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Compound 4880 is a potent inhibitor of human platelet aggregation antagonizing the calmodulin-dependent platelet reaction in vitro
THROMBOSIS RESEARCH 36; 475-480, 1984 0049-3848/84 $3.00 + .CO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.
BRIEF
COMMUNICATION
COMPOUND 48/80 IS A POTENT INHIBITOR OF HUMAN PLATELET AGGREGATION ANTAGONIZING THE CALMODULIN-DEPENDENT PLATELET REACTION IN VITRO
J. Heinrich
and R. E . Zimmermann
Department of Physiology, University of Mtinster, Robert -Koch -Str. 28, 44 Miinster, FRG (Received 18.12.1983; Accepted in revised form 17.7.1984 by Editor D.L. Heene) (Received in final form by Executive Editorial Office 4.9.1984) INTRODUCTION Calmodulin, a nearly ubiquitous small molecular weight protein, plays an important role in the regulation of many calcium-dependent enzymes (1). Human platelets contain large quantities of calmodulin, which modulates the action of platelet phosphodiesterase, phospholipase A2 and myosin light chain kinase (2). Compound 48/80, a mixture of condensation products of N-methyl-pmethoxyphenethylamine with formaldehyde, is known as a mastocyte degranulating agent. It was recently reported to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and erythrocyte Ca2+-transport ATPase. It does not affect the basal activity of both enzymes in concentrations up to 300pg/ml. This fact characterizes compound 48180 as the most specific inhibitor of the Ca2+-calmodulin complex known thus far (3,4). The present study exhibits results, which show the effect of compound 48/80 on the action of several platelet stimuli, using distinct pathways of aggregation. MATERIALS
AND METHODS
Adenosine-5’-diphosphate (ADP) was obtained from E. Merck, Darmstadt Compound 48/80, ristocetin and heparin from porcine intestinal mucosa (gr. I) were purchased from Sigma Chemie, Munchen. Collagen was obtained from Hormon-Chemie, Munchen and thrombin from Behringwerke, Marburg. PAF-Acether (l-O-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) was kindly provided by Prof. H. K. Mangold (Institute for Biochemistry and Technology, Miinster). It was semisynthetically prepared from ratfish liver oil (5). PAF-Acether was dissolved in 60% ethanol as stock solution, which was kept at -2OOC. Collagen solution was diluted prior to the test with isotoKey words:
Platelet
aggregation,
MDA formation, 475
calmodulin
antagonist,
476
CALMODULIN ANTAGONIST
Vo1.36, No.5
nit glucose solution; ADP stock solution was diluted for daily use with 0. 9% saline. In the applied quantities (1 - 10 pi/O. 3 ml PRP) the solvents did not effect detectable aggregation of platelets. Platelet rich plasma (PRP) was prepared from titrated whole blood (1: 9), obtained from healthy volunteers, who had not ingested acetylsalicic acid or other nonsteroidal antiinflammatory drugs for the last 10 days. Blood was carefully collected by venipuncture and centrifuged at 1OOxg for 15 minutes, Platelet counts of the PRP were adjusted to 300 OOO/pl using a Thrombocounter-C (Coulter Electronics, Krefeld). Each aggregation test tube was fi.lled with 300~1 PRP, sealed in order to minimize loss of CO2 and allowed to stand at room temperature for 30 minutes to counteract platelet preactivation by blood sampling. Platelet aggregation tests were performed according to G. V. R. Born (6) at 3’7OC and 500 rpm using a dual channel aggregometer (LAbor GmbH, Hamburg) with microcuvet tes. PRP was incubated with the respective amount of compound 48/80 for 10 minutes at 37OC before adding the aggregating agent, Malondialdehyde (MDA) formation was determined according to M. J. Stuart et al (7) by measuring the absorption at 532 nm of the reaction product of the plateof malondialdehyde and 2 -thiobarbituric acid. Before incubation let suspension in phosphate buffered saline, 0.15 m, pH 7.4 with 0.5 U/ml thrombin, a preincubation with different concentrations of compound 48180 for lominutes was performed. MDA production of platelets without calmodulin antagonist were assessed 100 percent and that of plasma residue after aspiration of supernatant 0 per cent, All tests were carried out within 3 hours after venipuncture. Inhibition of each stimulant was studied with platelets from 5 different donors.
RESULTS FIG. 1 shows the effect of incubation time of 40ug/ml compound 48/80 with PRP prior to the addition of 5 pM ADP. Pretreatment of platelets with compound48/80 for 1 minute before inducing aggregation resulted in 24% inhibition of aggregation, incubation for 5 minutes in 85. 7% inhibition. Adding the aggregating agent after an incubation time of 10 minutes showed complete inhition. The inhibition of human platelet aggregation induced by 0. 2 U/ml thrombin depending on the concentration of compound48/80 as a typical example for one of the dose-response curves is shown in FIG. 2. The extent of inhibition was expressed as percentage of maximal unblocked aggregation. The amount of compound48/80, which inhibits 50 per cent of normal aggregation (IC50), was ascertained graphically; the IC50 in case of 0.2 U/ml thrombin is 4.2 pg/ml. Platelets, which were incubated with 45ug/ml compound 48/80 for 10 minutes at 37oC, could not be aggregated by 5 uM ADP; addition of another 15 uM ADP to the PRP resulted in 36.4% of maximal aggregation. This indicates that platelets are not irreversibly blocked and IC50-values are only valid for the applied concentrations of the aggregating agents, which were chosen so that complete aggregation was just achieved. On the contrary platelets, which were completely aggregated by 5 uM ADP, deaggregated within 20 minutes
477
CALMODULIN ANTAGONIST
Vo1.36, No.5
Transmission
5 min.incubation
IminI
t FIG. 1 Effect of time of PRP platelet aggregation.
treatment
with compound48/80
on 5 uM ADP-induced
100 _
Rrcenia of nwma$_ aggregation
20 -
1
f
1 I 0
5
IO FIG.
15
2
Inhibition of 0. 2 U/ml thrombin-induced platelet aggregation with compound 48/80 for 10 minutes. Mea&SD; five dondors. after
B
20 Compound 48/80 Tpg/mll by preincubation
the addition of 1 mg/ml compound48/80. Although calcium plays an important role in the regulation of cell activicomplexed by citrate for blood anticoagulation, the ties and Ca2+ was partially usage of heparin (2U/ml), which does not alter physiological Ca2+-concentraagent. tion, did not result in a change of IC50 using 2 uM ADP as aggregating Various stimuli for human platelet aggregation following different mecha and the used aggreganism were applied. The IC50- values for compound48/80 ting drugs are listed in TABLE 1 . Malondialdehyde (MDA) formation of platelet suspensions in phosphate which were stimulated by 0.5 U/ml buffered saline containing 3 x 108 platelets, thrombin, was found to be 0. 98k 0. 11 nM. Its inhibition by preincubation with
478
CALMODULIN ANTAGONIST
compound48/80
was dose dependent;
IC50 was 43.8 ug/ml
TABLE
Vo1.36, No.5 as shown in FIG. 3.
1
Amounts of Compound 48/80 for halfmaximal Inhibition (IC50) of Platelet Aggregation induced by different Stimuli Aggregating Agent
ADP
Thrombin 0.2 U/ml
Collagen
PAF -Acether
3uM
2 ug/ml
2uM
IC50 ug/ml
12.9
4.2
7.3
15.4
MDA Inmol/ 3*10*p1at.1
Q5
FIG. 3 Inhibition of 0.5 U/ml thrombin-stimulated bation with compound 48/80 for 10 minutes.
[ug/mlI platelet MDA formation Mean&SD; five donors.
by preincu-
which was observable by a short decrease The shape change of platelets, of light transmission immediately after the addition of the stimulants ADP, PAF-Acether and thrombin, was not altered by pretreatment with amounts of compound 48/80 causing complete inhibition of aggregation. The antibiotic ristocetin is necessary for the in vitro aggregation of human platelets, involving the binding of F VIII/van Willebrand factor to its platelet membrane receptor. Platelet aggregation, induced by 2 mg/ml ristocetin, was not affected by concentrations of compound 48/80 between 10v4and lmg/ml
DISCUSSION which binds to platelet calmoCompound 48/80 is a cationic amphiphile, dulin. According to a hypothesis of K. Gietzen et al (8) calmodulin-regulated enzymes possess an inhibitory pepetide sequence. The effect of this unit that is assumed to be positively charged and to have a hydrophobic site is suppressed
Vo1.36, No.5
479
CALMODULIN ANTAGONIST
by interaction with its anionic activator calmodulin, in its active form in complex with Ca2+. Compound 48 / 80 acts in competing with the enzyme for the regulator protein. It is composed of a great number of condensation products of N-methyl-pmethoxyphenethylamine with formaldehyde with different chain length. The ave rage molecular weight was determined to be 1300 (9). The most active oligomers in degranulating histamin from mastocytes are the hexamer and heptamer which corresponds with the inhibitory effect upon calmodulin dependent enzyme activities (10). Human platelet aggregation induced by ADP, thrombin, collagen and PAF Acether was shown to be inhibitable in a dose dependent manner by compound 48/80. Though thrombin and collagen are stronger aggregating agentsthanADP, for the applied concentrations lie in the same their corresponding IC 50-values range. Addition of the drug together with the platelet stimulant yielded in nearly no reduction of aggregation; pretreatment of PRP with the inhibitor for 10 minutes showed the total effect. Complete aggregation, induced by any of the agents, was reversible by 1 mg/ml compound48/80. Exact mechanism of platelet deaggregation still remains to be elucidated. It seems to be more likely that the highly concentrated cationic amphiphile disrupts the association of fibrinogen to its platelet receptor, than the inhibition of calmodulin-dependent processes being responsible for deaggregation. The variety of aggregating agents was chosen in order to study the effect of compound48/80 on different pathways of aggregation. The results of the aggregation tests as well as the measurement of MDA formation show that not only platelet aggregation depending on arachidonic metabolism product thromboxane A2, which may trigger the contractile system, but also prostanoid-independent pathways like that one stimulated by PAF-Acether were blocked. K. Kao et al (11) investigated the effect of the calmodulin inhibitors trifluoperazine and chlorpromazine on binding of F VIII/van Willebrand factor to its platelet membrane receptor. They found that the total number of accessible binding sites on each platelet was significantly reduced. This suggests that the ristocetin-induced formation of platelet plug, which was not inhibited by compound 48/80 up to 1 mg/ml, is not exclusively mediated by F VIII/vWF, but may take place via a calmodulin-independent passive agglutination mechanism. The fact that shape change, occuring after the addition of thrombin, ADP and PAF-Acether to PRP, is not inhibited by compound48/80, was also observed in thrombinand PAF -Acether-stimulated platelets pretreated with trifluoperazine (12, 13). These findings seem to be in contradiction with studies of K. Kao et al (ll), who reported that platelets, incubated with 50 uM trifluoperazine at 370C for 5 minutes , became spheroid in shape. These results might be explained by an additional effect of a shape change mechanism, whichis not dependent on the calmodulin-activated platelet enzymes. It may be associated with the calmodulin-independent formation of phosphatidic acid and phosphorylation of a 40 000 dalton protein (13). ACKNOWLEDGEMENT The authors wish to thank Berthold B. Baldus study and the preparation of the manuscript.
for helpful
suggestions
in the
480
CALMODULIN ANTAGONIST
Vo1.36, No.5
REFERENCES 1.
CHEUNG, W. Y. Calmodulin plays a pivotal role in cellular Science -207, 19-27, 1980.
2.
MUSZBEK, L., KUZNICKI, J., SZABO, T. and DRABIKOWSKI, W. Troponin C like protein of blood platelets. FEBS Lett. 80, 308-312, 1977.
3.
GIETZEN, K. , SANCHEZ-DELGADO, E. and BADER, H. Compound48/ 80: A powerful and specific inhibitor of calmodulin-dependent Ca2+-transport ATPase. IRCS Medical Science: Biochemistry -’ 11 12-13, 1983.
4.
GIETZEN, K. and ADAMCZYK-ENGELMANN, P. Compound48/80: A highly specific and powerful anti-calmodulin drug. Naunyn-Schmiedebergs Arch. Pharmacol. 322 (Supplement), R4, 1983.
5.
MURAMATSU, T., TOTANI, N. and MANGOLD, H. K. A facile method for the preparation of 1-0-alkyl-2-O-acetoyl-sn-glycero-3-phosphocholine (platelet activating factor). Chem. Phys. Lipids -29, 121-127, 1981.
6.
BORN, G. V. R. Quantitative investigations into the aggregation platelets. J. Physiol. (London) -J 162 67-68, 1962.
7.
STUART, M. J., MURPHY, S. and OSKI, F. A. A simple nonradioisotope technic for the determination of platelet life-span. New Engl. J. Med. 292, 1310-1313, 1975.
8.
GIETZEN, K., SADORF, I. and BADER, H. A model for the regulation of the calmodulin-dependent enzyme erythrocyte Ca2+-transport ATPase and brain phosphodiesterase by activators and inhibitors. Biochem. J. 207 541-548,1982. -’
9.
READ, G. W. and LENNEN, J. F. Molecular weight studies on the active constituents of compound48/80, J. Med. Chem. -15, 320-323, 1972.
10.
P., WUTHRICH, A., KONGIETZEN, K., ADAMCZYK-ENGELMANN, STANTINOVA, A. and BADER, H. Compound48/80 is a selective and powerful inhibitor of calmodulin-regulated functions. Biochim. Biophys. Acta 736,109-118, 1983.
11.
KAO, K.J., SOMMER, J. R. and PIZZO, S. Modulation of platelet shape Nature 292, 82-84, and membrane receptor by Ca2’ -calmodulin complex. -1981.
12.
WHITE, G. C. II and RAYNOR, S. T. The effects of trifluoperazine, an inhibitor of calmodulin, on platelet function. Thromb. Res. .-18, 279-284, 1980.
13.
LAPETINA, E. G. and SIEGEL, F. L. Shape change induced in human platelets by platelet-activating factor. J. Biol. Chem. 258, 7241-7244, 1983.
regulation.
of blood
BRIEF
COMMUNICATION
COMPOUND 48/80 IS A POTENT INHIBITOR OF HUMAN PLATELET AGGREGATION ANTAGONIZING THE CALMODULIN-DEPENDENT PLATELET REACTION IN VITRO
J. Heinrich
and R. E . Zimmermann
Department of Physiology, University of Mtinster, Robert -Koch -Str. 28, 44 Miinster, FRG (Received 18.12.1983; Accepted in revised form 17.7.1984 by Editor D.L. Heene) (Received in final form by Executive Editorial Office 4.9.1984) INTRODUCTION Calmodulin, a nearly ubiquitous small molecular weight protein, plays an important role in the regulation of many calcium-dependent enzymes (1). Human platelets contain large quantities of calmodulin, which modulates the action of platelet phosphodiesterase, phospholipase A2 and myosin light chain kinase (2). Compound 48/80, a mixture of condensation products of N-methyl-pmethoxyphenethylamine with formaldehyde, is known as a mastocyte degranulating agent. It was recently reported to be a potent inhibitor of the calmodulin-activated fraction of brain phosphodiesterase and erythrocyte Ca2+-transport ATPase. It does not affect the basal activity of both enzymes in concentrations up to 300pg/ml. This fact characterizes compound 48180 as the most specific inhibitor of the Ca2+-calmodulin complex known thus far (3,4). The present study exhibits results, which show the effect of compound 48/80 on the action of several platelet stimuli, using distinct pathways of aggregation. MATERIALS
AND METHODS
Adenosine-5’-diphosphate (ADP) was obtained from E. Merck, Darmstadt Compound 48/80, ristocetin and heparin from porcine intestinal mucosa (gr. I) were purchased from Sigma Chemie, Munchen. Collagen was obtained from Hormon-Chemie, Munchen and thrombin from Behringwerke, Marburg. PAF-Acether (l-O-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) was kindly provided by Prof. H. K. Mangold (Institute for Biochemistry and Technology, Miinster). It was semisynthetically prepared from ratfish liver oil (5). PAF-Acether was dissolved in 60% ethanol as stock solution, which was kept at -2OOC. Collagen solution was diluted prior to the test with isotoKey words:
Platelet
aggregation,
MDA formation, 475
calmodulin
antagonist,
476
CALMODULIN ANTAGONIST
Vo1.36, No.5
nit glucose solution; ADP stock solution was diluted for daily use with 0. 9% saline. In the applied quantities (1 - 10 pi/O. 3 ml PRP) the solvents did not effect detectable aggregation of platelets. Platelet rich plasma (PRP) was prepared from titrated whole blood (1: 9), obtained from healthy volunteers, who had not ingested acetylsalicic acid or other nonsteroidal antiinflammatory drugs for the last 10 days. Blood was carefully collected by venipuncture and centrifuged at 1OOxg for 15 minutes, Platelet counts of the PRP were adjusted to 300 OOO/pl using a Thrombocounter-C (Coulter Electronics, Krefeld). Each aggregation test tube was fi.lled with 300~1 PRP, sealed in order to minimize loss of CO2 and allowed to stand at room temperature for 30 minutes to counteract platelet preactivation by blood sampling. Platelet aggregation tests were performed according to G. V. R. Born (6) at 3’7OC and 500 rpm using a dual channel aggregometer (LAbor GmbH, Hamburg) with microcuvet tes. PRP was incubated with the respective amount of compound 48/80 for 10 minutes at 37OC before adding the aggregating agent, Malondialdehyde (MDA) formation was determined according to M. J. Stuart et al (7) by measuring the absorption at 532 nm of the reaction product of the plateof malondialdehyde and 2 -thiobarbituric acid. Before incubation let suspension in phosphate buffered saline, 0.15 m, pH 7.4 with 0.5 U/ml thrombin, a preincubation with different concentrations of compound 48180 for lominutes was performed. MDA production of platelets without calmodulin antagonist were assessed 100 percent and that of plasma residue after aspiration of supernatant 0 per cent, All tests were carried out within 3 hours after venipuncture. Inhibition of each stimulant was studied with platelets from 5 different donors.
RESULTS FIG. 1 shows the effect of incubation time of 40ug/ml compound 48/80 with PRP prior to the addition of 5 pM ADP. Pretreatment of platelets with compound48/80 for 1 minute before inducing aggregation resulted in 24% inhibition of aggregation, incubation for 5 minutes in 85. 7% inhibition. Adding the aggregating agent after an incubation time of 10 minutes showed complete inhition. The inhibition of human platelet aggregation induced by 0. 2 U/ml thrombin depending on the concentration of compound48/80 as a typical example for one of the dose-response curves is shown in FIG. 2. The extent of inhibition was expressed as percentage of maximal unblocked aggregation. The amount of compound48/80, which inhibits 50 per cent of normal aggregation (IC50), was ascertained graphically; the IC50 in case of 0.2 U/ml thrombin is 4.2 pg/ml. Platelets, which were incubated with 45ug/ml compound 48/80 for 10 minutes at 37oC, could not be aggregated by 5 uM ADP; addition of another 15 uM ADP to the PRP resulted in 36.4% of maximal aggregation. This indicates that platelets are not irreversibly blocked and IC50-values are only valid for the applied concentrations of the aggregating agents, which were chosen so that complete aggregation was just achieved. On the contrary platelets, which were completely aggregated by 5 uM ADP, deaggregated within 20 minutes
477
CALMODULIN ANTAGONIST
Vo1.36, No.5
Transmission
5 min.incubation
IminI
t FIG. 1 Effect of time of PRP platelet aggregation.
treatment
with compound48/80
on 5 uM ADP-induced
100 _
Rrcenia of nwma$_ aggregation
20 -
1
f
1 I 0
5
IO FIG.
15
2
Inhibition of 0. 2 U/ml thrombin-induced platelet aggregation with compound 48/80 for 10 minutes. Mea&SD; five dondors. after
B
20 Compound 48/80 Tpg/mll by preincubation
the addition of 1 mg/ml compound48/80. Although calcium plays an important role in the regulation of cell activicomplexed by citrate for blood anticoagulation, the ties and Ca2+ was partially usage of heparin (2U/ml), which does not alter physiological Ca2+-concentraagent. tion, did not result in a change of IC50 using 2 uM ADP as aggregating Various stimuli for human platelet aggregation following different mecha and the used aggreganism were applied. The IC50- values for compound48/80 ting drugs are listed in TABLE 1 . Malondialdehyde (MDA) formation of platelet suspensions in phosphate which were stimulated by 0.5 U/ml buffered saline containing 3 x 108 platelets, thrombin, was found to be 0. 98k 0. 11 nM. Its inhibition by preincubation with
478
CALMODULIN ANTAGONIST
compound48/80
was dose dependent;
IC50 was 43.8 ug/ml
TABLE
Vo1.36, No.5 as shown in FIG. 3.
1
Amounts of Compound 48/80 for halfmaximal Inhibition (IC50) of Platelet Aggregation induced by different Stimuli Aggregating Agent
ADP
Thrombin 0.2 U/ml
Collagen
PAF -Acether
3uM
2 ug/ml
2uM
IC50 ug/ml
12.9
4.2
7.3
15.4
MDA Inmol/ 3*10*p1at.1
Q5
FIG. 3 Inhibition of 0.5 U/ml thrombin-stimulated bation with compound 48/80 for 10 minutes.
[ug/mlI platelet MDA formation Mean&SD; five donors.
by preincu-
which was observable by a short decrease The shape change of platelets, of light transmission immediately after the addition of the stimulants ADP, PAF-Acether and thrombin, was not altered by pretreatment with amounts of compound 48/80 causing complete inhibition of aggregation. The antibiotic ristocetin is necessary for the in vitro aggregation of human platelets, involving the binding of F VIII/van Willebrand factor to its platelet membrane receptor. Platelet aggregation, induced by 2 mg/ml ristocetin, was not affected by concentrations of compound 48/80 between 10v4and lmg/ml
DISCUSSION which binds to platelet calmoCompound 48/80 is a cationic amphiphile, dulin. According to a hypothesis of K. Gietzen et al (8) calmodulin-regulated enzymes possess an inhibitory pepetide sequence. The effect of this unit that is assumed to be positively charged and to have a hydrophobic site is suppressed
Vo1.36, No.5
479
CALMODULIN ANTAGONIST
by interaction with its anionic activator calmodulin, in its active form in complex with Ca2+. Compound 48 / 80 acts in competing with the enzyme for the regulator protein. It is composed of a great number of condensation products of N-methyl-pmethoxyphenethylamine with formaldehyde with different chain length. The ave rage molecular weight was determined to be 1300 (9). The most active oligomers in degranulating histamin from mastocytes are the hexamer and heptamer which corresponds with the inhibitory effect upon calmodulin dependent enzyme activities (10). Human platelet aggregation induced by ADP, thrombin, collagen and PAF Acether was shown to be inhibitable in a dose dependent manner by compound 48/80. Though thrombin and collagen are stronger aggregating agentsthanADP, for the applied concentrations lie in the same their corresponding IC 50-values range. Addition of the drug together with the platelet stimulant yielded in nearly no reduction of aggregation; pretreatment of PRP with the inhibitor for 10 minutes showed the total effect. Complete aggregation, induced by any of the agents, was reversible by 1 mg/ml compound48/80. Exact mechanism of platelet deaggregation still remains to be elucidated. It seems to be more likely that the highly concentrated cationic amphiphile disrupts the association of fibrinogen to its platelet receptor, than the inhibition of calmodulin-dependent processes being responsible for deaggregation. The variety of aggregating agents was chosen in order to study the effect of compound48/80 on different pathways of aggregation. The results of the aggregation tests as well as the measurement of MDA formation show that not only platelet aggregation depending on arachidonic metabolism product thromboxane A2, which may trigger the contractile system, but also prostanoid-independent pathways like that one stimulated by PAF-Acether were blocked. K. Kao et al (11) investigated the effect of the calmodulin inhibitors trifluoperazine and chlorpromazine on binding of F VIII/van Willebrand factor to its platelet membrane receptor. They found that the total number of accessible binding sites on each platelet was significantly reduced. This suggests that the ristocetin-induced formation of platelet plug, which was not inhibited by compound 48/80 up to 1 mg/ml, is not exclusively mediated by F VIII/vWF, but may take place via a calmodulin-independent passive agglutination mechanism. The fact that shape change, occuring after the addition of thrombin, ADP and PAF-Acether to PRP, is not inhibited by compound48/80, was also observed in thrombinand PAF -Acether-stimulated platelets pretreated with trifluoperazine (12, 13). These findings seem to be in contradiction with studies of K. Kao et al (ll), who reported that platelets, incubated with 50 uM trifluoperazine at 370C for 5 minutes , became spheroid in shape. These results might be explained by an additional effect of a shape change mechanism, whichis not dependent on the calmodulin-activated platelet enzymes. It may be associated with the calmodulin-independent formation of phosphatidic acid and phosphorylation of a 40 000 dalton protein (13). ACKNOWLEDGEMENT The authors wish to thank Berthold B. Baldus study and the preparation of the manuscript.
for helpful
suggestions
in the
480
CALMODULIN ANTAGONIST
Vo1.36, No.5
REFERENCES 1.
CHEUNG, W. Y. Calmodulin plays a pivotal role in cellular Science -207, 19-27, 1980.
2.
MUSZBEK, L., KUZNICKI, J., SZABO, T. and DRABIKOWSKI, W. Troponin C like protein of blood platelets. FEBS Lett. 80, 308-312, 1977.
3.
GIETZEN, K. , SANCHEZ-DELGADO, E. and BADER, H. Compound48/ 80: A powerful and specific inhibitor of calmodulin-dependent Ca2+-transport ATPase. IRCS Medical Science: Biochemistry -’ 11 12-13, 1983.
4.
GIETZEN, K. and ADAMCZYK-ENGELMANN, P. Compound48/80: A highly specific and powerful anti-calmodulin drug. Naunyn-Schmiedebergs Arch. Pharmacol. 322 (Supplement), R4, 1983.
5.
MURAMATSU, T., TOTANI, N. and MANGOLD, H. K. A facile method for the preparation of 1-0-alkyl-2-O-acetoyl-sn-glycero-3-phosphocholine (platelet activating factor). Chem. Phys. Lipids -29, 121-127, 1981.
6.
BORN, G. V. R. Quantitative investigations into the aggregation platelets. J. Physiol. (London) -J 162 67-68, 1962.
7.
STUART, M. J., MURPHY, S. and OSKI, F. A. A simple nonradioisotope technic for the determination of platelet life-span. New Engl. J. Med. 292, 1310-1313, 1975.
8.
GIETZEN, K., SADORF, I. and BADER, H. A model for the regulation of the calmodulin-dependent enzyme erythrocyte Ca2+-transport ATPase and brain phosphodiesterase by activators and inhibitors. Biochem. J. 207 541-548,1982. -’
9.
READ, G. W. and LENNEN, J. F. Molecular weight studies on the active constituents of compound48/80, J. Med. Chem. -15, 320-323, 1972.
10.
P., WUTHRICH, A., KONGIETZEN, K., ADAMCZYK-ENGELMANN, STANTINOVA, A. and BADER, H. Compound48/80 is a selective and powerful inhibitor of calmodulin-regulated functions. Biochim. Biophys. Acta 736,109-118, 1983.
11.
KAO, K.J., SOMMER, J. R. and PIZZO, S. Modulation of platelet shape Nature 292, 82-84, and membrane receptor by Ca2’ -calmodulin complex. -1981.
12.
WHITE, G. C. II and RAYNOR, S. T. The effects of trifluoperazine, an inhibitor of calmodulin, on platelet function. Thromb. Res. .-18, 279-284, 1980.
13.
LAPETINA, E. G. and SIEGEL, F. L. Shape change induced in human platelets by platelet-activating factor. J. Biol. Chem. 258, 7241-7244, 1983.
regulation.
of blood