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Effects of monovalent cations and anions on ADP-induced aggregation of bovine platelets, and mechanism thereof
Biochimica et Biophysica Acta 840 (1985) 371-376 Elsevier
371
BBA 22066
E f f e c t s of m o n o v a l e n t c a t i o n s and anions on A D P - i n d u c e d a g g r e g a t i o n of b o v i n e platelets, and m e c h a n i s m s t h e r e o f Shuji K i t a g a w a *, H i d e a k i Seki a n d F u j i o K a m e t a n i Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi 1, Tokushima 770 (Japan) (Received November 6th, 1984) (Revised manuscript received March 25th, 1985)
Key words: Monovalent ion; ADP; Platelet aggregation; (Bovine)
The effects of monovalent cations - inorganic alkali metal cations and organic quaternary ammonium cations - and monovalent inorganic anions on ADP-induced aggregation of bovine platelets were investigated. In the presence of K +, Rb +, Cs +, choline or tetramethylammonium, aggregation proceeded. However, aggregation was markedly restricted in media containing Li +, Na +, tetrabutylammonium or dimethyldibenzylammonium. With anions, aggregation proceeded in the order C I - > Br - > I - > CIO4-- > SCN -. The effects of cations significantly depended on Ca 2+ concentration, whereas those of the anions depended little on Ca 2+. Anions such as S C N - and 0 0 4 markedly decreased the fluorescence of the sudaee charge probe 2-p-toluidinylnaphthalene-6-sulfonate, whereas cations had less pronounced effects. The relative effects of the anions on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that inhibition of platelet aggregation by the anions is due to a change in the surface change of the platelet plasma membrane. On the other hand, kinetic analysis suggests that the effects of monovalent cations on platelet aggregation are due to their competition with Ca 2+ during the process of aggregation.
Introduction
Platelet aggregation is affected by the ionic milieu, and has been shown to depend on the concentration of monovalent cations such as K ÷ [1-3] and divalent cations such as Ca 2+ [1]. We have previously reported, by using concentrated platelet-rich plasma, that ADP-induced aggregation is also affected by inorganic anions in the medium [4]. However, their inhibitory mechanism is still unknown. It has been shown that platelet * To whom correspondence should be addressed. Abbreviations: THIS, 2-p-toluidinylnaphthalene-6-sulfonate; TMA, tetramethylammonium; TEA, tetraethylamrnonium; TMBA, trimethylbenzylammonium; TBA, tetrabutylammonium; DDA, dimethyldibenzylammonium.
functions are inhibited by various organic substances [5,6]. Among these substances, we have shown that fatty acids (organic anions) inhibit platelet aggregation due to modification of membrane surface change or membrane fluidity [5,6]. In this work we examined the effects of five alkali metal cations, six quaternary ammonium cations and five inorganic anions on ADP-induced aggregation. It is the first time that the effects of quaternary ammonium cations on platelet aggregation have been studied. In the present investigation we aimed at a further understanding of the mechanisms by which both cations and anions affect the platelet aggregation, in particular in relation to their effects on membrane surface charge and their competition with Ca 2+ during the process.
0304-4165/85/$03.30 © Elsevier Science Publishers B.V. (Biomedical Division)
372
Materials and Methods
Materials. Bovine fibrinogen a n d 2-p-toluidinyln a p h t h a l e n e - 6 - s u l f o n a t e ( T N S ) were p u r c h a s e d f r o m Sigma (St. Louis, MO). A D P was p u r c h a s e d from O r i e n t a l Yeast Co. (Tokyo, Japan). Potassium salts of anions a n d chloride salts of c a t i o n s were p u r c h a s e d from W a k o Pure C h e m i c a l Industries (Osaka, J a p a n ) or T o k y o C h e m i c a l Ind u s t r y Co. (Tokyo, Japan). Preparation of platelet suspension. Platelet-rich p l a s m a of bovine (Holstein) b l o o d was o b t a i n e d as d e s c r i b e d in a previous p a p e r [4]. The platelet-rich plasma, which c o n t a i n e d a b o u t 10% ( v / v ) A C D c o a g u l a n t solution (74.8 m M s o d i u m citrate, 38.1 m M citric acid a n d 122 m M dextrose), was centrifuged at 1000 × g for 10 min a n d the platelets were s u s p e n d e d in a solution of 150 m M KCI c o n t a i n i n g 2 m g / m l fibrinogen buffered with 10 m M Tris-HC1, a d j u s t e d to p H 7.4 for the use of aggregation m e a s u r e m e n t . F o r the use of fluorescence m e a s u r e m e n t , the platelets were s u s p e n d e d in the same m e d i u m without fibrinogen. S p o n t a neous platelet aggregation d u r i n g storage was prevented by a d d i n g 129 m M citrate (adjusted to p H 7.4) to these suspensions at a v o l u m e ratio of 1:19. Measurement of aggregation. The platelet susp e n s i o n o b t a i n e d as d e s c r i b e d a b o v e was m i x e d with 9 vol. of solutions of 150 m M c o n c e n t r a t i o n s of various cations a n d anions buffered with 10 m M Tris-HC1 ( p H 7.4). The final platelet conc e n t r a t i o n was a b o u t 9 . 104/btl. The suspension was mixed with CaC12 a n d then A D P , a n d the a b s o r b a n c e change at 600 nm was r e c o r d e d at 37°C in a UV-180 s p e c t r o p h o t o m e t e r ( S h i m a d z u Seisakusho Co., K y o t o , J a p a n ) e q u i p p e d with a stirrer and t h e r m o s t a t . The rate of aggregation was m e a s u r e d as the steepest tangential slope of the d o w n w a r d deflection of the records. Measurement of TNS fluorescence T N S was e m p l o y e d as a p r o b e to m e a s u r e the surface charge c h a n g e in platelet m e m b r a n e s as d e s c r i b e d p r e v i o u s l y [5]. T N S was a d d e d at a final c o n c e n t r a t i o n of 5 ~ M to a platelet suspension as d e s c r i b e d above. T h e fluorescence was m e a s u r e d in the s p e c t r o f l u o r o m e t e r 650-40 ( H i t a c h i Seisakusho, Co., T o k y o , J a p a n ) at 37°C using exciation
a n d emission wavelengths of 323 nm and 433 nm, respectively.
Measurement of ionized Ca concentration The c o n c e n t r a t i o n of ionized Ca in the m e d i u m was m e a s u r e d with a C a electrode from H N U System Inc. ( N e w t o n , MA).
Results
Effects of monovalent cations on ADP-induced aggregation First we m e a s u r e d the aggregation in m e d i a c o n t a i n i n g 150 m M c o n c e n t r a t i o n s of the chloride salts of alkali metal cations a n d q u a t e r n a r y amm o n i u m cations and the p o t a s s i u m salts of ino r g a n i c anions to d e t e r m i n e the effects of m o n o v a lent cations on A D P - i n d u c e d aggregation. The c a t i o n s affected the aggregation rates a n d maxim u m aggregation to similar degrees. T a b l e I lists the relative aggregation rates in m e d i a c o n t a i n i n g five alkali metal c a t i o n s and six q u a t e r n a r y amm o n i u m cations. A t the lower C a 2 + c o n c e n t r a t i o n , there were m a r k e d differences in aggregations with
TABLE I EFFECTS OF CATIONS ON 10 ~M ADP-INDUCED AGGREGATION OF BOVINE PLATELETS The platelet suspension obtained as described in Materials and Methods was mixed with 9 vol. of solutions of 150 mM of each cation, buffered with 10 mM Tris-HCl (pH 7.4). The suspension was mixed with 200 btM or 5 mM CaC12 and then ADP, and the absorbance change at 600 nm was recorded at 37°C. Values are means± S.D. for three experiments. Cation Li ÷ Na + K~ Rb + Cs + Choline TMA TEA TBA TMBA DDA
Relative aggregation 200 ttM CaCI 2
5 mM CaCI 2
31.6 + 12.1 27.8+ 7.3 100 a 103.8 ± 13.2 87.3± 3.3 103.5 _+ 3.5 86.6± 4.0 73.0_+ 2.6 13.9_+ 0.7 43.0:t: 0.7 12.5± 0.2
77.4 + 3.6 71.4_+1.1 100 ~ 105.7 + 8.9 95.6±1.0 93.4±9.2 115.5 ± 7.0 103.6±3.0 36.6_+0.2 81.5±1.4 35.9_+0.5
The rate of aggregation in KC1 medium at each CaCI 2 concentration was defined as 100.
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different ions. Among the alkali metal cations, aggregation was greatest with K ÷ and Rb ÷, slightly less with Cs ÷ medium and least with Na ÷ and Li ÷. These results are consistent with those on ADPinduced aggregation of human platelets [2]. Among the quaternary ammonium cations, the order of aggregation was choline > TMA > TEA > TMBA > TBA > DDA. Aggregation decreased with increase in the hydrocarbon chain length of the ammonium ions or with the addition of a benzyl group. Aggregation in medium with TMA or choline proceeded to almost the same extent as that in medium with K ÷. At the high Ca 2+ concentration, the differences in aggregations with different ions were less. The aggregations with K ÷, Rb ÷, Cs ÷, choline, TMA and TEA were almost the same, and those with Li +, Na + and TMBA were only slightly less than with K +. However, the aggregations with TBA and DDA were still much less than that with K ÷, although their differences from that with K ÷ were less.
Effects of monovalent anions on ADP-induced aggregation Next we measured the aggregation in media containing 150 mM concentrations of the potassium salts of inorganic anions, CI-, Br-, I-, S C N and CIO4-, to determine the effects of the anions on platelet aggregation. Like cations, anions affected the aggregation rates and maximum aggregation to similar degrees. Table II lists the relative
aggregations in media containing the anions. At the lower Ca 2÷ concentration, the order of aggregation was CI- > B r - > I - > C104- > SCN-, which resembles with that of the lyotropic effects of the anions [7]. These results are also consistent with those on the effects of the same anions on ADPinduced aggregation with concentrated bovine platelet-rich plasma [4]. At the high Ca 2÷ concentration, the differences in aggregations with different ions were little changed. Only the difference between C1- and B r - disappeared. These results are quite different from those on the effects of monovalent cations described above.
Effects of ions on membrane surface charge The membrane surface charge or surface potential has been suggested to have important roles in membrane stability and cellular functions [8,9]. We have already suggested the relationship between the inhibition of platelet aggregation by saturated fatty acids and a change in the surface charge of the platelet plasma membrane [5]. To investigate the mechanism of the effects of monovalent cations and anions on platelet aggregation, we then examined the effects of the ions on the membrane surface charge by measuring the fluorescence of TNS as described previously [5]. As shown in Table III, anions affected the intensities of TNS fluorescence. The order of fluorescence in five anion media was C I - > B r - > I - > S C N - > CIO)4. This order is consistent with that of aggregation, except for the order of S C N - and C1Oa-. These results suggest that these anions in-
TABLE 11 EFFECTS OF A N I O N S ON 10 /tM A D P - I N D U C E D AGG R E G A T I O N OF BOVINE PLATELETS Experimental procedure was similar to that in Table 1. Values are means _+S.D. for three experiments. Anion
CIBrI SCNCIO~
relative aggregation 200 ~M CaCI 2
5 mM CaCI 2
100 a 82.1_+5.0 50.3_+5.6 19.5_+3.4 30.8_+1.8
100 a 99.4_+1.4 60.5_+7.0 21.4_+1.2 29.2_+1.0
The rate of aggregation in KCI medium at each CaC12 concentration was defined as 100.
TABLE Ill EFFECTS OF ANIONS ON TNS FLUORESCENCE Platelet suspension as described in the Materials and Methods were mixed with 9 vol. of 150 mM of the various anion media. After incubation of platelets with 5 p.M TNS for 1 min, fluorescence intensities were measured. Values are means _+S.D. for two experiments. CIBr I SCN CIO4-
100 a 75.5 -+ 1.4 38.9_+1.3 32.8 _+0.6 25.4 _+0.6
Fluorescence intensity in KCI medium was defined as 100.
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creased the negative charge on the membrane, repelling the anion TNS from the membrane in the reverse order. To further investigate the relationship between the change of membrane negative charge and aggregation ability, we examined the effects of various concentrations of anions on TNS fluorescence and aggregation. As shown in Fig. 1 for S C N - , anions affected the fluorescence and aggregation over almost the same concentration range. On the other hand, cations affected the fluorescence intensities less than anions, as shown in Table IV. The order of fluorescence in four alkali metal cations was L i + > N a + > K + > Cs ÷, which is similar to that in erythrocyte membranes [10]. However, the differences in fluorescence among these cations were not significant compared with those among the anions described above, although inhibitory effects of N a ÷ and Li ÷ on aggregation resemble with those of SCN and CIO4- at least at low Ca 2+ concentration. As for quaternary a m m o n i u m cations, their in-
100 o v
8
80
60 C
.o
n~
n~
40
teraction with organic anion TNS should be considered at the fluorescence analysis especially when their concentrations are high. We examined the effect of 10 mM DDA, which concentration was high enough to exhibit marked inhibitory effects on aggregation as shown in Table V. However, as shown in Table IV, it also affected TNS fluorescence little.
Kinetic analysis of the interaction between Ca" + and monovalent cations Experimental results on TNS fluorescence suggested a relationship between change in membrane surface charge and aggregation inhibition for anions, but not for cations. Then, to obtain information on the mechanism of the effects of monovalent cations on platelet aggregation, we studied the kinetics of the interaction between Ca e+ and monovalent cations, because the effects of cations on aggregation were dependent on the Ca 2+ concentrations as described above. As in the study of Anderson and Foulks [11] on the effects of Li ÷ and H ÷ on aggregation, we examined the Michaelis-Menten kinetics of the interaction for cations which exhibited marked inhibitory effects on aggregation. Ca 2÷ was assumed to be a substrate ion for the aggregation reaction and monovalent cations as the inhibitors. The control Linewaver-Burk plot gave a straight line and provided a value of (1.42 + 0.13). 10 -4 M for the K,, + S.D. of the ADP-induced aggregation reaction of bovine platelets. This value is quite similar with that for the same stimulant-induced aggregation of rabbit platelets [11 ], although
20
0
I
I
I
50
100
150
Concentration of SCN-
(raM)
Fig. 1. Effect of SCN - on 10 # M A D P - i n d u c e d aggregation (e) a n d T N S fluorescence ( O ) . The platelet s u s p e n s i o n o b t a i n e d as described in Materials a n d M e t h o d s was m i x e d with 9 vol. of s o l u t i o n s c o n t a i n i n g various ratios of p o t a s s i u m salts of C1and SCN-, whose total c o n c e n t r a t i o n was 150 mM. The suspension was m i x e d with 200 # M CaCI 2 and then A D P , a n d the a b s o r b a n c e c h a n g e at 600 n m was recorded at 37°C. T N S fluorescence in the same platelet s u s p e n s i o n w i t h o u t fibrinogen was m e a s u r e d as described in T a b l e III. The rate of aggregation a n d fluorescence i n t e n s i t y of T N S in KCI m e d i u m were each defined as 100%. D a t a are m e a n s _+ S.D. for two or three experiments.
T A B L E IV EFFECTS OF CATIONS ON TNS FLUORESCENCE E x p e r i m e n t a l p r o c e d u r e was as for T a b l e III. Values are m e a n s _+ S.D. for three experiments. Cation
Relative fluorescence
Li + Na + K÷ Cs ÷ D D A (10 m M ) b
118.3___4.0 108.9_+1.7 100 a 93.6_+5.1 97.4_+2.7
a Fluorescence intensity in KCI m e d i u m was defined as 100. h Platelet suspension c o n t a i n e d 140 m M KCI.
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TABLE V INHIBITORY CONSTANTS, K i, OF MONOVALENT CATIONS Inhibitory constant of each cations was determined by the Dixon plots of the effect of each cations. Values are means +_ S.D. for duplicate assays on duplicate samples. Cation
K i (mM)
Li + Na ÷ TBA DDA
29.8 + 1.1 28.0 + 7.1 12.3 + 3.9 2.0 _+0.4
the aggregation abilities of these two mammalian platelets are different. Lineweaver-Burk plots and Dixon plots on the effects of Li ÷, Na ÷, TBA and relatively low concentrations of DDA suggest that these cations interact competitively with Ca 2÷ in the aggregation process. Dixon plots gave the inhibitory constants. K~ for these cations, which are shown in Table V. These results suggest that amphiphilic cation DDA strongly competes with Ca 2+ at some process in aggregation, resulting in inhibition of aggregation. The g i values of Li ÷ and Na ÷ were almost the same. The value for Li ÷ was also similar to that for rabbit platelets [11]. At much higher concentrations of DDA, its inhibitory effects did not increase so much, as shown in Table I, indicating that the effects are different from those at the lower concentrations. Discussion
In this work we investigated the effects of both monovalent cations and anions on ADP-induced aggregation of bovine platelets and tried to reveal the mechanisms of their effects. Our results presented here suggest that the mechanisms of the effects of cations and anions were different from each other, because the dependencies of their effects on Ca 2÷ concentration and their effects on membrane surface charge were quite different. Inorganic anions seem to affect aggregation at least partly due to their effect in changing the surface charge of the platelet plasma membranes just like saturated fatty acids, as reported previously [5], because a relationship between their effects on
membrane surface charge and those on aggregation was observed. S C N - and CIO4 are known to produce negative electrostatic potential at the surface of artificial phosphatidylethanolamine membrane [12]. In platelet membrane, since neutral phospholipids such as phosphatidylcholine and sphingomyelin are mainly located in the outer leaflet [13], anions such as S C N - and C10 4 are also thought to interact with the phospholipids, especially with their choline groups on the other surface, thus increasing the net negative charge on the outer surface. The order of the effects of anions on platelet aggregation probably reflects the order of their binding capacities to the membrane surface. A different order of the effects of S C N - and C10 4 on surface charge than the order of their effects on aggregation may be due to oxidative power of
c1o4. It is unknown why change in surface charge should affect platelet function. Since the change in surface charge is suggested to modify various membrane-associated functions such as enzymatic activities [9] and ionic transport [14] as well as cellular shape [8], as a result of their changes, it may affect platelet function. Our results presented here and previously [5] also suggest that amphiphilic anions generally affect aggregation by the same mechanism. On the other hand, according to our kinetic analysis, the effects of monovalent cations on platelets seem to be due to their competition with Ca 2 + at some step in platelet aggregation. Aggregation proceeded in media with K ÷, Rb ÷, Cs ÷, choline, TMA or TEA, because of their weak competition with Ca 2÷, but was restricted in media with Li ÷, Na ÷, TMBA, TBA or DDA, because of their strong competition with Ca 2÷. In this sense the effects of the latter cations resemble the effect of the trivalent cation Gd 3÷, whose inhibitory effect on platelet aggregation and serotonin release has been reported [15]. In platelets, Ca 2+ plays essential roles at several steps in their activating process. On the outside of the plasma membrane, Ca 2+ is necessary for the complex formation of glycoproteins lib and Ilia which have been suggested to be fibrinogen receptor proteins [16,17]. Moreover, extracellular Ca 2÷ is also essential for fibrinogen binding itself to
376
stimulated platelets [18]. On the other hand, as is a well-known fact, cytoplasmic Ca 2+ plays essential roles in regualtion of platelet functions by modifying a lot of enzymes such as phospholipase A 2 and myosin light-chain kinase [19]. Since quaternary ammonium cations seem to have much lower permeabilities through plasma membrane than alkali metal cations, and since preincubation of platelets with monovalent cations did not alter their effects significantly (data not shown), it is more probable that the effects of monovalent cations on platelets were pronounced due to their action at some Ca2+-dependent process on the outside of the plasma membrane rather than its cytoplasmic side. these cations may affect fibrinogen binding to the platelet plasma membrane due to competition with C a 2 +.
Several reports suggest that depolarization of platelets is necessary for their activition [3,20,21] and the ionic dependency of platelet aggregation is often explained in relation to this, especially for cations [3]. However, some workers do not believe that change in membrane potential is involved in platelet activation [22]. The present results suggest that monovalent cations affect platelet aggregation mainly by competition with Ca 2 + at some stage in the aggregation reaction. However, since the effects of monovalent cations on platelet adenylate cyclase must also be considered with alkali metal cations [23], further studies on the mechanism of the effects of the cations as well as anions on platelet aggregation is now in progress. Acknowledgement This work was supported in part by a grant from the Japanese Ministry of Education, Science and Culture (No. 58771623 and 59771674).
References 1 Born, G.V.R. and Cross, M.J. (1964) J. Physiol. (Lond.) 170, 397-414 2 Greil, W., Patscheke, H. and Brossmer, R. (t972) FEBS Lett. 26, 271 273 3 Friedhoff, L.T., Kim, E., Priddle, M. and Sonenberg, M. (1981) Biochem. Biophys. Res. Commun. 102, 832 837 4 Kitagawa, S., Hongu, Y. and Kametani, F. (1982) Biochem. Biophys. Res. Commun. 104, 1371-1375 5 Kitagawa, S., Nishitama, H. and Kametani, F. (1984) Biochim. Biophys. Acta 775, 197-202 6 Kitagawa, S., Endo, J. and Kametani, F. (1984) Biochim. Biophys. Acta 798, 210 215 7 McBain, J.W. (1950) Colloid Science, Heath, Boston 8 Fortes, P.A.G. and EIIory, J.C. (1975) Biochim. Biophys. Acta 413, 65-78 9 Wojtczak, L. and Nalecz. M.J. (1979) Eur. J. Biochem. 94, 99-107 10 Handa, T., Ichihashi, C., Matsumoto, M. and Nakagaki, M. (1984) Yakugaku Zasshi 104, 576-582 11 Anderson, E.R. and Foulks, J.G. (1976) Thrombos. Haemostas. 36, 343 359 12 McLaughlin, S., Bruder, A., Chen, S. and Moser, C. (1975) Biochim. Biophys. Acta 394, 304 313 13 Chap, H.J., Zwaal, R.F.A. and Van Deenen, L.L.M. (1977) Biochim. Biophys. Acta 467, 146 164 14 Cabantchik, Z.I., Knauf, P.A. and Rothstein, A. (1978) Biochim. Biophys. Acta 515, 239-302 15 Brass, L.F. and Shattil, S.J. (1984) J. Clin. Invest. 73, 626 632 16 Fujimura, K. and Phillips, D.R. (1982) Circulation 66 (Suppl. 2), 11-174 17 Jennings, L.K. and Phillips, D.R. (1982) J. Biol. Chem. 257, 10458 10466 18 Marguerie, G.A. and Plow, E.F. (1983) Ann. N.Y. Acad. Sci. 408, 556-566 19 Feinstein, M.B. and Hadjian, R.A. (1982) Mol. Pharmacol. 21,422-431 20 Friedhoff, L.T. and Sonenberg, M. (1983) Blood 61,180-185 21 Greenberg-Sepersky, S.M. and Simons, E.R. (1984) J. Biol. Chem. 259, 1502-1508 22 Maclntyre, D.E. and Rink, T.J. (1982) " Thrombos. Haemostas. 47, 22-26 23 Steer, M.L. and Wood, A. (1981) J. Biol. Chem. 256, 9990-9993
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BBA 22066
E f f e c t s of m o n o v a l e n t c a t i o n s and anions on A D P - i n d u c e d a g g r e g a t i o n of b o v i n e platelets, and m e c h a n i s m s t h e r e o f Shuji K i t a g a w a *, H i d e a k i Seki a n d F u j i o K a m e t a n i Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi 1, Tokushima 770 (Japan) (Received November 6th, 1984) (Revised manuscript received March 25th, 1985)
Key words: Monovalent ion; ADP; Platelet aggregation; (Bovine)
The effects of monovalent cations - inorganic alkali metal cations and organic quaternary ammonium cations - and monovalent inorganic anions on ADP-induced aggregation of bovine platelets were investigated. In the presence of K +, Rb +, Cs +, choline or tetramethylammonium, aggregation proceeded. However, aggregation was markedly restricted in media containing Li +, Na +, tetrabutylammonium or dimethyldibenzylammonium. With anions, aggregation proceeded in the order C I - > Br - > I - > CIO4-- > SCN -. The effects of cations significantly depended on Ca 2+ concentration, whereas those of the anions depended little on Ca 2+. Anions such as S C N - and 0 0 4 markedly decreased the fluorescence of the sudaee charge probe 2-p-toluidinylnaphthalene-6-sulfonate, whereas cations had less pronounced effects. The relative effects of the anions on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that inhibition of platelet aggregation by the anions is due to a change in the surface change of the platelet plasma membrane. On the other hand, kinetic analysis suggests that the effects of monovalent cations on platelet aggregation are due to their competition with Ca 2+ during the process of aggregation.
Introduction
Platelet aggregation is affected by the ionic milieu, and has been shown to depend on the concentration of monovalent cations such as K ÷ [1-3] and divalent cations such as Ca 2+ [1]. We have previously reported, by using concentrated platelet-rich plasma, that ADP-induced aggregation is also affected by inorganic anions in the medium [4]. However, their inhibitory mechanism is still unknown. It has been shown that platelet * To whom correspondence should be addressed. Abbreviations: THIS, 2-p-toluidinylnaphthalene-6-sulfonate; TMA, tetramethylammonium; TEA, tetraethylamrnonium; TMBA, trimethylbenzylammonium; TBA, tetrabutylammonium; DDA, dimethyldibenzylammonium.
functions are inhibited by various organic substances [5,6]. Among these substances, we have shown that fatty acids (organic anions) inhibit platelet aggregation due to modification of membrane surface change or membrane fluidity [5,6]. In this work we examined the effects of five alkali metal cations, six quaternary ammonium cations and five inorganic anions on ADP-induced aggregation. It is the first time that the effects of quaternary ammonium cations on platelet aggregation have been studied. In the present investigation we aimed at a further understanding of the mechanisms by which both cations and anions affect the platelet aggregation, in particular in relation to their effects on membrane surface charge and their competition with Ca 2+ during the process.
0304-4165/85/$03.30 © Elsevier Science Publishers B.V. (Biomedical Division)
372
Materials and Methods
Materials. Bovine fibrinogen a n d 2-p-toluidinyln a p h t h a l e n e - 6 - s u l f o n a t e ( T N S ) were p u r c h a s e d f r o m Sigma (St. Louis, MO). A D P was p u r c h a s e d from O r i e n t a l Yeast Co. (Tokyo, Japan). Potassium salts of anions a n d chloride salts of c a t i o n s were p u r c h a s e d from W a k o Pure C h e m i c a l Industries (Osaka, J a p a n ) or T o k y o C h e m i c a l Ind u s t r y Co. (Tokyo, Japan). Preparation of platelet suspension. Platelet-rich p l a s m a of bovine (Holstein) b l o o d was o b t a i n e d as d e s c r i b e d in a previous p a p e r [4]. The platelet-rich plasma, which c o n t a i n e d a b o u t 10% ( v / v ) A C D c o a g u l a n t solution (74.8 m M s o d i u m citrate, 38.1 m M citric acid a n d 122 m M dextrose), was centrifuged at 1000 × g for 10 min a n d the platelets were s u s p e n d e d in a solution of 150 m M KCI c o n t a i n i n g 2 m g / m l fibrinogen buffered with 10 m M Tris-HC1, a d j u s t e d to p H 7.4 for the use of aggregation m e a s u r e m e n t . F o r the use of fluorescence m e a s u r e m e n t , the platelets were s u s p e n d e d in the same m e d i u m without fibrinogen. S p o n t a neous platelet aggregation d u r i n g storage was prevented by a d d i n g 129 m M citrate (adjusted to p H 7.4) to these suspensions at a v o l u m e ratio of 1:19. Measurement of aggregation. The platelet susp e n s i o n o b t a i n e d as d e s c r i b e d a b o v e was m i x e d with 9 vol. of solutions of 150 m M c o n c e n t r a t i o n s of various cations a n d anions buffered with 10 m M Tris-HC1 ( p H 7.4). The final platelet conc e n t r a t i o n was a b o u t 9 . 104/btl. The suspension was mixed with CaC12 a n d then A D P , a n d the a b s o r b a n c e change at 600 nm was r e c o r d e d at 37°C in a UV-180 s p e c t r o p h o t o m e t e r ( S h i m a d z u Seisakusho Co., K y o t o , J a p a n ) e q u i p p e d with a stirrer and t h e r m o s t a t . The rate of aggregation was m e a s u r e d as the steepest tangential slope of the d o w n w a r d deflection of the records. Measurement of TNS fluorescence T N S was e m p l o y e d as a p r o b e to m e a s u r e the surface charge c h a n g e in platelet m e m b r a n e s as d e s c r i b e d p r e v i o u s l y [5]. T N S was a d d e d at a final c o n c e n t r a t i o n of 5 ~ M to a platelet suspension as d e s c r i b e d above. T h e fluorescence was m e a s u r e d in the s p e c t r o f l u o r o m e t e r 650-40 ( H i t a c h i Seisakusho, Co., T o k y o , J a p a n ) at 37°C using exciation
a n d emission wavelengths of 323 nm and 433 nm, respectively.
Measurement of ionized Ca concentration The c o n c e n t r a t i o n of ionized Ca in the m e d i u m was m e a s u r e d with a C a electrode from H N U System Inc. ( N e w t o n , MA).
Results
Effects of monovalent cations on ADP-induced aggregation First we m e a s u r e d the aggregation in m e d i a c o n t a i n i n g 150 m M c o n c e n t r a t i o n s of the chloride salts of alkali metal cations a n d q u a t e r n a r y amm o n i u m cations and the p o t a s s i u m salts of ino r g a n i c anions to d e t e r m i n e the effects of m o n o v a lent cations on A D P - i n d u c e d aggregation. The c a t i o n s affected the aggregation rates a n d maxim u m aggregation to similar degrees. T a b l e I lists the relative aggregation rates in m e d i a c o n t a i n i n g five alkali metal c a t i o n s and six q u a t e r n a r y amm o n i u m cations. A t the lower C a 2 + c o n c e n t r a t i o n , there were m a r k e d differences in aggregations with
TABLE I EFFECTS OF CATIONS ON 10 ~M ADP-INDUCED AGGREGATION OF BOVINE PLATELETS The platelet suspension obtained as described in Materials and Methods was mixed with 9 vol. of solutions of 150 mM of each cation, buffered with 10 mM Tris-HCl (pH 7.4). The suspension was mixed with 200 btM or 5 mM CaC12 and then ADP, and the absorbance change at 600 nm was recorded at 37°C. Values are means± S.D. for three experiments. Cation Li ÷ Na + K~ Rb + Cs + Choline TMA TEA TBA TMBA DDA
Relative aggregation 200 ttM CaCI 2
5 mM CaCI 2
31.6 + 12.1 27.8+ 7.3 100 a 103.8 ± 13.2 87.3± 3.3 103.5 _+ 3.5 86.6± 4.0 73.0_+ 2.6 13.9_+ 0.7 43.0:t: 0.7 12.5± 0.2
77.4 + 3.6 71.4_+1.1 100 ~ 105.7 + 8.9 95.6±1.0 93.4±9.2 115.5 ± 7.0 103.6±3.0 36.6_+0.2 81.5±1.4 35.9_+0.5
The rate of aggregation in KC1 medium at each CaCI 2 concentration was defined as 100.
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different ions. Among the alkali metal cations, aggregation was greatest with K ÷ and Rb ÷, slightly less with Cs ÷ medium and least with Na ÷ and Li ÷. These results are consistent with those on ADPinduced aggregation of human platelets [2]. Among the quaternary ammonium cations, the order of aggregation was choline > TMA > TEA > TMBA > TBA > DDA. Aggregation decreased with increase in the hydrocarbon chain length of the ammonium ions or with the addition of a benzyl group. Aggregation in medium with TMA or choline proceeded to almost the same extent as that in medium with K ÷. At the high Ca 2+ concentration, the differences in aggregations with different ions were less. The aggregations with K ÷, Rb ÷, Cs ÷, choline, TMA and TEA were almost the same, and those with Li +, Na + and TMBA were only slightly less than with K +. However, the aggregations with TBA and DDA were still much less than that with K ÷, although their differences from that with K ÷ were less.
Effects of monovalent anions on ADP-induced aggregation Next we measured the aggregation in media containing 150 mM concentrations of the potassium salts of inorganic anions, CI-, Br-, I-, S C N and CIO4-, to determine the effects of the anions on platelet aggregation. Like cations, anions affected the aggregation rates and maximum aggregation to similar degrees. Table II lists the relative
aggregations in media containing the anions. At the lower Ca 2÷ concentration, the order of aggregation was CI- > B r - > I - > C104- > SCN-, which resembles with that of the lyotropic effects of the anions [7]. These results are also consistent with those on the effects of the same anions on ADPinduced aggregation with concentrated bovine platelet-rich plasma [4]. At the high Ca 2÷ concentration, the differences in aggregations with different ions were little changed. Only the difference between C1- and B r - disappeared. These results are quite different from those on the effects of monovalent cations described above.
Effects of ions on membrane surface charge The membrane surface charge or surface potential has been suggested to have important roles in membrane stability and cellular functions [8,9]. We have already suggested the relationship between the inhibition of platelet aggregation by saturated fatty acids and a change in the surface charge of the platelet plasma membrane [5]. To investigate the mechanism of the effects of monovalent cations and anions on platelet aggregation, we then examined the effects of the ions on the membrane surface charge by measuring the fluorescence of TNS as described previously [5]. As shown in Table III, anions affected the intensities of TNS fluorescence. The order of fluorescence in five anion media was C I - > B r - > I - > S C N - > CIO)4. This order is consistent with that of aggregation, except for the order of S C N - and C1Oa-. These results suggest that these anions in-
TABLE 11 EFFECTS OF A N I O N S ON 10 /tM A D P - I N D U C E D AGG R E G A T I O N OF BOVINE PLATELETS Experimental procedure was similar to that in Table 1. Values are means _+S.D. for three experiments. Anion
CIBrI SCNCIO~
relative aggregation 200 ~M CaCI 2
5 mM CaCI 2
100 a 82.1_+5.0 50.3_+5.6 19.5_+3.4 30.8_+1.8
100 a 99.4_+1.4 60.5_+7.0 21.4_+1.2 29.2_+1.0
The rate of aggregation in KCI medium at each CaC12 concentration was defined as 100.
TABLE Ill EFFECTS OF ANIONS ON TNS FLUORESCENCE Platelet suspension as described in the Materials and Methods were mixed with 9 vol. of 150 mM of the various anion media. After incubation of platelets with 5 p.M TNS for 1 min, fluorescence intensities were measured. Values are means _+S.D. for two experiments. CIBr I SCN CIO4-
100 a 75.5 -+ 1.4 38.9_+1.3 32.8 _+0.6 25.4 _+0.6
Fluorescence intensity in KCI medium was defined as 100.
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creased the negative charge on the membrane, repelling the anion TNS from the membrane in the reverse order. To further investigate the relationship between the change of membrane negative charge and aggregation ability, we examined the effects of various concentrations of anions on TNS fluorescence and aggregation. As shown in Fig. 1 for S C N - , anions affected the fluorescence and aggregation over almost the same concentration range. On the other hand, cations affected the fluorescence intensities less than anions, as shown in Table IV. The order of fluorescence in four alkali metal cations was L i + > N a + > K + > Cs ÷, which is similar to that in erythrocyte membranes [10]. However, the differences in fluorescence among these cations were not significant compared with those among the anions described above, although inhibitory effects of N a ÷ and Li ÷ on aggregation resemble with those of SCN and CIO4- at least at low Ca 2+ concentration. As for quaternary a m m o n i u m cations, their in-
100 o v
8
80
60 C
.o
n~
n~
40
teraction with organic anion TNS should be considered at the fluorescence analysis especially when their concentrations are high. We examined the effect of 10 mM DDA, which concentration was high enough to exhibit marked inhibitory effects on aggregation as shown in Table V. However, as shown in Table IV, it also affected TNS fluorescence little.
Kinetic analysis of the interaction between Ca" + and monovalent cations Experimental results on TNS fluorescence suggested a relationship between change in membrane surface charge and aggregation inhibition for anions, but not for cations. Then, to obtain information on the mechanism of the effects of monovalent cations on platelet aggregation, we studied the kinetics of the interaction between Ca e+ and monovalent cations, because the effects of cations on aggregation were dependent on the Ca 2+ concentrations as described above. As in the study of Anderson and Foulks [11] on the effects of Li ÷ and H ÷ on aggregation, we examined the Michaelis-Menten kinetics of the interaction for cations which exhibited marked inhibitory effects on aggregation. Ca 2÷ was assumed to be a substrate ion for the aggregation reaction and monovalent cations as the inhibitors. The control Linewaver-Burk plot gave a straight line and provided a value of (1.42 + 0.13). 10 -4 M for the K,, + S.D. of the ADP-induced aggregation reaction of bovine platelets. This value is quite similar with that for the same stimulant-induced aggregation of rabbit platelets [11 ], although
20
0
I
I
I
50
100
150
Concentration of SCN-
(raM)
Fig. 1. Effect of SCN - on 10 # M A D P - i n d u c e d aggregation (e) a n d T N S fluorescence ( O ) . The platelet s u s p e n s i o n o b t a i n e d as described in Materials a n d M e t h o d s was m i x e d with 9 vol. of s o l u t i o n s c o n t a i n i n g various ratios of p o t a s s i u m salts of C1and SCN-, whose total c o n c e n t r a t i o n was 150 mM. The suspension was m i x e d with 200 # M CaCI 2 and then A D P , a n d the a b s o r b a n c e c h a n g e at 600 n m was recorded at 37°C. T N S fluorescence in the same platelet s u s p e n s i o n w i t h o u t fibrinogen was m e a s u r e d as described in T a b l e III. The rate of aggregation a n d fluorescence i n t e n s i t y of T N S in KCI m e d i u m were each defined as 100%. D a t a are m e a n s _+ S.D. for two or three experiments.
T A B L E IV EFFECTS OF CATIONS ON TNS FLUORESCENCE E x p e r i m e n t a l p r o c e d u r e was as for T a b l e III. Values are m e a n s _+ S.D. for three experiments. Cation
Relative fluorescence
Li + Na + K÷ Cs ÷ D D A (10 m M ) b
118.3___4.0 108.9_+1.7 100 a 93.6_+5.1 97.4_+2.7
a Fluorescence intensity in KCI m e d i u m was defined as 100. h Platelet suspension c o n t a i n e d 140 m M KCI.
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TABLE V INHIBITORY CONSTANTS, K i, OF MONOVALENT CATIONS Inhibitory constant of each cations was determined by the Dixon plots of the effect of each cations. Values are means +_ S.D. for duplicate assays on duplicate samples. Cation
K i (mM)
Li + Na ÷ TBA DDA
29.8 + 1.1 28.0 + 7.1 12.3 + 3.9 2.0 _+0.4
the aggregation abilities of these two mammalian platelets are different. Lineweaver-Burk plots and Dixon plots on the effects of Li ÷, Na ÷, TBA and relatively low concentrations of DDA suggest that these cations interact competitively with Ca 2÷ in the aggregation process. Dixon plots gave the inhibitory constants. K~ for these cations, which are shown in Table V. These results suggest that amphiphilic cation DDA strongly competes with Ca 2+ at some process in aggregation, resulting in inhibition of aggregation. The g i values of Li ÷ and Na ÷ were almost the same. The value for Li ÷ was also similar to that for rabbit platelets [11]. At much higher concentrations of DDA, its inhibitory effects did not increase so much, as shown in Table I, indicating that the effects are different from those at the lower concentrations. Discussion
In this work we investigated the effects of both monovalent cations and anions on ADP-induced aggregation of bovine platelets and tried to reveal the mechanisms of their effects. Our results presented here suggest that the mechanisms of the effects of cations and anions were different from each other, because the dependencies of their effects on Ca 2÷ concentration and their effects on membrane surface charge were quite different. Inorganic anions seem to affect aggregation at least partly due to their effect in changing the surface charge of the platelet plasma membranes just like saturated fatty acids, as reported previously [5], because a relationship between their effects on
membrane surface charge and those on aggregation was observed. S C N - and CIO4 are known to produce negative electrostatic potential at the surface of artificial phosphatidylethanolamine membrane [12]. In platelet membrane, since neutral phospholipids such as phosphatidylcholine and sphingomyelin are mainly located in the outer leaflet [13], anions such as S C N - and C10 4 are also thought to interact with the phospholipids, especially with their choline groups on the other surface, thus increasing the net negative charge on the outer surface. The order of the effects of anions on platelet aggregation probably reflects the order of their binding capacities to the membrane surface. A different order of the effects of S C N - and C10 4 on surface charge than the order of their effects on aggregation may be due to oxidative power of
c1o4. It is unknown why change in surface charge should affect platelet function. Since the change in surface charge is suggested to modify various membrane-associated functions such as enzymatic activities [9] and ionic transport [14] as well as cellular shape [8], as a result of their changes, it may affect platelet function. Our results presented here and previously [5] also suggest that amphiphilic anions generally affect aggregation by the same mechanism. On the other hand, according to our kinetic analysis, the effects of monovalent cations on platelets seem to be due to their competition with Ca 2 + at some step in platelet aggregation. Aggregation proceeded in media with K ÷, Rb ÷, Cs ÷, choline, TMA or TEA, because of their weak competition with Ca 2÷, but was restricted in media with Li ÷, Na ÷, TMBA, TBA or DDA, because of their strong competition with Ca 2÷. In this sense the effects of the latter cations resemble the effect of the trivalent cation Gd 3÷, whose inhibitory effect on platelet aggregation and serotonin release has been reported [15]. In platelets, Ca 2+ plays essential roles at several steps in their activating process. On the outside of the plasma membrane, Ca 2+ is necessary for the complex formation of glycoproteins lib and Ilia which have been suggested to be fibrinogen receptor proteins [16,17]. Moreover, extracellular Ca 2÷ is also essential for fibrinogen binding itself to
376
stimulated platelets [18]. On the other hand, as is a well-known fact, cytoplasmic Ca 2+ plays essential roles in regualtion of platelet functions by modifying a lot of enzymes such as phospholipase A 2 and myosin light-chain kinase [19]. Since quaternary ammonium cations seem to have much lower permeabilities through plasma membrane than alkali metal cations, and since preincubation of platelets with monovalent cations did not alter their effects significantly (data not shown), it is more probable that the effects of monovalent cations on platelets were pronounced due to their action at some Ca2+-dependent process on the outside of the plasma membrane rather than its cytoplasmic side. these cations may affect fibrinogen binding to the platelet plasma membrane due to competition with C a 2 +.
Several reports suggest that depolarization of platelets is necessary for their activition [3,20,21] and the ionic dependency of platelet aggregation is often explained in relation to this, especially for cations [3]. However, some workers do not believe that change in membrane potential is involved in platelet activation [22]. The present results suggest that monovalent cations affect platelet aggregation mainly by competition with Ca 2 + at some stage in the aggregation reaction. However, since the effects of monovalent cations on platelet adenylate cyclase must also be considered with alkali metal cations [23], further studies on the mechanism of the effects of the cations as well as anions on platelet aggregation is now in progress. Acknowledgement This work was supported in part by a grant from the Japanese Ministry of Education, Science and Culture (No. 58771623 and 59771674).
References 1 Born, G.V.R. and Cross, M.J. (1964) J. Physiol. (Lond.) 170, 397-414 2 Greil, W., Patscheke, H. and Brossmer, R. (t972) FEBS Lett. 26, 271 273 3 Friedhoff, L.T., Kim, E., Priddle, M. and Sonenberg, M. (1981) Biochem. Biophys. Res. Commun. 102, 832 837 4 Kitagawa, S., Hongu, Y. and Kametani, F. (1982) Biochem. Biophys. Res. Commun. 104, 1371-1375 5 Kitagawa, S., Nishitama, H. and Kametani, F. (1984) Biochim. Biophys. Acta 775, 197-202 6 Kitagawa, S., Endo, J. and Kametani, F. (1984) Biochim. Biophys. Acta 798, 210 215 7 McBain, J.W. (1950) Colloid Science, Heath, Boston 8 Fortes, P.A.G. and EIIory, J.C. (1975) Biochim. Biophys. Acta 413, 65-78 9 Wojtczak, L. and Nalecz. M.J. (1979) Eur. J. Biochem. 94, 99-107 10 Handa, T., Ichihashi, C., Matsumoto, M. and Nakagaki, M. (1984) Yakugaku Zasshi 104, 576-582 11 Anderson, E.R. and Foulks, J.G. (1976) Thrombos. Haemostas. 36, 343 359 12 McLaughlin, S., Bruder, A., Chen, S. and Moser, C. (1975) Biochim. Biophys. Acta 394, 304 313 13 Chap, H.J., Zwaal, R.F.A. and Van Deenen, L.L.M. (1977) Biochim. Biophys. Acta 467, 146 164 14 Cabantchik, Z.I., Knauf, P.A. and Rothstein, A. (1978) Biochim. Biophys. Acta 515, 239-302 15 Brass, L.F. and Shattil, S.J. (1984) J. Clin. Invest. 73, 626 632 16 Fujimura, K. and Phillips, D.R. (1982) Circulation 66 (Suppl. 2), 11-174 17 Jennings, L.K. and Phillips, D.R. (1982) J. Biol. Chem. 257, 10458 10466 18 Marguerie, G.A. and Plow, E.F. (1983) Ann. N.Y. Acad. Sci. 408, 556-566 19 Feinstein, M.B. and Hadjian, R.A. (1982) Mol. Pharmacol. 21,422-431 20 Friedhoff, L.T. and Sonenberg, M. (1983) Blood 61,180-185 21 Greenberg-Sepersky, S.M. and Simons, E.R. (1984) J. Biol. Chem. 259, 1502-1508 22 Maclntyre, D.E. and Rink, T.J. (1982) " Thrombos. Haemostas. 47, 22-26 23 Steer, M.L. and Wood, A. (1981) J. Biol. Chem. 256, 9990-9993