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Spin Label Study of the Effect of Ticlopidine on Platelets
Spin
Label
Study Toshio
Faculty
of
of the HONGO,
Effect Morio
Pharmaceutical
of Ticlopidine
SETAKA
Sciences,
Kanagawa Accepted
Teikyo
199-01, June
and Takao University,
on Platelets KWAN Sagamiko,
Japan 3, 1985
Abstract-The effect of ticlopidine on platelet membrane fluidity was investigated using a spin label technique. Ticlopidine, when orally administered to rats, in creased both the order parameter and the motion parameter, indicating a decrease in the membrane fluidity of platelets. On the other hand, the order parameter and the motion parameter decreased markedly when the platelets were aggregated by thrombin. Ticlopidine inhibited the thrombin-induced aggregation of platelets and caused a slight increase in order parameter and motion parameter in thrombin aggregated platelets. Judging from sodium dodecyl sulfate polyacrylamide gel electrophoresis, ticlopidine did not modify the electrophoretic pattern of platelet proteins appreciably. Ticlopidine decreased cholesterol/phospholipids molar ratio and increased slightly total amounts of proteins of the platelets. These results indicate that the inhibitory action by ticlopidine was accompanied by changes in membrane fluidity, and these changes were due to a perturbation of the mem brane phospholipid core of the platelets by ticlopidine and/or its metabolites. antithrombotic
drug.
For this
purpose, we have investigated the ticlopidine on platelet membrane using a spin label technique.
effect
of
the
effect of fluidity
Materials and Methods Animals and treatment: Male Sprague Dawley rats weighing 200-300 g were used. They were fed a standard diet (CE-2, Clea Japan, Inc., Tokyo), except for fasting for 18 hr prior to the experimental dosing and water ad libitum. Ticlopidine, 5-(2 chlorobenzyl)-4, 5, 6, 7-tetrahydrothieno (3, 2,-c) pyridine hydrochloride, was orally administered in 0.5% Tween 80 solution at 3 and 24 hr before blood collection. The vehicle alone was orally given to control rats. Washed platelet preparation: The blood was collected from the inferior vena-cava under light ether anesthesia into 1 volume of 3.13% trisodium citrate-2H20. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 170xg for 10 min at room temperature, and a platelet pellet was obtained by centrifuging PRP at 645xg for 10 min. The pellet was washed in a 25 mM Tris-HCI,
pH 7.3, containing 125 mM NaCI and 5 mM glucose. Platelets were counted with a Sysmex Platelet Counter PL-100 (TOA Medical Electro Co., Ltd, Kobe, Japan). Electron spin resonance (ESR) and platelet aggregation study: The ethanol solution of 5-doxylstearic acid (5-DSA) or 16-doxylstearic acid (16-DSA) was added to a cuvette and dried under a stream of nitrogen. Aliquots of washed platelets (2x109/ml) were added to yield a final concentration of 5-DSA and 16-DSA (40 80 uM). After 5 min incubation at 37'C, aliquots of the suspensions were taken up into a glass capillary, and the ESR spectra were recorded on a JEOL JM-FE 3A spectrometer at 25'C. The temperature was controlled by the nitrogen gas stream. Simultaneously, the remaining suspensions were employed to perform aggregation studies by the turbidimetric method of Born and Cross (16), utilizing a Rikadenki HUSM System Platelet Aggregometer (Rikadenki Kogyo Co., Ltd, Tokyo, Japan), modified to equip a continuous stirring and thermosta tically controlled cuvette holder (37°C). The instrument was adjusted to 0% transmission with a platelet suspension and 100% with a buffer. The increase in light transmission was recorded as a function of time, and ag gregation responses were represented by the percent aggregation and the time required to reach half the maximal aggregation (T1;2). The order parameter, S, characterizing the molecular alignment of lipids in the mem brane, was calculated according to the following equation:
and ho and h_1 refer to the heights of the mid and high field lines, respectively (18). This equation is valid only when the rotational correlation time (r,) was >10-9 sec. Lipid extraction: Lipid extraction was carried out with chloroform-methanol-H20 by the method of Bligh and Dyer (19). The organic solvents were evaporated, and the lipids were dissolved in chloroform/methanol (1 :1, v/v) and stored at -20°C until analyzed. The quantities of phospholipids and choles terol were determined by the method of Ames (20) and Ott et al. (21), respectively. Determination of protein: Protein was measured by the method of Lowry et al. (22) using bovine serum albumin as a standard. Sodium dodecyl sulfate (SDS) poly acrylamide gel electrophoresis: Gel electro phoresis was carried out by the method of Laemmli (23) using 10-20% polyacrylamide gel plates in the presence of 0.1% SDS. Gels were stained for protein with Coomassie brilliant blue R-250 and for carbohydrate by a periodic acid-Schiff reagent according to Fairbanks et al. (24). Drugs used: 5-doxylstearic acid, 2-(3 carboxypropyl) -4,4-d imethyl-2-tridecyl-3 oxazolidinyloxyl and 16-doxylstearic acid, 2 (14-carboxytetradecyl) -2-ethyl -4,4-d imethyl -3-oxazolidinyloxyl were obtained from Syva Corp., CA, U.S.A. Thrombin was purchased from Parke-Davis, NJ, U.S.A. Ticlopidine was generously supplied by Daiichi Seiyaku Co., Ltd., Tokyo. All other chemicals were of the highest purity available commercially. Statistical analysis: Values are expressed as the mean±S.E.M. The statistical significance of differences between mean values was assessed by Student's t-test.
AS_ (A„ A~_) a zz-(Axx+Ayr)/2 a' Results where a= (Axx+AYY+Azz)/3, a'= (A„+2A)1/ 3, Axx (6.3 gauss), AYE (5.8 gauss) and Azz Platelet aggregation: The experimental (33.6 gauss) are the hyperfine principal results given in Fig. 1 show that in ex vivo values of nitroxide radical (17). A„ and A i_ experiments, ticlopidine (300 mg/kg, p.o.) were measured from the spectrum. inhibited the extent of aggregation induced by The motion parameter, vo, giving the thrombin (1 unit/ml) at 3 and 24 hr after apparent rotation frequency of the nitroxide administration and also prolonged T1;2. In radical, were calculated using the following in vitro experiments, ticlopidine in concen equation: trations up to 200 PM did not affect the z0=6.5X10-1o.W0• [(h0/h-1)1i2-1] where WD is the width of the mid field line,
platelet aggregation (Fig. 2). Effect of ticlopidine
elicited
by
thrombin
on platelet membrane
Fig. 1. Platelet aggregation in washed platelets from control and ticlopidine-treated rats. Values are given as the mean+S.E.M. of 11 experiments. Platelet aggregation was initiated by adding thrombin (1 unit/ml). Ticlopidine (300 mg/kg, p.o.) or vehicle (0.5% Tween 80) was orally administered to rats before the test. *P<0.01 compared with the control.
Fig. 2. Effect of ticlopidine on platelet aggregation in washed platelets. Values are given as the mean +S.E.M. of 5 experiments. Inhibition studies were carried out by addition of ticlopidine to the stirring platelet preparation 5 min prior to the addition of thrombin (1 unit/ml).
fluidity: Figure 3 shows changes in the fluidity of the platelet membrane induced by ticlopidine, obtained with the probe 5-DSA, at 3 and 24 hr after ticlopidine administration. Marked decreases in order parameters of the platelets aggregated by thrombin were observed for both control and ticlopidine treatment. At 3 and 24 hr after 300 mg/kg ticlopidine administration, values of the order
Fig. 3. Changes in order parameter of washed platelets from control and ticlopidine-treated rats. Values are given as the mean+S.E.M. of 4-11 experiments. Platelet aggregation was initiated by adding thrombin (1 unit/ml). Ticlopidine (100 and 300 mg/kg, p.o.) or vehicle (0.5% Tween 80) was orally administered to rats before the test. Experi mental details are given in the text. *P<0.05 com pared with the control.
parameter of the platelets increased con siderably, and changes in the order parameter of thrombin-aggregated platelets were very small. On the other hand, after 100 mg/kg ticlopidine administration, values of the order parameter are almost the same as compared with the control. Marked changes in the order parameter after 300 mg/kg ticlopidine administration were also indicated by motion parameter measurement using 16-DSA as a probe (Fig. 4). The motion parameter of the platelets also decreased considerably by thrombin-induced aggregation. At 3 and 24 hr after 300 mg/kg ticlopidine administration, the motion para meter of the platelets before thrombin elicited aggregation increased as compared with the control in the same manner as the results obtained with 5-DSA as a probe. However, there were no significant variations in the motion parameter between the control and ticlopidine-treatment after thrombin induced aggregation. In in vitro experiments, as shown in Fig. 5, after the 5-DSA-labeled platelets were
Fig. 4. Alterations in motion parameter of washed platelets from control and ticlopidine-treated rats. Values are given as the mean±S.E.M. of 5 experi ments. Platelet aggregation was initiated by adding thrombin (1 unit/ml). Ticlopidine (300 mg/kg, p.o.) or vehicle (0.5% Tween 80) was orally administered to rats before the test. Experimental details are given in the text. *P<0.05 compared with the control.
Fig. 5. Effect of ticlopidine on order parameter of washed platelets. Values are given as the mean ±S .E.M. of 5 experiments. Inhibition studies were carried out by addition of ticlopidine to the stirring platelet preparation 5 min prior to the addition of thrombin (1 unit/ml). Experimental details are given in the text.
Fig. 6. Phospholipids and cholesterol determination in washed platelets from control and ticlopidine treated rats. Values are given as the mean±S.E.M. of 6 experiments. A fresh platelet sample was washed three times with a 25 mM Tris-HCI buffer, containing 125 mM NaCI and 5 mM glucose, pH 7.3. Lipids were extracted according to Bligh and Dyer (19), and the amounts of phospholipids and cholesterol were measured as described in the text.
treated for 5 min with ticlopidine, the values of the order parameter were found not to change remarkably before and after thrombin treatment as compared with controls.
Effects of ticlopidine on the contents of cholesterol, phospholipids, and proteins in the platelets and electrophoretic pattern of platelet proteins: As shown in Fig. 6, the amounts of cholesterol of the platelets tended to decrease at 3 and 24 hr after ticlopidine administration as compared with the control. On the contrary, there was a small increase in the amounts of phospholipids of the platelets. Therefore, cholesterol/phospholipids (C L/ PL) molar ratio decreased at 3 and 24 hr after ticlopidine was administered orally to rats. Ticlopidine, when orally administered, induced a slight increase in quantities of total proteins (Fig. 7). Figure 8 shows typical patterns of SDS polyacrylamide gel electro phoresis of platelet proteins. Changes in the electrophoretic pattern were not observed at 3 and 24 hr after ticlopidine administration as
Fig. from
7.
Protein
control
given platelets mination
as the
determination and
in
ticlopidine-treated
mean±S.E.M.
were prepared was measured
washed rats.
of 6 experiments. as in Fig. as described
platelets Values
are
Washed
6. Protein deter in the text.
Fig. 8. Electrophoretic pattern of washed platelets from control and ticlopidine-treated rats. Ticlopidine (300 mg/kg, p.o.) or vehicle (0.5% Tween 80) was orally administered to rats before the test. Washed platelets were prepared as in Fig. 6.
compared with the control. Furthermore, in gels stained by the periodic acid-Schiff procedure, the electrophoretic pattern was not modified (data not shown). Discussion The spin label technique has been established as a valuable approach to investigate the physical state of the biological
membrane (18, 25-28). Stearic acid spin labels inlaid in the biological membrane exhibit their freedom of anisotropic motion in proportion to the position of the nitroxide radical on the alkyl fatty acid chain (18, 26, 27). This anisotropic motion characterizes the molecular alignment and motion of lipids in the membrane: the so-called membrane fluidity (18, 25-27, 29, 30). The values measured with 5-DSA reflect the fluidity of the region located nearer the surface of the membrane, whereas those of 16-DSA re present the fluidity of the hydrophobic region. Also, it is well known that membrane fluidity is involved in various membrane functions such as permeability, ion transport, membrane-associated enzyme activities, and so on (29, 30). Furthermore, the major contributing factors to changes in the mem brane fluidity are lipid composition and lipid-protein interactions (18, 29-32). In ex vivo experiments, our results showed that platelet membrane fluidity was decreased by orally administered ticlopidine as deter mined by measurements using 5-DSA or 1 6-DSA as a probe and that it was increased by thrombin-induced aggregation, whereas the membrane fluidity of the platelets ag gregated by thrombin was decreased slightly by ticolpidine. These facts suggest that ticlopidine and/or its metabolites are respon sible for observed changes in the platelet membrane fluidity. Membrane CL/PL molar ratios were known to be important for regulating changes of membrane fluidity. Generally, it is sug gested that in liposomes prepared from egg phosphatidylcholine decreasing the CL/PL ratio causes an increase in membrane fluidity (33). In the present results, the amount of cholesterol was reduced slightly by ticlopidine, while that of phospholipids showed a tendency to increase, leading to the decrease of CL/PL molar ratio. Never theless, ticlopidine induced the decrease in the platelet membrane fluidity observed with 5-DSA and 16-DSA, therefore suggesting that changes in the membrane fluidity elicited by ticlopidine were regulated by factors other than the CL/PL molar ratio. On the other hand, lipid-protein inter actions are considered to be one of the major
factors contributing to alterations in the platelet membrane fluidity (34). The mem brane fluidity of rat platelets became much smaller than that of liposomes prepared by the total lipids extracted from platelets (T. Hongo et al., unpublished observations), as previously reported on human platelets (34), suggesting that the interaction between lipids and proteins could reduce membrane fluidity markedly. Our results showed that the values of the phospholipid/protein ratio of ticlopidine-treated platelets were almost equal to that of the control. Accordingly, there was little possibility that the cause of marked decreases in the platelet membrane fluidity is due to an immobilizing effect induced by proteins. Moreover, ticlopidine, a highly hydro phobic molecule, binds on the erythrocyte membrane (35) and the platelet membrane (36). This is a non-saturable and irreversible binding. Especially, in erythrocytes (35), the highest concentration (1 mM ticlopidine) has a non-specific denaturating effect on the membrane proteins; new peptide bands appear between bands 2 and 3, band 3 intensity is reduced, and so on. This protein denaturation causes a partial perturbation of the lipid bilayer (35). However, the observed decreases in the membrane fluidity by ticlopidine-treatment was not caused by such a denaturating effect since no variations in the electrophoretic pattern between control and ticlopidine-treatment were observed. In in vitro experiments, our results show that ticlopidine at concentrations of less than 200 pM did not affect platelet aggregation appreciably nor the platelet membrane fluidity, although the possibility of a partial perturbation of the lipidic bilayer due to binding of ticlopidine and/or its metabolites to platelet membrane proteins still remained at higher concentrations of ticlopidine (i.e., more than 200 ,FPM). Although the exact mechanism for the inhibitory effect of ticlopidine on platelet aggregation remains unclear, it may involve the production of cyclic AMP (2, 13, 14). Ticlopidine has been found to potentiate the stimulation by prostaglandin El of adenylate cyclase activity in rat platelets, suggesting that ticlopidine stimulates the adenylate
cyclase system by its action on the N regulatory subunit of the enzyme as GTP does (37). Our present understanding of the regulation of the adenylate cyclase system indicates that it involves the interaction of at least three membrane-associated proteins, namely, the receptor, the so-called G/F or N regulatory subunit, and the catalytic unit (38). The binding of hormone to receptor stimulates its coupling with the N regulatory subunit, which in turn associates the activated N regulatory subunit with the catalytic unit, resulting in the production of cyclic AMP. Since these events can occur through lateral diffusion of the subunits in the plane of the membranes (38-40), changes in the physical state of the membrane lipids must he considered to play an important role in the regulation of these interactions. It may be very significant to debate the relationship between alterations in the membrane fluidity and that of adenylate cyclase activity, thereby suggesting a possible involvement of the N regulatory subunit in this system. Our present results showed that orally administered ticlopidine induced marked changes in the platelet membrane fluidity. Since ticlopidine could act on the N regulatory subunit of the enzyme (37), it is suggested that ticlopidine and/or its metabolites could be inserted into the lipid region of the membrane, resulting in the increased as sociation of subunits necessary for adenylate cyclase activity, or that ticlopidine and/or its metabolites could bind the N regulatory subunit, causing marked alterations in the lipid bilayer fluidity. Recently, ticlopidine, when orally admin istered to rats, has been found to inhibit platelet phospholipases, resulting in the inhibition of platelet aggregation (Hirai, A., Personal communication). In in vitro experi ments, ticlopidine affects the metabolism of human platelet phospholipids by inhibiting the incorporation of arachidonic acid (41). These observations indicate that ticlopidine may influence the length and the degree of unsaturation of phospholipid acyl chains as well as phospholipid species in the platelet membrane. Since these factors are also important determinants of membrane fluidity, further detailed investigation will be necessary
in this respect. Acknowledgements: The authors wish to thank Dr. Y. Abiko, Dr. S. Ashida and Dr . T. Takayanagi for useful suggestions and comments on this manu script. Secretarial assistance by Ms . N. Satoh is gratefully acknowledged.
12
References 1 Johnson, M., Walton, P.L., Cotton, R.C. and Strachan, C.J.L.: Pharmacological evaluation of ticlopidine, a novel inhibitor of platelet function. 6th Int. Congr. Thrombosis and Haemostasis , Philadelphia 1977, Abstracts. Thromb. Haemost. 38, 64 (1977) 2 Ashida, S. and Abiko, Y.: Inhibition of platelet aggregation by a new agent , ticlopidine. Thromb. Haemost. 40, 542-550 (1978) 3 O'Brien, J.R., Etherington, M.D. and Shut tleworth, R.D.: Ticlopidine- An antiplatelet drug: effects in human volunteers. Thromb. Res. 13, 245-254 (1978) 4 Knudsen, J.B. and Gormsen , J.: The effect of ticlopidine on platelet function in normal volunteers and in patients with platelet hyper aggregability in vitro. Thromb. Res. 16, 663-671 (1979) 5 Lips, J.P.M., Sixma, J.J. and Schiphorst, M.E.: The effect of ticlopidine administration to humans on the binding of adenosine diphosphate to blood platelets. Thromb. Res. 17, 19-27 (1980) 6 Kirstein, P., Jogestrand, T., Johnson, H. and Olsson, A.G.:Anti-aggregatory, physiological and clinical effects of ticlopidine in subjects with peripheral atherosclerosis. Atherosclerosis 36, 471-480 (1980) 7 Nunn, B. and Lindsay, R.: Effect of ticlopidine on human platelet responsiveness ex vivo: com parison with aspirin. Thromb. Res. 18, 807-813 (1980) 8 Lecrubier, C., Conard, J., Horellou, M.H. and Samama, M.: Study of platelet aggregation induced by platelet activating factor (PAF) after administration of ticlopidine or aspirin . Agents Actions 13, 77-80 (1983) 9 Ebihara, A., Aoki, N., Yoshida, N., Sano, M ., Matsubayashi, K. and Suzuki, T.: Clinical phar macology of a new antithrombotic agent , ticlopidine. Japan. J. Clin. Pharmacol. 9, 395 402 (1978) 10 David, J.L., Monfort, F., Herion, F. and Raskinet, R.: Compared effects of three dose-levels of ticlopidine on platelet function in normal subjects . Thromb. Res. 14, 35-49 (1979) 11 Conard, J., Lecrubier, C., Scarabin, P. Y.,
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14
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18
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21
22
23
24
25
Horellou, M.H., Samama, M. and Bousser, M.G.: Effects of long term administration of ticlopidine on platelet function and hemostatic variables. Thromb. Res. 20. 143-148 (1980) Johnson, M. and Heywood, J.B.: Possible mode of action of ticlopidine: a novel inhibitor of platelet aggregation. 7th Int. Congr. Thrombosis and Haemostasis. London 1979, Abstract. Thromb. Haemost. 42, 367 (1979) Ashida, S. and Abiko, Y.: Mode of action of ticlopidine in inhibition of platelet aggregation in the rat. Thromb. Haemost. 41, 436-449 (1979) Panak, E., Maffrand, J.P., Picard-Fraire, C., Vallee, E., Blanchard, J. and Roncucci, R.: Ticlopidine-a promise for the prevention and treatment of thrombosis and its complications. Haemostasis 13 Supp. 1, 1-54 (1983) Lee, H., Paton, R.C., Ruan, C. and Caen, J.P.: The in vitro effect of ticlopidine on fibrinogen and factor VIII binding to human platelets. Thromb. Haemost. 46, 590-592 (1981) Born, G.V.R. and Cross, M.J.: The aggregation of blood platelets. J. Physiol. (Lend.) 168, 178-195 (1963) Gaffney, B.J. and McConnell, H.M.: The para magnetic resonance spectra of spin labels in phospholipid membrane. J. Magn. Resonance 16. 1-28 (1974) Henry, S.A. and Keith, A.D.: Membrane pro perties of saturated fatty acid mutants of yeast revealed by spin labels. Chem. Phys. Lipids 7, 245-265 (1971) Bligh, E.G. and Dyer, W.J.: A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917 (1959) Ames, B.N.: Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol. 8, 115-118 (1966) Ott, P., Binggeli, Y. and Brodbeck, U.: A rapid and sensitive assay for determination of choles terol in membrane lipid extracts. Biochim. Biophys. Acta 685, 211-213 (1982) Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265 275 (1951) Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacterio phage T4. Nature 227 680-685 (1970) Farbanks, G., Steck T.L. and Wallach, D.F.H.: Electrophoretic analysis of the major polype ptides of the human erythrocyte membrane. Biochemistry 10, 2606-2617 (1971) Hubbell, W.L. and McConnell, H.M.: Orientation
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29
30
31
32
33
and motion of amphiphilic spin labels in mem branes. Proc. Natl. Acad. Sci. U.S.A. 64, 20-27 (1969) Hubbell, W.L. and McConnell, H.M.: Molecular motion in spin labeled phospholipids and membranes. J. Am. Chem. Soc. 93, 314-326 (1971) Keith, A.D., Sharnoff, M. and Cohn, G.E.: A summary and evaluation of spin labels used as probes for biological membrane structure. Biochim. Biophys. Acta 300, 379-419 (1973) McConnell, H.M.: Molecular motion in biological membranes. In Spin Labeling Theory and Applicaitons, Edited by Berliner, L. J., p. 525 560, Academic Press, New York, San Francisco and London (1976) Stubbs, C.D. and Smith, A.D.: The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim. Biophys. Acta 779, 89-137 (1984) Goldstein, D.B.: The effects of drugs on mem brane fluidity, Annu. Rev. Pharmacol. Toxicol. 24, 43-64 (1984) Schreier, S., Polnaszek, C.F. and Smith, I.C.P.: Spin labels in membranes-problems in practice. Biochim. Biophys. Acta 515, 395-436 (1978) Emmelot, P. and Van Hoeven, R.P.: Phospholipid unsaturation and plasma membrane organization. Chem. Phys. Lipids 14, 236-246 (1975) Schreier-Muccillo, S., Marsh, D., Dugas, H., Schneider, H. and Smith, I.C.P.: A spin probe study of the influence of chelesterol on motion and orientation of phospholipids in oriented
multibilayers 10, 34
Ohki,
11-27 K.,
and
vesicles.
Chem.
Phys.
(1973) Imai,
A.
and
Nozawa,
Y.:
Aggregation
Lipids
Label
Study Toshio
Faculty
of
of the HONGO,
Effect Morio
Pharmaceutical
of Ticlopidine
SETAKA
Sciences,
Kanagawa Accepted
Teikyo
199-01, June
and Takao University,
on Platelets KWAN Sagamiko,
Japan 3, 1985
Abstract-The effect of ticlopidine on platelet membrane fluidity was investigated using a spin label technique. Ticlopidine, when orally administered to rats, in creased both the order parameter and the motion parameter, indicating a decrease in the membrane fluidity of platelets. On the other hand, the order parameter and the motion parameter decreased markedly when the platelets were aggregated by thrombin. Ticlopidine inhibited the thrombin-induced aggregation of platelets and caused a slight increase in order parameter and motion parameter in thrombin aggregated platelets. Judging from sodium dodecyl sulfate polyacrylamide gel electrophoresis, ticlopidine did not modify the electrophoretic pattern of platelet proteins appreciably. Ticlopidine decreased cholesterol/phospholipids molar ratio and increased slightly total amounts of proteins of the platelets. These results indicate that the inhibitory action by ticlopidine was accompanied by changes in membrane fluidity, and these changes were due to a perturbation of the mem brane phospholipid core of the platelets by ticlopidine and/or its metabolites. antithrombotic
drug.
For this
purpose, we have investigated the ticlopidine on platelet membrane using a spin label technique.
effect
of
the
effect of fluidity
Materials and Methods Animals and treatment: Male Sprague Dawley rats weighing 200-300 g were used. They were fed a standard diet (CE-2, Clea Japan, Inc., Tokyo), except for fasting for 18 hr prior to the experimental dosing and water ad libitum. Ticlopidine, 5-(2 chlorobenzyl)-4, 5, 6, 7-tetrahydrothieno (3, 2,-c) pyridine hydrochloride, was orally administered in 0.5% Tween 80 solution at 3 and 24 hr before blood collection. The vehicle alone was orally given to control rats. Washed platelet preparation: The blood was collected from the inferior vena-cava under light ether anesthesia into 1 volume of 3.13% trisodium citrate-2H20. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 170xg for 10 min at room temperature, and a platelet pellet was obtained by centrifuging PRP at 645xg for 10 min. The pellet was washed in a 25 mM Tris-HCI,
pH 7.3, containing 125 mM NaCI and 5 mM glucose. Platelets were counted with a Sysmex Platelet Counter PL-100 (TOA Medical Electro Co., Ltd, Kobe, Japan). Electron spin resonance (ESR) and platelet aggregation study: The ethanol solution of 5-doxylstearic acid (5-DSA) or 16-doxylstearic acid (16-DSA) was added to a cuvette and dried under a stream of nitrogen. Aliquots of washed platelets (2x109/ml) were added to yield a final concentration of 5-DSA and 16-DSA (40 80 uM). After 5 min incubation at 37'C, aliquots of the suspensions were taken up into a glass capillary, and the ESR spectra were recorded on a JEOL JM-FE 3A spectrometer at 25'C. The temperature was controlled by the nitrogen gas stream. Simultaneously, the remaining suspensions were employed to perform aggregation studies by the turbidimetric method of Born and Cross (16), utilizing a Rikadenki HUSM System Platelet Aggregometer (Rikadenki Kogyo Co., Ltd, Tokyo, Japan), modified to equip a continuous stirring and thermosta tically controlled cuvette holder (37°C). The instrument was adjusted to 0% transmission with a platelet suspension and 100% with a buffer. The increase in light transmission was recorded as a function of time, and ag gregation responses were represented by the percent aggregation and the time required to reach half the maximal aggregation (T1;2). The order parameter, S, characterizing the molecular alignment of lipids in the mem brane, was calculated according to the following equation:
and ho and h_1 refer to the heights of the mid and high field lines, respectively (18). This equation is valid only when the rotational correlation time (r,) was >10-9 sec. Lipid extraction: Lipid extraction was carried out with chloroform-methanol-H20 by the method of Bligh and Dyer (19). The organic solvents were evaporated, and the lipids were dissolved in chloroform/methanol (1 :1, v/v) and stored at -20°C until analyzed. The quantities of phospholipids and choles terol were determined by the method of Ames (20) and Ott et al. (21), respectively. Determination of protein: Protein was measured by the method of Lowry et al. (22) using bovine serum albumin as a standard. Sodium dodecyl sulfate (SDS) poly acrylamide gel electrophoresis: Gel electro phoresis was carried out by the method of Laemmli (23) using 10-20% polyacrylamide gel plates in the presence of 0.1% SDS. Gels were stained for protein with Coomassie brilliant blue R-250 and for carbohydrate by a periodic acid-Schiff reagent according to Fairbanks et al. (24). Drugs used: 5-doxylstearic acid, 2-(3 carboxypropyl) -4,4-d imethyl-2-tridecyl-3 oxazolidinyloxyl and 16-doxylstearic acid, 2 (14-carboxytetradecyl) -2-ethyl -4,4-d imethyl -3-oxazolidinyloxyl were obtained from Syva Corp., CA, U.S.A. Thrombin was purchased from Parke-Davis, NJ, U.S.A. Ticlopidine was generously supplied by Daiichi Seiyaku Co., Ltd., Tokyo. All other chemicals were of the highest purity available commercially. Statistical analysis: Values are expressed as the mean±S.E.M. The statistical significance of differences between mean values was assessed by Student's t-test.
AS_ (A„ A~_) a zz-(Axx+Ayr)/2 a' Results where a= (Axx+AYY+Azz)/3, a'= (A„+2A)1/ 3, Axx (6.3 gauss), AYE (5.8 gauss) and Azz Platelet aggregation: The experimental (33.6 gauss) are the hyperfine principal results given in Fig. 1 show that in ex vivo values of nitroxide radical (17). A„ and A i_ experiments, ticlopidine (300 mg/kg, p.o.) were measured from the spectrum. inhibited the extent of aggregation induced by The motion parameter, vo, giving the thrombin (1 unit/ml) at 3 and 24 hr after apparent rotation frequency of the nitroxide administration and also prolonged T1;2. In radical, were calculated using the following in vitro experiments, ticlopidine in concen equation: trations up to 200 PM did not affect the z0=6.5X10-1o.W0• [(h0/h-1)1i2-1] where WD is the width of the mid field line,
platelet aggregation (Fig. 2). Effect of ticlopidine
elicited
by
thrombin
on platelet membrane
Fig. 1. Platelet aggregation in washed platelets from control and ticlopidine-treated rats. Values are given as the mean+S.E.M. of 11 experiments. Platelet aggregation was initiated by adding thrombin (1 unit/ml). Ticlopidine (300 mg/kg, p.o.) or vehicle (0.5% Tween 80) was orally administered to rats before the test. *P<0.01 compared with the control.
Fig. 2. Effect of ticlopidine on platelet aggregation in washed platelets. Values are given as the mean +S.E.M. of 5 experiments. Inhibition studies were carried out by addition of ticlopidine to the stirring platelet preparation 5 min prior to the addition of thrombin (1 unit/ml).
fluidity: Figure 3 shows changes in the fluidity of the platelet membrane induced by ticlopidine, obtained with the probe 5-DSA, at 3 and 24 hr after ticlopidine administration. Marked decreases in order parameters of the platelets aggregated by thrombin were observed for both control and ticlopidine treatment. At 3 and 24 hr after 300 mg/kg ticlopidine administration, values of the order
Fig. 3. Changes in order parameter of washed platelets from control and ticlopidine-treated rats. Values are given as the mean+S.E.M. of 4-11 experiments. Platelet aggregation was initiated by adding thrombin (1 unit/ml). Ticlopidine (100 and 300 mg/kg, p.o.) or vehicle (0.5% Tween 80) was orally administered to rats before the test. Experi mental details are given in the text. *P<0.05 com pared with the control.
parameter of the platelets increased con siderably, and changes in the order parameter of thrombin-aggregated platelets were very small. On the other hand, after 100 mg/kg ticlopidine administration, values of the order parameter are almost the same as compared with the control. Marked changes in the order parameter after 300 mg/kg ticlopidine administration were also indicated by motion parameter measurement using 16-DSA as a probe (Fig. 4). The motion parameter of the platelets also decreased considerably by thrombin-induced aggregation. At 3 and 24 hr after 300 mg/kg ticlopidine administration, the motion para meter of the platelets before thrombin elicited aggregation increased as compared with the control in the same manner as the results obtained with 5-DSA as a probe. However, there were no significant variations in the motion parameter between the control and ticlopidine-treatment after thrombin induced aggregation. In in vitro experiments, as shown in Fig. 5, after the 5-DSA-labeled platelets were
Fig. 4. Alterations in motion parameter of washed platelets from control and ticlopidine-treated rats. Values are given as the mean±S.E.M. of 5 experi ments. Platelet aggregation was initiated by adding thrombin (1 unit/ml). Ticlopidine (300 mg/kg, p.o.) or vehicle (0.5% Tween 80) was orally administered to rats before the test. Experimental details are given in the text. *P<0.05 compared with the control.
Fig. 5. Effect of ticlopidine on order parameter of washed platelets. Values are given as the mean ±S .E.M. of 5 experiments. Inhibition studies were carried out by addition of ticlopidine to the stirring platelet preparation 5 min prior to the addition of thrombin (1 unit/ml). Experimental details are given in the text.
Fig. 6. Phospholipids and cholesterol determination in washed platelets from control and ticlopidine treated rats. Values are given as the mean±S.E.M. of 6 experiments. A fresh platelet sample was washed three times with a 25 mM Tris-HCI buffer, containing 125 mM NaCI and 5 mM glucose, pH 7.3. Lipids were extracted according to Bligh and Dyer (19), and the amounts of phospholipids and cholesterol were measured as described in the text.
treated for 5 min with ticlopidine, the values of the order parameter were found not to change remarkably before and after thrombin treatment as compared with controls.
Effects of ticlopidine on the contents of cholesterol, phospholipids, and proteins in the platelets and electrophoretic pattern of platelet proteins: As shown in Fig. 6, the amounts of cholesterol of the platelets tended to decrease at 3 and 24 hr after ticlopidine administration as compared with the control. On the contrary, there was a small increase in the amounts of phospholipids of the platelets. Therefore, cholesterol/phospholipids (C L/ PL) molar ratio decreased at 3 and 24 hr after ticlopidine was administered orally to rats. Ticlopidine, when orally administered, induced a slight increase in quantities of total proteins (Fig. 7). Figure 8 shows typical patterns of SDS polyacrylamide gel electro phoresis of platelet proteins. Changes in the electrophoretic pattern were not observed at 3 and 24 hr after ticlopidine administration as
Fig. from
7.
Protein
control
given platelets mination
as the
determination and
in
ticlopidine-treated
mean±S.E.M.
were prepared was measured
washed rats.
of 6 experiments. as in Fig. as described
platelets Values
are
Washed
6. Protein deter in the text.
Fig. 8. Electrophoretic pattern of washed platelets from control and ticlopidine-treated rats. Ticlopidine (300 mg/kg, p.o.) or vehicle (0.5% Tween 80) was orally administered to rats before the test. Washed platelets were prepared as in Fig. 6.
compared with the control. Furthermore, in gels stained by the periodic acid-Schiff procedure, the electrophoretic pattern was not modified (data not shown). Discussion The spin label technique has been established as a valuable approach to investigate the physical state of the biological
membrane (18, 25-28). Stearic acid spin labels inlaid in the biological membrane exhibit their freedom of anisotropic motion in proportion to the position of the nitroxide radical on the alkyl fatty acid chain (18, 26, 27). This anisotropic motion characterizes the molecular alignment and motion of lipids in the membrane: the so-called membrane fluidity (18, 25-27, 29, 30). The values measured with 5-DSA reflect the fluidity of the region located nearer the surface of the membrane, whereas those of 16-DSA re present the fluidity of the hydrophobic region. Also, it is well known that membrane fluidity is involved in various membrane functions such as permeability, ion transport, membrane-associated enzyme activities, and so on (29, 30). Furthermore, the major contributing factors to changes in the mem brane fluidity are lipid composition and lipid-protein interactions (18, 29-32). In ex vivo experiments, our results showed that platelet membrane fluidity was decreased by orally administered ticlopidine as deter mined by measurements using 5-DSA or 1 6-DSA as a probe and that it was increased by thrombin-induced aggregation, whereas the membrane fluidity of the platelets ag gregated by thrombin was decreased slightly by ticolpidine. These facts suggest that ticlopidine and/or its metabolites are respon sible for observed changes in the platelet membrane fluidity. Membrane CL/PL molar ratios were known to be important for regulating changes of membrane fluidity. Generally, it is sug gested that in liposomes prepared from egg phosphatidylcholine decreasing the CL/PL ratio causes an increase in membrane fluidity (33). In the present results, the amount of cholesterol was reduced slightly by ticlopidine, while that of phospholipids showed a tendency to increase, leading to the decrease of CL/PL molar ratio. Never theless, ticlopidine induced the decrease in the platelet membrane fluidity observed with 5-DSA and 16-DSA, therefore suggesting that changes in the membrane fluidity elicited by ticlopidine were regulated by factors other than the CL/PL molar ratio. On the other hand, lipid-protein inter actions are considered to be one of the major
factors contributing to alterations in the platelet membrane fluidity (34). The mem brane fluidity of rat platelets became much smaller than that of liposomes prepared by the total lipids extracted from platelets (T. Hongo et al., unpublished observations), as previously reported on human platelets (34), suggesting that the interaction between lipids and proteins could reduce membrane fluidity markedly. Our results showed that the values of the phospholipid/protein ratio of ticlopidine-treated platelets were almost equal to that of the control. Accordingly, there was little possibility that the cause of marked decreases in the platelet membrane fluidity is due to an immobilizing effect induced by proteins. Moreover, ticlopidine, a highly hydro phobic molecule, binds on the erythrocyte membrane (35) and the platelet membrane (36). This is a non-saturable and irreversible binding. Especially, in erythrocytes (35), the highest concentration (1 mM ticlopidine) has a non-specific denaturating effect on the membrane proteins; new peptide bands appear between bands 2 and 3, band 3 intensity is reduced, and so on. This protein denaturation causes a partial perturbation of the lipid bilayer (35). However, the observed decreases in the membrane fluidity by ticlopidine-treatment was not caused by such a denaturating effect since no variations in the electrophoretic pattern between control and ticlopidine-treatment were observed. In in vitro experiments, our results show that ticlopidine at concentrations of less than 200 pM did not affect platelet aggregation appreciably nor the platelet membrane fluidity, although the possibility of a partial perturbation of the lipidic bilayer due to binding of ticlopidine and/or its metabolites to platelet membrane proteins still remained at higher concentrations of ticlopidine (i.e., more than 200 ,FPM). Although the exact mechanism for the inhibitory effect of ticlopidine on platelet aggregation remains unclear, it may involve the production of cyclic AMP (2, 13, 14). Ticlopidine has been found to potentiate the stimulation by prostaglandin El of adenylate cyclase activity in rat platelets, suggesting that ticlopidine stimulates the adenylate
cyclase system by its action on the N regulatory subunit of the enzyme as GTP does (37). Our present understanding of the regulation of the adenylate cyclase system indicates that it involves the interaction of at least three membrane-associated proteins, namely, the receptor, the so-called G/F or N regulatory subunit, and the catalytic unit (38). The binding of hormone to receptor stimulates its coupling with the N regulatory subunit, which in turn associates the activated N regulatory subunit with the catalytic unit, resulting in the production of cyclic AMP. Since these events can occur through lateral diffusion of the subunits in the plane of the membranes (38-40), changes in the physical state of the membrane lipids must he considered to play an important role in the regulation of these interactions. It may be very significant to debate the relationship between alterations in the membrane fluidity and that of adenylate cyclase activity, thereby suggesting a possible involvement of the N regulatory subunit in this system. Our present results showed that orally administered ticlopidine induced marked changes in the platelet membrane fluidity. Since ticlopidine could act on the N regulatory subunit of the enzyme (37), it is suggested that ticlopidine and/or its metabolites could be inserted into the lipid region of the membrane, resulting in the increased as sociation of subunits necessary for adenylate cyclase activity, or that ticlopidine and/or its metabolites could bind the N regulatory subunit, causing marked alterations in the lipid bilayer fluidity. Recently, ticlopidine, when orally admin istered to rats, has been found to inhibit platelet phospholipases, resulting in the inhibition of platelet aggregation (Hirai, A., Personal communication). In in vitro experi ments, ticlopidine affects the metabolism of human platelet phospholipids by inhibiting the incorporation of arachidonic acid (41). These observations indicate that ticlopidine may influence the length and the degree of unsaturation of phospholipid acyl chains as well as phospholipid species in the platelet membrane. Since these factors are also important determinants of membrane fluidity, further detailed investigation will be necessary
in this respect. Acknowledgements: The authors wish to thank Dr. Y. Abiko, Dr. S. Ashida and Dr . T. Takayanagi for useful suggestions and comments on this manu script. Secretarial assistance by Ms . N. Satoh is gratefully acknowledged.
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