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Antagonistic effects of N-ethylmaleimide on platelets treated with agents that are known to increase levels of cyclic AMP
THROMBOSIS RESEARCH 21; 201-206, 1981 0049-3848/81/010201-06$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
ANTAGONISTIC EFFECTS OF N-ETHYLMALEIMIDE ON PLATELETS TREATED WITH AGENTS THAT ARE KNOWN TO INCREASE LEVELS OF CYCLICAMP.
P. Wiirnerand H. Patscheke Abtlg. fUr Irnnunologieu. Serologie am Institut fi.lr Hygiene u. Med. Mikrobiologie des Klinikum Mannheim der UniversitBt Heidelberg, D 6, 5; D-68 Mannheim
(Received 1.10.1980; in revised form 9.12.1980. Accepted by Editor Hans Reuter)
INTRODUCTION The penetrating sulfhydryl-blocking agent N-ethylmaleimide (NEM) has been reported to interfere with platelet aggregation (1). Besides its inhibitory action, NEM at a low concentration promotes platelet shape change (2-4) which is an early event in platelet activation (5). At higher concentrations, NEM can also evoke prostaglandin synthesis (6) and, as its consequence, the release of serotonin (4). In addition to its ability to interfere with platelet aggregation, NEM exerts an anti-activating effect when platelets have been treated with agents known to elevate cyclic AMP. This anti-activation does not prevent NEM-induced shape change but postpones its onset. An elevation of cyclic AMP inhibits shape change induced by other stimuli such as ADP, thrombin, arachidonic acid, and A 23187 (7,8). A lag phase and an anti-activating effect prior to shape change does not occur with these stimuli of platelet activation. METHODS AND MATERIALS Platelet preparation. Discoid washed platelets were prepared from human ACDblood as has been described (8) with the exception that prostaglandin El was omitted throughout the preparation of platelets. The final platelet medium consisted of 103 mM NaCl, 45 mM Tris-HCl adjusted to pH 7.4 at 370 C, 5 mM KCl, 5 mM glucose, 1 mM sodium citrate, 1 mM CaC12, 0.5 mg albumin/ml and 50 ug apyrase/ml. Shape change of discoid platelets (2.108/ml) was followed in a 6-channel aggregometer equipped with interference filters 600 nm. The optical signal was amplified 5-fold and recorded after appropriate zero suppression. Three minutes prior to addition of a stimulus, 2 mM EDTA was added to prevent aggre201
202
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gation. The stirring mechanism (250 rpm) was switched off prior to the addition of a stimulus and at the end of an experiment to evaluate the stir-dependent change in light transmission (LT) which decreases the more, the more the platelets transform from a discoid to an irregular shape (8). Materials. Apyrase from potato (grade I), prostaglandin Ei and diamide were obtained from Sigma GmbH MUnchen, D 8021 Taufkirchen. Papaverinium chloride and N-ethylmaleimide were achieved from Merck, D 6100 Darmstadt. Dipyridamole was from Dr. Karl Thomae GmbH, D 7g5D Biberach. Prostaglandin D2 was a gift of the Upjohn Co, Kalamazoo, Michigan, USA. RESULTS Addition of NEM at a concentration of 50-100 uM to a suspension of discoid platelets resulted in changes of the optical signal which indicate the shape change of platelets (5,7,8). Shape change occurred at lower concentrations of NEM than those (>0.5 mM) which were required to evoke indcmethacin-sensitive formation of malondialdehyde and the release of serotonin. NEM-induced shape change is not inhibited by indomethacin or acetylsalicylic acid (4). Prostaglandin Ei (PGEi), PGD2, adenosine, papaverine and dipyridamole hardly inhibited NEM-induced shape change but postponed its onset (Fig. 1,I and Table I). During the lag phase prior to NEM-promoted shape change, the optical signal changed in a manner opposite to that characteristic for the transformation of discoid platelets to irregular spheres: light transmission (LT) and oscillation amplitude of the stirred platelet suspension increased (Fig. 1,I) as did the decrease in LT upon interruption of stirring. Treatment of platelets with PGEi (1 uM) and papaverine (10 uM) did not mimick this effect in the absence of NEM. A small lag phase prior to the shape change induced by 50 uM NEM was even observed when platelets had been treated with such a low concentration of PGEi as 0.5 nM. PGEi at 10 nM prolonged the time lapse to about 3 minutes. However, this lag phase did not exceed 3.5 minutes even when platelets were treated with 1 uM of PGEl combined with IO uM papaverine or 20 uM dipyridamole. When the concentration of NEM was increased, the time lapse up to the onset of shape change was shorter and disappeared at an NEM-concentration of about 2 mM. Shape change induced by diamide was also postponed after treatment of platelets with PGEi, adenosine, papaverine or dipyridamole. During the time lapse up to the onset of shape change, the oscillation amplitude and the LT as well as did the decrease in LT when the stirring increased (Fig. 1,II) mechanism was interrupted. PGEi at a high concentration (1 1 uM) causes changes in the optical signal which imitate a partial shape change (8). The anti-activating effect which was observed when diamide or NEM was added after PGEl was not only due to the reversal of the slight decrease in LT and oscillation amplitude promoted by PGEi, but did exceed the original level of LT. Moreover, the antiactivating effect of NEM or diamide was observed also at low concentrations of PGEi which per se had no influnece on platelet shape. DISCUSSION The transformation of discoid platelets to an irregular spheroid shape (shape change) can be followed by monitoring changes in optical signals. During shape change, the light transmission (LT) and the oscillation amplitude of a stirred platelet sample diminuish as well as the decrease in LT on stopping the stirrer (8). The change in LT which occurs on interruption of stirring seems to be particularly suited to estimate the extent to which platelets have transformed to a spheroid shape. Changes in the optical signal opposite to those observed during disc-to-sphere transformation indicate that
vo1.21,
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203
NEM AND PLATELET SHAPE
TABLE 1 Time Lapse prior to NEM-Induced Shape Change after Treatment of Platelets with Agents Known to Increase Levels of Cyclic AMP. control
0 min*
5 nM PGD2
2.6 min
5 nM PGE,
2.4 min
5 nM PGEl + 5 pM papaverine
2.9 min
0.5 uM adenosine
2.0 min
5 nM PGEi + 20 uM dipyridamole
3.5 min
5 uM papaverine
0.8 min
0.5 uM adenosine + 5 uM papaverine3.0
20 uM dipyridamole
0.2 min
0.5 uM adenosine + 20 pM dipyridamole
min
3.3 min
* = time from the addition of 50 uM NEM to the onset of the decrease in LT. PGEi, PGD2, adenosine, papaverine, and dipyridamole were added 2 min prior to NEM to the stirred platelet suspension (2*108/ml, 370 C). Single values from one platelet preparation out of a series of qualitatively similar results.
I
‘01 mM NEM
0.5 mM diamide
l0 pM odenosin
Fig. 1. Shape change induced by NEM and diamide. Effect of agents which elevate platelet CAMP. I: NEM (0.1 mM) evokes shape change without serotonin release (upper curve). After treatment of platelets with PGEi and papaverine a lag phase up to the onset of the decrease in light transmission (LT) is observed. During this lag phase oscillation amplitude and LT increase (lower curve). II: Diamide (0.5 mM) evokes shape change without serotonin release (upper curve). Treatment of platelets with adenosine and dipyridamole postponed the onset of shape change (lower curve). During this lag phase changes in the,optical signal opposite to those during shape change occur.
discoid platelets have become more flat. This has been observed with acid citrate (pH 6.5) which exerts an anti-activating effect on platelets (9). Such an anti-activating effect also occurs prior to NEM-induced shape change when platelets had been exposed to PGE1, PGD2, adenosine, papaverine, or dipyridamole. PGEl (lo)',PGD2 (11) and adenosine(12) increase cAMPby stimulating ade-
204
NEM AND PLATELET SHAPE
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nylcyclase. Papaverine and dipyridamole interfere with c/VlP-degradation by inhibiting platelet phosphodiesterase (12). Since agents from both groups have similar effects and act synergistic when combined, it is most likely that an elevation of CAMP is the common cause for the anti-activating effect of NEM in platelets treated with these agents. NEM has been reported to enhance the PGEj-induced stimulation of adenylcyclase and to increase the steady state level of CAMP (13). However, an elevated level of CAMP alone seems not to be sufficient to mediate this anti-activation: discoid platelets do not become more flat upon an elevation of CAMP, e.g. by their exposure to the combined action of papaverine and PGEl, unless NEM is added. An anti-activating effect similar to that which preceeds NEM-induced shape change occurs when platelets with elevated CAMP are exposed to the thiol-oxidizing agent diamide. The similarity of the anti-activating effects of NEM and diamide may indicate that these agents mediate anti-activation and possibly also the following shape change by their interaction with common targets, presumably thiols. However, diamide-induced platelet activation differs in several aspects from that promoted by NEM. Diamide does, unlike NEM, induce or enhance platelet aggregation prior to its inhibitory effect (14,15). Moreover, diamide does not evoke serotonin release or prostaglandin synthesis even at a high concentration. In addition, diamide-induced shape change, but hardly that promoted by NEM, can be completely suppressed by a high concentration of PGE1. These differences may be the result of different reactivities of various target molecules towards the thiol-alkylating agent NEM and the bivalent thiol-oxidizing substance diamide. It is noteworthy that NEM (and diamide) can evoke shape change after a lag phase during which PGEj-treated platelets become even more discoid. When PGEltreated platelets are exposed to other platelet stimuli such as thrombin, shape change is reduced or completely prevented but no lag phase and no antiactivating effect occurs prior to the onset of shape change (7,8). The ability of NEM to evoke shape change after a time lapse can not be due to a decrease in CAMP mediated by NEM since the concentrations of NEM employed even augment the PGEl-induced increase in platelet CAMP (13). An alternative explanation is more plausible: besides its contribution to the anti-activation, NEM creates a long-lasting platelet-activating signal which may even increase with time. This long-lasting signal may finally overcome the initial antiactivation by CAMP and NEM. Platelet stimuli such as thrombin may only create a short-lasting triggering signal which is not maintained or.does at least not increase with time. The long-lasting activating signal produced by NEM could consist in an interference of NEM with the compartimentation of calcium. Platelet shape change has been assumed to be mediated by a low and the release reaction by a higher increase in the concentration of cytoplasmic calcium (16-18). Cyclic AMP may interfere with shape change and release by stimulating the removal of calcium: it greatly accelerates the uptake of calcium into platelet membrane vesicles (19). An interference by NEM with calcium compartimentation would lead to an accumulation of cytoplasmic calcium which may be antagonized for some time but not completely prevented by CAMP-stimulated removal of cytoplasmic calcium. Inhibition of calcium uptake into sarcoplasmic reticulum (ZO), an equivalent of which may represent the dense tubular system of platelets (21), and the NEM-induced release of calcium from mitochondria (22) indicate, that NEM may act by such a mechanism. The extent of an accumulation of calcium mediated by NEM may be sufficient to evoke shape change but does not lead to serotonin release when platelets had been treated with indomethacin (4). In this aspect, the action of NEM differs from that of A 23187 which is suggested to stimulate the platelets by its direct access to internal calcium stores: A 23187 induces a release reaction also from platelets treated with indotnethacin. In contrast, NEM-induced serotonin release,but not the shape change,seems to depend on the formation
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of prostaglandin endoperoxides and thromboxane A2 which may act as calcium ionophores (23). Acknowledgements. The authors are indebted to Miss M. Jesper for expert technical assistance. This study was supported by the Deutsche Forschungsgemeinscgaft, Bad Godesberg, within the SFB 90 "Cardiovascultires System". REFERENCES 1.
ROBISON, C.W., MASON, R.G. and WAGNER, R.H. Effect of sulfhydryl inhibitors on platelet agglutinability. Proc. Sot. Exptl. Biol. Med. 113, 857861, 1963.
2.
HARBURY, C.B. and SCHRIER. S.L. Modification of platelet sulfhydryl groups. Thromb. Diath. haemorrh. 31, 469-484, 1974.
3.
BOOYSE, F.M. and RAFELSON, M.E. Regulation and mechanism of platelet aggregation. Annals N.Y. Acad. Sci. 201, 37-60, 1972.
4.
WURNER, P. and PATSCHEKE, H. Chemiluminescence in washed human platelets during prostaglandin-thromboxanesynthesis induced by N-ethylmaleimide and thimerosal. Thromb. Res. -19, 277-282, 1980.
5.
BORN, G.V.R. Quantitative investigations of the rapid swelling reaction of blood platelets. J. Physiol. 202, 93-94P, 1969.
6.
JAFARI, E., SALEEM, A., SHAIKH, B.S. and DEMERS, L.M. Effect of aspirin on prostaglandin synthesis by human platelets. Prostaglandins -12, 829835, 1976.
7.
BORN, G.V.R., DEARNLEY, R., FOULKS, J.G. and SHARP, D.E. Quantification of the morphological reaction of platelets to aggregating agents and of its reversal by aggregation inhibitors. J. Physiol. 280, 193-212, 1978.
8.
PATSCHEKE, H. and WURNER, P. Platelet activation detected by turbidometric shape-change analysis. Differential influence of cytochalasin B and prostaglandin EI. Thromb. Res. 12, 485-496, 1978.
9.
PATSCHEKE, H. Shape and functional properties of human platelets washed with acid citrate. Haemostasis, in press.
IO. WOLFE, S.M. and SHULMAN, N.R. Adenylcyclase activity in human platelets. Biochem. Biophys. Res. Commun. -35, 265-272, 1969. II. MILLS, D.C.B., McFARLANE, P.E. Stimulation of human platelet adenylate cyclase by prostaglandin D2. Thromb. Res. 5, 401-412, 1974. 12. MILLS, D.C.B. and SMITH, J.B. The influence on platelet aggregation of drugs that affect the accumulation of adensosine 3':5'-cyclic monophosphate in platelets. Biochem. J. 121, 185-196, 1971. 13. MILLS, D.C.B. Factors influencing the adenylate cyclase system in human blood platelets. In: Platelets and thrombosis. S.Sherry and A. Scriabine (eds.) Urban und Schwarzenberg: MUnchen - Berlin - Wien. 1974, p.45. 14. PATSCHEKE, H. and WURNER, P. Sequential effects of the thiol-oxidizing agent, diamide, on human platelets. Thromb. Res. -12, 609-618, 1978. 15. WURNER, P. and PATSCHEKE, H. Influence of the glutathion-oxidizing agent, diamide, on human platelet aggregation. Serono Symposia Vol. 15. G.G. Neri Serneri and C.R.M. Prentice (eds.) Academic Press: New York - London, p. 649, 1979.
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16. HOLMSEN, H. Are platelet shape change, aggregation and release reaction tangible manifestations of one basic platelet function? In: Platelets: production, function, transfusion and storage. M.G. Baldini and S. Ebbe (eds.) Grune & Stratton: New York, p. 207, 1974. 17. LeBRETON, G.C., SANDLER, W. and FEINBERG, H. The effect of D20 and chlorotatracycline on ADP-induced platelet shape change and aggregation. Thromb. Res. 8_,477-485, 1976. 18. MASSINI, P., KASER-GLANZMANN, R. and LDSCHER, E.F. Movement of calcium ions and their role in the activation of platelets. Thromb. haem. -40, 212-218, 1978. 19. KASER-GLANZMANN, R., JAKABOVA, M., GEORGE, J.N. and LUSCHER, E.F. Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3'5' cyclic monophosphate and proteinkinase. Biochim. Biophys. Acta 406, 429440, 1977. 20. HASSELBACH, W. and SERAYDARIAN, K. The role of sulfhydryl groups in calcium transport through the sarcoplasmic membranes of skeletal muscle. Biothem. Zeitschrift 345, $69-172, 1966. 21. WHITE, J.G. Interaction of membrane systems in blood platelets. Am. J. Pathol. 66, 295-312, 1972. -_ 22. PFEIFFER, D.R., KAUFFMANN, R.F. and ARDY, H.A. Effects of N-ethylmaleimide on the limited uptake of Ca2+, Mnb+ , and Sr2+ by rat liver mitochondria. J. Biol. Chem. 253, 4165-4171, 1978. 23. GERRARD, J.M., BUTLER, A.M., GRAFF, G., STODDARD, S.F. and WHITE, J.G. Prostaglandin endoperoxides promote calcium release from a platelet membrane fraction in vitro. Prostaglandins and Med. l_, 373-385, 1978.
BRIEF
COMMUNICATION
ANTAGONISTIC EFFECTS OF N-ETHYLMALEIMIDE ON PLATELETS TREATED WITH AGENTS THAT ARE KNOWN TO INCREASE LEVELS OF CYCLICAMP.
P. Wiirnerand H. Patscheke Abtlg. fUr Irnnunologieu. Serologie am Institut fi.lr Hygiene u. Med. Mikrobiologie des Klinikum Mannheim der UniversitBt Heidelberg, D 6, 5; D-68 Mannheim
(Received 1.10.1980; in revised form 9.12.1980. Accepted by Editor Hans Reuter)
INTRODUCTION The penetrating sulfhydryl-blocking agent N-ethylmaleimide (NEM) has been reported to interfere with platelet aggregation (1). Besides its inhibitory action, NEM at a low concentration promotes platelet shape change (2-4) which is an early event in platelet activation (5). At higher concentrations, NEM can also evoke prostaglandin synthesis (6) and, as its consequence, the release of serotonin (4). In addition to its ability to interfere with platelet aggregation, NEM exerts an anti-activating effect when platelets have been treated with agents known to elevate cyclic AMP. This anti-activation does not prevent NEM-induced shape change but postpones its onset. An elevation of cyclic AMP inhibits shape change induced by other stimuli such as ADP, thrombin, arachidonic acid, and A 23187 (7,8). A lag phase and an anti-activating effect prior to shape change does not occur with these stimuli of platelet activation. METHODS AND MATERIALS Platelet preparation. Discoid washed platelets were prepared from human ACDblood as has been described (8) with the exception that prostaglandin El was omitted throughout the preparation of platelets. The final platelet medium consisted of 103 mM NaCl, 45 mM Tris-HCl adjusted to pH 7.4 at 370 C, 5 mM KCl, 5 mM glucose, 1 mM sodium citrate, 1 mM CaC12, 0.5 mg albumin/ml and 50 ug apyrase/ml. Shape change of discoid platelets (2.108/ml) was followed in a 6-channel aggregometer equipped with interference filters 600 nm. The optical signal was amplified 5-fold and recorded after appropriate zero suppression. Three minutes prior to addition of a stimulus, 2 mM EDTA was added to prevent aggre201
202
NEM AND PLATELET SHAPE
Vo1.21, No.1/2
gation. The stirring mechanism (250 rpm) was switched off prior to the addition of a stimulus and at the end of an experiment to evaluate the stir-dependent change in light transmission (LT) which decreases the more, the more the platelets transform from a discoid to an irregular shape (8). Materials. Apyrase from potato (grade I), prostaglandin Ei and diamide were obtained from Sigma GmbH MUnchen, D 8021 Taufkirchen. Papaverinium chloride and N-ethylmaleimide were achieved from Merck, D 6100 Darmstadt. Dipyridamole was from Dr. Karl Thomae GmbH, D 7g5D Biberach. Prostaglandin D2 was a gift of the Upjohn Co, Kalamazoo, Michigan, USA. RESULTS Addition of NEM at a concentration of 50-100 uM to a suspension of discoid platelets resulted in changes of the optical signal which indicate the shape change of platelets (5,7,8). Shape change occurred at lower concentrations of NEM than those (>0.5 mM) which were required to evoke indcmethacin-sensitive formation of malondialdehyde and the release of serotonin. NEM-induced shape change is not inhibited by indomethacin or acetylsalicylic acid (4). Prostaglandin Ei (PGEi), PGD2, adenosine, papaverine and dipyridamole hardly inhibited NEM-induced shape change but postponed its onset (Fig. 1,I and Table I). During the lag phase prior to NEM-promoted shape change, the optical signal changed in a manner opposite to that characteristic for the transformation of discoid platelets to irregular spheres: light transmission (LT) and oscillation amplitude of the stirred platelet suspension increased (Fig. 1,I) as did the decrease in LT upon interruption of stirring. Treatment of platelets with PGEi (1 uM) and papaverine (10 uM) did not mimick this effect in the absence of NEM. A small lag phase prior to the shape change induced by 50 uM NEM was even observed when platelets had been treated with such a low concentration of PGEi as 0.5 nM. PGEi at 10 nM prolonged the time lapse to about 3 minutes. However, this lag phase did not exceed 3.5 minutes even when platelets were treated with 1 uM of PGEl combined with IO uM papaverine or 20 uM dipyridamole. When the concentration of NEM was increased, the time lapse up to the onset of shape change was shorter and disappeared at an NEM-concentration of about 2 mM. Shape change induced by diamide was also postponed after treatment of platelets with PGEi, adenosine, papaverine or dipyridamole. During the time lapse up to the onset of shape change, the oscillation amplitude and the LT as well as did the decrease in LT when the stirring increased (Fig. 1,II) mechanism was interrupted. PGEi at a high concentration (1 1 uM) causes changes in the optical signal which imitate a partial shape change (8). The anti-activating effect which was observed when diamide or NEM was added after PGEl was not only due to the reversal of the slight decrease in LT and oscillation amplitude promoted by PGEi, but did exceed the original level of LT. Moreover, the antiactivating effect of NEM or diamide was observed also at low concentrations of PGEi which per se had no influnece on platelet shape. DISCUSSION The transformation of discoid platelets to an irregular spheroid shape (shape change) can be followed by monitoring changes in optical signals. During shape change, the light transmission (LT) and the oscillation amplitude of a stirred platelet sample diminuish as well as the decrease in LT on stopping the stirrer (8). The change in LT which occurs on interruption of stirring seems to be particularly suited to estimate the extent to which platelets have transformed to a spheroid shape. Changes in the optical signal opposite to those observed during disc-to-sphere transformation indicate that
vo1.21,
No.1/2
203
NEM AND PLATELET SHAPE
TABLE 1 Time Lapse prior to NEM-Induced Shape Change after Treatment of Platelets with Agents Known to Increase Levels of Cyclic AMP. control
0 min*
5 nM PGD2
2.6 min
5 nM PGE,
2.4 min
5 nM PGEl + 5 pM papaverine
2.9 min
0.5 uM adenosine
2.0 min
5 nM PGEi + 20 uM dipyridamole
3.5 min
5 uM papaverine
0.8 min
0.5 uM adenosine + 5 uM papaverine3.0
20 uM dipyridamole
0.2 min
0.5 uM adenosine + 20 pM dipyridamole
min
3.3 min
* = time from the addition of 50 uM NEM to the onset of the decrease in LT. PGEi, PGD2, adenosine, papaverine, and dipyridamole were added 2 min prior to NEM to the stirred platelet suspension (2*108/ml, 370 C). Single values from one platelet preparation out of a series of qualitatively similar results.
I
‘01 mM NEM
0.5 mM diamide
l0 pM odenosin
Fig. 1. Shape change induced by NEM and diamide. Effect of agents which elevate platelet CAMP. I: NEM (0.1 mM) evokes shape change without serotonin release (upper curve). After treatment of platelets with PGEi and papaverine a lag phase up to the onset of the decrease in light transmission (LT) is observed. During this lag phase oscillation amplitude and LT increase (lower curve). II: Diamide (0.5 mM) evokes shape change without serotonin release (upper curve). Treatment of platelets with adenosine and dipyridamole postponed the onset of shape change (lower curve). During this lag phase changes in the,optical signal opposite to those during shape change occur.
discoid platelets have become more flat. This has been observed with acid citrate (pH 6.5) which exerts an anti-activating effect on platelets (9). Such an anti-activating effect also occurs prior to NEM-induced shape change when platelets had been exposed to PGE1, PGD2, adenosine, papaverine, or dipyridamole. PGEl (lo)',PGD2 (11) and adenosine(12) increase cAMPby stimulating ade-
204
NEM AND PLATELET SHAPE
Vo1.21, No.112
nylcyclase. Papaverine and dipyridamole interfere with c/VlP-degradation by inhibiting platelet phosphodiesterase (12). Since agents from both groups have similar effects and act synergistic when combined, it is most likely that an elevation of CAMP is the common cause for the anti-activating effect of NEM in platelets treated with these agents. NEM has been reported to enhance the PGEj-induced stimulation of adenylcyclase and to increase the steady state level of CAMP (13). However, an elevated level of CAMP alone seems not to be sufficient to mediate this anti-activation: discoid platelets do not become more flat upon an elevation of CAMP, e.g. by their exposure to the combined action of papaverine and PGEl, unless NEM is added. An anti-activating effect similar to that which preceeds NEM-induced shape change occurs when platelets with elevated CAMP are exposed to the thiol-oxidizing agent diamide. The similarity of the anti-activating effects of NEM and diamide may indicate that these agents mediate anti-activation and possibly also the following shape change by their interaction with common targets, presumably thiols. However, diamide-induced platelet activation differs in several aspects from that promoted by NEM. Diamide does, unlike NEM, induce or enhance platelet aggregation prior to its inhibitory effect (14,15). Moreover, diamide does not evoke serotonin release or prostaglandin synthesis even at a high concentration. In addition, diamide-induced shape change, but hardly that promoted by NEM, can be completely suppressed by a high concentration of PGE1. These differences may be the result of different reactivities of various target molecules towards the thiol-alkylating agent NEM and the bivalent thiol-oxidizing substance diamide. It is noteworthy that NEM (and diamide) can evoke shape change after a lag phase during which PGEj-treated platelets become even more discoid. When PGEltreated platelets are exposed to other platelet stimuli such as thrombin, shape change is reduced or completely prevented but no lag phase and no antiactivating effect occurs prior to the onset of shape change (7,8). The ability of NEM to evoke shape change after a time lapse can not be due to a decrease in CAMP mediated by NEM since the concentrations of NEM employed even augment the PGEl-induced increase in platelet CAMP (13). An alternative explanation is more plausible: besides its contribution to the anti-activation, NEM creates a long-lasting platelet-activating signal which may even increase with time. This long-lasting signal may finally overcome the initial antiactivation by CAMP and NEM. Platelet stimuli such as thrombin may only create a short-lasting triggering signal which is not maintained or.does at least not increase with time. The long-lasting activating signal produced by NEM could consist in an interference of NEM with the compartimentation of calcium. Platelet shape change has been assumed to be mediated by a low and the release reaction by a higher increase in the concentration of cytoplasmic calcium (16-18). Cyclic AMP may interfere with shape change and release by stimulating the removal of calcium: it greatly accelerates the uptake of calcium into platelet membrane vesicles (19). An interference by NEM with calcium compartimentation would lead to an accumulation of cytoplasmic calcium which may be antagonized for some time but not completely prevented by CAMP-stimulated removal of cytoplasmic calcium. Inhibition of calcium uptake into sarcoplasmic reticulum (ZO), an equivalent of which may represent the dense tubular system of platelets (21), and the NEM-induced release of calcium from mitochondria (22) indicate, that NEM may act by such a mechanism. The extent of an accumulation of calcium mediated by NEM may be sufficient to evoke shape change but does not lead to serotonin release when platelets had been treated with indomethacin (4). In this aspect, the action of NEM differs from that of A 23187 which is suggested to stimulate the platelets by its direct access to internal calcium stores: A 23187 induces a release reaction also from platelets treated with indotnethacin. In contrast, NEM-induced serotonin release,but not the shape change,seems to depend on the formation
Vo1.21,
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205
of prostaglandin endoperoxides and thromboxane A2 which may act as calcium ionophores (23). Acknowledgements. The authors are indebted to Miss M. Jesper for expert technical assistance. This study was supported by the Deutsche Forschungsgemeinscgaft, Bad Godesberg, within the SFB 90 "Cardiovascultires System". REFERENCES 1.
ROBISON, C.W., MASON, R.G. and WAGNER, R.H. Effect of sulfhydryl inhibitors on platelet agglutinability. Proc. Sot. Exptl. Biol. Med. 113, 857861, 1963.
2.
HARBURY, C.B. and SCHRIER. S.L. Modification of platelet sulfhydryl groups. Thromb. Diath. haemorrh. 31, 469-484, 1974.
3.
BOOYSE, F.M. and RAFELSON, M.E. Regulation and mechanism of platelet aggregation. Annals N.Y. Acad. Sci. 201, 37-60, 1972.
4.
WURNER, P. and PATSCHEKE, H. Chemiluminescence in washed human platelets during prostaglandin-thromboxanesynthesis induced by N-ethylmaleimide and thimerosal. Thromb. Res. -19, 277-282, 1980.
5.
BORN, G.V.R. Quantitative investigations of the rapid swelling reaction of blood platelets. J. Physiol. 202, 93-94P, 1969.
6.
JAFARI, E., SALEEM, A., SHAIKH, B.S. and DEMERS, L.M. Effect of aspirin on prostaglandin synthesis by human platelets. Prostaglandins -12, 829835, 1976.
7.
BORN, G.V.R., DEARNLEY, R., FOULKS, J.G. and SHARP, D.E. Quantification of the morphological reaction of platelets to aggregating agents and of its reversal by aggregation inhibitors. J. Physiol. 280, 193-212, 1978.
8.
PATSCHEKE, H. and WURNER, P. Platelet activation detected by turbidometric shape-change analysis. Differential influence of cytochalasin B and prostaglandin EI. Thromb. Res. 12, 485-496, 1978.
9.
PATSCHEKE, H. Shape and functional properties of human platelets washed with acid citrate. Haemostasis, in press.
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