preparation of the Platelet Membrane Fraction A platelet membrane fraction which actively sequesters calcium was prepared using a modification (39) of the methods of Robblee (40) and Kaser-Glanzmann (41). Blood was drawn from the antecubital vein of normal donors after obtaining informed consent according to the declaration of Helsinki, and mixed inmediately with citrate-citric acid dextrose, pH 6.5, to achieve final concentrations of 9.3mM sodium citrate, 0.7mM citric acid, and 0.14 M dextrose. For some experiments, platelet concentrates less than 12 hours old, obtained from the St. Paul Regional Red Cross were used. Platelet rich plasma (PRP) was separated by centrifugation at 100 g for 20 minutes at room temperature. Ethyleneglycol-bis (B-amino-ethyl ether) N,N'-tetra-acetic acid (EGTA) was then added to the PRP to a final concentration of 26mM and the platelets were pelleted by centrifugation at 4°C for 10 minutes at 1500 g. The platelets were then washed twice using 0.15 M NaCl containing 13mM EGTA, and then 0.15 M NaCl without EGTA. The washed platelets were resuspended in 3 mls homogenizing medium (30mM KCl, 2.5mM MgC12, 1OmM potassium oxalate, 20mM Tris-HCl, 1OmM Pipes pH 7.0), frozen-thawed and sonicated under nitrogen. The resulting platelet homogenate was centrifuged at 13,000 g for 10 minutes at 4°C and the 204

The pellet (membrane) supernatant centrifuged at 40,000 g for 60 minutes. fraction was resuspended in homogenizing medium at a protein concentration of Calcium uptake experiments were performed at 37OC approximately 1.5 mg/ml. using a shaking incubator to agitate the incubation mixture during uptake and Uptake was started by adding 1 volume of the membrane susrelease studies. pension to 9 volumes 8; an incubation mixture containg 1OOmM KCl, 2.5mM MgC12, 20mM Tris-HCL, 10 uM CaC12 (specific activity 5 uCi/umol), 2mM ATP, and 1OmM Pipes buffer pH 7.0 and vitamin E or soybean oil in 10 ul ethanol where inTo assess release of calcium f m inside the membrane vesicles, the dicated. membrane fraction was equilibrated with zg CaC12 in the presence of ATP for 10 minutes, so that the vesicles could accumulate calcium, before addition of A control to which buffer alone was added, was the calcium ionophore A23187. studied in parallel to each experimental sample. 0.5 ml aliquots from the incubation mixture were removed just before and at various time points after the addition of the A23187 or buffer, and filtered through a millipore sampling unit using 0.22 u pore size filters. Filters were air dried, solubilized in 10 mls aquasol, and counted in a Tri-carb liquid scintillation counter. Calculation of calcium uptake by the membrane fraction was done allowing for endogenous calcium in the membrane fraction suspension which raised the total calcium contentration in the final incubation solution to about 60 uM (40i5 Calculation of the release of calcium was done by dividing the amount of Calcium remaining in the vesicles at 2 or 5 minutes after A23187, by the calcium content of the vesicles just before addition of the addition of the A23187. To obtain a final figure for calcium release, the per cent release of a control sample to which buffer alone had been added was then subtracted. Statistical comparison was done using the Students t test. The protein concentration of the membrane fraction was determined by the method of Lowry (42). Release ofl-14C-arachidonic acid from platelet phospholipids was measured using,! modification (34) of the method of Bills et al (43). Conversion of the 1- C-arachidonic acid to hydroxy fatty acids and thromboxane B2 was evaluated as described previously (34) employing thin layer chromatographic separation of the methyl esters on silica gel G using the organic layer of 100:100:50 iso-pctane: water:ethyl acetate as the eluting solvent (system A). Conversion of l- 4C-arachidonic acid to thromboxane B2 was also evaluated by thin layer chromatography of the free acids on silica gel G using diethyl ether:methanol: acetic acid (135:5:3 v/v) as the eluting solvent (system B). Aggregation and electron microscopic studies of platelets were performed as described previously (44,45). Vitamin E, as a pure oil (Sigma Product No. T3634) or as a 70% solution in ATP as the Na salt, and Pipes were soybean oil (Sigma product No. T3251), obtained from the Sigma Chemical Company (St. Louis Missouri). A23187 was a generous gift from Dr. Hamill, Eli Lilly (Indianapolis, Indiana). 45CaCl2 was obtained from New England Nuclear (Boston, Mass), l-14C-arachidonic acid from New England Nuclear (Boston, Mass). The vitamin E in soybean oil (Sigma product No. T3251) was diluted one part to nine parts ethanol. The pure vitamin E was used dissolved in ethanol, or where indicated in soybean oil (2 volumes vitamin E to one volume soybean oil) and then diluted in ethanol as above.

205

RESULTS 1)

The influence of vitamin E on calcium uptake

When the platelet membrane fraction was incubated with 45Ca in the presence of ATP and Mg, the membrane vesicles took up an average of 123 + 35" nanomoles calcium per mg protein in 10 minutes (n = 23). Addition of TTitaminE at a concentration of 0.56mM to the membrane vesicles and comparison with con;r;'h;ptake showed that uptake was 99.6 + 7.6 % of control (n = 3), and with . calcium uptake was 104.4 + 9.2 % of control (n = 18). These values are no; significantly different from control levels of uptake, and show that vitamin E has no significant effect on calcium uptake at these concentrations. 2)

The influence of vitamin E on calcium release from the vesicles

Addition of the calcium ionophore A23187 at a concentration of 10 uM to the membrane fraction which had taken up calcium for 10 minutes caused a significant release of calcium from the vesicles. This release was 32.7% at 2 minutes and 47.8% at 5 minutes. Incubation of the membranes with vitamin E caused a concentration dependent inhibition of A23187 induced calcium release (Table 1). Since these studies were done using vitamin E which was solubilized in soybean oil, we felt it essential to be sure that the effects seen TABLE 1. THE INFLUENCE OF VITAMIN E ON CALCIUM RELEASE FROM A PLATELET MEMBRANE FRACTION STIMULATED BY A23187 Number of Experiments 2 min. 10 PM A23187

13

45Calcium Release (Per cent of control)

2 min. 5min

5 min. 8

Per cent Inhibition

32.7 +2.5*

47.8 + 6.4

Vitamin E O.llmM + A23187 10 uM

4

27.7 24.3

ND

15

-

Vitamin E 0.23nt+l + A23187 10 uM

5

17.0 + 0.9

ND

48

-

Vitamin E 0.56mM + A23187 10 uM

4

11.8 + 3.0

ND

64

-

Vitamin E l.lmM + A23187 10 uM

8

3

12.0 +2.0

14.0 + 6.2

63

71

Soybean oil + A23187 10 ?JM

6

6

28.8 t1.8

50.3 24.2

12

0

3

3

11.6 + 3.4

9.5 +4.1

65

80

Pure vitamin E 1 1mM in So bean o;l + A23187 10 uM *?tSE -

206

were not due to the presence of the soybean oil or to a contaminant in the soybean oil. Pure vitamin E dissolved directly in absolute ethanol did have some inhibitory effect on calcium release, but it was relatively insoluble and tended to precipitate out of solution when added to the incubation medium. We therefore dissolved pure vitamin E in soybean oil and compared this to soybean oil alone and to the original vitamin E in soybean oil. Soybean oil by itself was completely ineffective in inhibiting the release of calcium stimuPure vitamin E dissolved in soybean oil at a concentration lated by A23187. of l.lmM caused 65% inhibition of calcium release as stimulated by the calcium ionophore A23187 which was as effective as the vitamin E in soybean oil used in the initial experiments (63% inhibition). Therefore, vitamin E appeared to be the active agent in causing inhibition of calcium release. 3) The influence of vitamin E on the transformation of arachidonic acid by platelets l-14C-arachidonic acid was added to washed platelets and its conversion to 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2 was assessed in the presence and ab. sence of vitamin E (Table 2). Vitamin E inhibited by 70% the aggregation induced by arachidonic acid in this experiment but had no effect on the transformation of arachidonic acid. Since other prostaglandins co-migrate with the TABLE 2. THE INFLUENCE OF l.lmM VITAMIN E ON THE CONVERSION OF 14C-ARACHIDONIC ACID (14C-AA) TO METABOLITES

BY WASHED PLATELETS

Per cent of Product

Washed platelets + 14C-AA

AA

HETE

HHT

Thromboxane B2

a)

3.7

52.3

21.0

23.0

b)

3.6

52.1

21.6

22.6

a)

3.6

52.4

20.7

23.2

b)

5.1

55.0

19.7

20.2

Washed platelets + l.llT+l yi;a;Ln E +

thromboxane B2 , on solvent system A, one half of the reaction mixture from each of the above experiments was extracted into diethyl ether and the relative production of prostaglandins and thromboxane B2 assessed using a different system (B). In each case a single peak was seen in the prostaglandin region corresponding to authentic thromboxane B2. The size of this peak accounted for essentially all the radioactivity in the thromboxane B2 region seen on system A. Thus vitamin E had no inhibitory effect on the thromboxane synthetase enzymes or on the cycle-oxygenaze enzyme, even though it did inhibit the 207

platelet aggregation stimulated by this agent. 4)

The influence of vitamin E on phospholipase A2 activity

Vitamin E was evaluated for an effect as an inhibitor of phospholipase A2 activity stimulated by the calcium ionophore A23187 and thrombin. Vitamin E, in each case, failed to inhibit release of 1-14C-arachidonic acid from platelet phospholipids even though it markedly inhibited aggregation and secretion of serotonin (Tables 3 and 4). Indeed vitamin E enhanced phospholipase A activity, an effect which was particularly apparent at low concentrations o? W&in. TABLE 3. THE INFLUENCE OF VITAMIN E ON PLATELET AGGREGATION,

PHOSPHOLIPASE A2

ACTIVITY AND 14C-SEROTONIN SECRETION STIMULATED BY A23187 Per Cent Aggregation

Platelets + 10 PM A23187 Platelets + vitamin E 2.3mM + 10 JJM A23187 *

Per Cent 14Carachidonic acid release from platelet phospholipids

Per Cent serotonin secretion

100

18.7 +0.5*

56.0 + 4.0

10

22.1 + 1.8

20.2 f. 8.1

Results are the mean + SE of 4 experiments.

Conversion of released arachidonic acid to HETE, HHT and thromboxane B2 also remained intact in vitamin E treated samples. 5)

The influence of vitamin E on platelet ultrastructure

Vitamin E alone added to platelets at concentrations up to2.3rrftland incubated with the platelets for up to 30 minutes had no effect on platelet ultrastructure as reported previously (46). The effect of vitamin E on the ultrastructure changes produced when A23187 was stirred with platelets on an A23187 by itself produced a concentration deaggregometer was next examined. pendent internal contraction as described previously (44). Internal contraction was characterized by centralization of the granules surrounded by a band Incubation of the platelets of microtubules and contractile microfilaments. with 2.3mM vitamin E for 15 minutes before addition of the A23187 inhibited both internal contraction and platelet-platelet stickiness (aggregation) stimulated by the A23187. The degree of inhibition of internal contraction and aggregation depended on the concentration of A23187. At a threshold concentration of A23187 just sufficient to cause platelet aggregation (5-13 uM depending on the experiment), vitamin E completely inhibited the internal con(Figure 1). At higher concentrations of A23187 the traction and aggregation ionophore was able to partially overcome the inhibitory effect of vitamin E and internal contraction and partial platelet aggregation could be identified.

208

TABLE 4. THE INFLUENCE OF VITAMIN E ON THROMBIN-INDUCED

AGGREGATION,

PHOSPHOLIPASE A2 ACTIVATION, AND METABOLISM OF RELEASED ARACHIDONIC ACID Percent aggregation

Percent Percent of released release of 14C-arachidonic acid 14C-arachidonic converted to from platelet HETE & HHT ThromboaeB2 phospholipids

Thrombin 1 U/ml

100

17.9, 16.5

43

11

Vitamin E 2.3mM + thrombin lU/ml

100

20.4, 20.5

45

12

Thrombin 0.1 U/ml

100

Vitamin E 2.3mM + thrombin 0.1 U/ml Thrombin 0.01 U/ml

5.0 +_ 0.4 (3)*

N.D.

35

12.1 + 2.6 (3)

N.D.

100

3.0 f_ 2.8 (8)

N.D.

Vitamin E 2.3rrfyI + thrombin 0.01 U/ml

0

13.3 51.8

(8)

36

Vitamin E 2.3mM

0

0.5 LO.2

(5)

N.D.

24

* j;+ SE; the number in parenthases represents the number of experiments. N.D. = not done DISCUSSION The present investigation has sought to elucidate the mechanism of action of vitamin E by combining studies of calcium flux, arachidonic acid metabolism and electron microscopy. The results demonstrate that vitamin E can inhibit the release of calcium from a platelet membrane fraction rich in elements of the dense tubular system which actively accumulates this cation. In addition, the results confirm earlier results of others (31) that in spite of markedly inhibiting aggregation, vitamin E had no effect in vitro on the conversion of arachidonic acid to HHT and thromboxane B2, the principal products of the active endoperoxides and thromboxane A Release of calcium from the dense tubular system to initiate intracellulgr processes is believed to be the final common pathway through which many aggregating agents including arachidonic acid and A23187 act (47-49). Inhibition of this release by vitamin E, could therefore adequately explain the ability of vitamin E to block platelet aggregation without inhibiting the conversion of arachidonic acid to prostaglandin endoperoxides and thromboxane B2. Intracellular platelet calcium flux is believed to play an important role in at least two separate processes - phospholipase A2 activation and internal contraction (34-37, 44). The present investigation has evaluated the influence of vitamin E on these two processes to determine which was dependent on efflux of calcium from the dense tubular system. Vitamin E was found to inhibit internal contraction stimulated by A23187 (and also the secretion produced by

209

Figur!&!2 Electron micrographs of platelets stirred with 10 urnA23187. Plate lets ln A were incubated for 15 minutes with 2.3mM vitamin E in 10 ul ethanol, where!as pl atelets in B were incubated for 15 minutes with 10 ul ethanol only. Vitamin E treated platelets remained discoid with randomly dispersed organelles and no internal contraction or aggregation. In contrast, A23187 added to platelets which had not seen vitamin E caused internal contraction with centralization of granules, shape change with pseudopod extension, and aggregation. (stained with uranyl acetate and lead citrate A x 22,000; b x 13,000).

210

A23187 which is dependent on the internal contraction). However, vitamin E failed to inhibit phospholipase A2 activation and indeed potentiated it. Thus we conclude that release of calcium from the dense tubular system is essential for stimulation of internal contraction and secretion but not for activation of phospholipase A2. There would appear to be two possible explanations 1) that phospholipase A2 activation is primarily dependent on an event other than calcium flux or 2) that the site of the calcium flux which stimulates the phospholipase A2 is different from that which stimulates internal contraction. The latter seems most likely, and indeed it has been suggested (49) that calcium flux essential for phospholipase A? activation occurs within the dense tubular system membrane, while that which IS essential to contraction occurs from the dense tubular system to the cytoplasm. Our present results suggest that the vitamin E can inhibit the second flux of calcium, but not the first. The block of the second of two such fluxes of calcium could result in the increased concentration of calcium in the area just proximal to the block (in the region of the phospholipase A2) with resulting potentiation by vitamin E of this enzyme action. (Figure 2).

insideof Dense Tubular

Calcium Binding

Profein

Dense Tubular System Membrane

Ca’/Mp” A Pose

PlateletCytoplasm

Figure 2. A proposal for the mechanism of action+ff vitamin E. The dense tubular system membrane is shown with its Ca++/Mg ATPase which can transport calcium from the platelet cytoplasm to a calcium binding site on a protein within the dense tubular system vesicle. The A23187 mobilizes the calcium from the binding site and transports it to the site of the phospholipase A2 and across the membrane into the platelet cytoplasm. Vitamin E,which is highly lipophilic,and probably therefore tends to go to the middle of the bilayer acts by preventing transport of calcium across the membrane causing a localized buildup of calcium in the vicinity of the phospholipase A2 thus enhancing the activity of this enzyme.

211

The mechanism by which vitamin E inhibits calcium flux is not clear. When viewed in the light of the two alternate proposals for the mechanism of action of vitamin E on cell physiology (inhibition of lipid peroxidation and stabilization of membranes) it is apparent that the effect of vitamin E to inhibit calcium flux would correspond most closely to a role in stabilizing membranes. The effect of vitamin E to inhibit calcium flux might be the molecular basis Alternatively, stabilization for its effect to produce membrane stabilization. of the membranes by vitamin E sitting within the bilayer could render the membrane less permeable to ions such as calcium. The present study conflicts directly with earlier evidence that vitamin E can inhibit prostaglandin synthesis in vivo (29,50). In the present study in vitro we have found no inhibition by vitamin E on either the phospholipase A2 or on metabolism of arachidonic acid to HHT and thromboxane B2. To resolve this discrepancy there would have to be in vivo 1) a metabolite of vitamin E which has an effect on one or both of these processes or 2) an intracellular chemical which can act together with vitamin E in the same fashion as NBT (33) to Both mechanisms are being explored currently, suppress prostaglandin synthesis. and evidence for the former has been found (Cox A.C., Rao G.H.R., Gerrard J.M., White J.G., unpublished results), and will be reported in a later publication.

In conclusion, the present study has shown that vitamin E has a direct effect on platelet intracellular calcium flux. Our results provide an explanation for the ability of this vitamin to suppress platelet activation, and provides a new concept for understanding the role of vitamin E as an essential human nutrient and potential antithrombotic agent. ACKNOWLEDGEMENTS We gratefully acknowledge the secretarial assistance of M. Prest, the artwork of L. Richter and the support of USPHS grants HL-11880, AM-06317, HL-06314, CA-12607, CA-08832, CA-11996, GM-AM-22167, HL-20695, HL-16833, AM-15317 and a grant from the Leukemia Task Force. JMG is the recipient of an Established Investigatorship from the American Heart Association.

212

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